Leishmania survives by exporting miR-146a from infected to resident cells to subjugate inflammation

Research Article

Leishmania survives by exporting miR-146a from infected

to resident cells to subjugate inﬂammation

Satarupa Ganguly

* , Bartika Ghoshal

*, Ishani Banerji

1,2,

* , Shreya Bhattacharjee

1,2

, Sreemoyee Chakraborty

1,2

Avijit Goswami

, Kamalika Mukherjee

, Suvendra N Bhattacharyya

1,2

Leishmania donovani, the causative agent of visceral leishman-

iasis, infects and resides within tissue m acropha ge cells. It is not

clear how the parasite infected cells crosstalk with t he non-

infected cells to regulate the infection process. During infection,

Leishmania adopts a dual strategy for its survival by regulating

the intercellular transport of host miRNAs to restrict inﬂam-

mation. The parasite, by preventing mitochondrial function of host

cells, restricts the entry of liver cell derived miR-122–containing

extracellular vesicles in infected macrophages to curtail the in-

ﬂammatory response associated with miR-122 entry. On contrary,

the parasite up-regulates the export of miR-146a from the infected

macrophages. The miR-146a, associated with the extracellular

vesicles released by infected cells, restricts miR-122 production

in hepatocytes while polarizing neighbouring na

ıve macro-

phages to the M2 state by affecting the cytokine expression. On

entering the recipient macrophages, miR-146a dominates the

miRNA antagonist RNA-binding protein HuR to inhibit the

expression of proinﬂammatory cytokine mRNAs having HuR-

interacting AU-rich elements whereas up-regulates anti-

inﬂammatory IL-10 by exporting the miR-21 to polarize the

recipient cells to M2 stage.

DOI 10.26508/lsa.202101229 | Received 8 September 2021 | Revised 7 February

2022 | Accepted 8 February 2022 | Published online 24 February 2022

Introduction

Leishmania donovani (Ld) is the causative agent of visceral leish-

maniasis that affects a large portion of the human population in the

Indian subcontinent and also in sub-Saharan Africa (Lenk et al, 2018).

The apicomplexan parasite has a dual-stage life cycle and lives in the

gut of the sandﬂy vector as promastigotes. The promastigote changes

to amastigote stage after entering the mammalian host macrophage

cells (Sunter & Gull, 2017). The promastigotes enter the mammalian

host with the saliva of the sandﬂy vector introduced during the blood

meal and subsequently make entry into the hepatic tissue where

they infect the Kupffer cells at the initial stage of infection before the

infection load is transferred to the spleen (Walker et al, 2014). The Ld

parasite lives in the tissue macrophages within a specialized sub-

cellular structure called parasitophorous vacuole and alters the

signalling components of the infected macrophages to polarize to

M2 stage. Ld ensures low expression of proinﬂammatory cytokines

like IL-1β and TNF-α, whereas the anti-inﬂammatory cytokines IL-10

and IL-4 expression get enhanced in the infected host (Mukherjee

et al, 2013). The status of the noninfected macrophages present in

the infection niche is not clear, but it is certain that the nonin-

fected macrophages present in the infection niche should not

get activated to ensure the overall dominance of an anti-

inﬂammatory res ponse that needs to b e maintained i n the in-

fected tissue. How the parasite, which remains within the specialized

vacuole structure o f the infected host macrophage, cross-

communicates with the resident noninfected macrophages to

suppress expression of inﬂammatory cytok ines is an important

question to explore. In this context, the extracellular signals derived

from resident noninfected macrophages and hepatocytes that can

activate the infected macrophage should also be counteracted

within the infected host cell milieu.

Extracellular vesicles (EVs) are released by different types of

mammalian cells that are used primarily by mammalian immune

cells to cross-communicate the cellular status across cell boundaries

(Regev-Rudzki et al, 2013; Fernandez-Messina et al, 2015). It is known

that the infected cell EVs can be used to transfer the parasite derived

and host factors to target speciﬁc pathways in neighbouring cells

to ensure establishment of systematic infection and its propaga-

tion (Regev-Rudzki et al, 2013; Kalluri & LeBleu, 2020). miRNAs are

important post-transcriptional regulators of gene expression that

are expressed in a cell ty pe and stage speciﬁcmannerinmam-

malian hosts (Bartel, 2018). miRNAs are also known to be com-

municated across the cell boundary to affect neigh bouring cell

fates. Therefor e, if communicat ed from the infected host cells,

miRNAs, a s an epigenetic signal, could alter the gene expression

process in neighbouring cells. The role of speciﬁc miRNAs in regu-

lation of expression of pro or anti-inﬂammatory pathway components

RNA Biology Research Laboratory, Molecular Genetics Division, Council of Scientiﬁc and Industrial Research (CSIR)-Indian Institute of Chemical Biology, Kolkata, India

Academy of Scientiﬁc and Innovative Research (AcSIR), CSIR-Human Resource Development Centre , (CSIR-HRDC) Campus, Ghaziabad, India

Correspondence: [email protected]; suvendra@ iicb.res.in

*Satarupa Ganguly, Bartika Ghoshal, and Ishani Banerji contrib uted equally to this work.

on 6 September, 2024life-science-alliance.org Downloaded from

http://doi.org/10.26508/lsa.202101229Published Online: 24 February, 2022 | Supp Info:

in mammalian immune cells has already been studied (Lindsay,

2008).

In this article, we have documented an extraordinary mechanism

that the internalized Ld adopts to ensure an anti-inﬂammatory

infection milieu in the infected liver of the mammalian host. The

internalized parasite prevents the proinﬂ ammatory response in the

infected macrophage by preventing the entry of hepatocyte derived

miR-122–containing EVs. The parasites achieve it through the de-

polarization of mitochondria of the host cells by enhancing the

expression of the uncoupler protein Ucp2 that restricts the entry of

the hepatic EVs into the infected cells and prevents miR-122 in-

duced inﬂammatory responses. We have also noted miR-146a up-

regulation in the infected cells and the excess miR-146a also gets

packaged into the EVs released by the infected macrophages. EVs

containing miR-146a enter the hepatocytes to restrict the pro-

duction of miR-122 there. The miR-146a–containing EVs are also

internalized by the noninfected macrophages that then get po-

larized to the M2 stage to express IL-10 in a miR-146a dependent

manner. HuR, the RNA binding protein with known miRNA antag-

onistic function, induces export of miR-21 out of macrophage cells

to cause high expression of miR-21 target IL-10 in the noninfected

cells in presence of miR-146a. Interestingly, HuR itself gets even-

tually repressed by high miR-146a. This in turn, reduces the HuR-

mediated stabilization and enhanced expression of ARE-containing

proinﬂammatory cytokines in the recipient neighbouring nonin-

fected macrophage cells. Thus, by cross communicating the in-

fected host cell–derived miR-146a, Leishmania ensures its own

survival by regulating both miR-122 and HuR in neighbouring he-

patocyte and na

ıve macrophage cells, respectively.

Results

Secretion of miR-122 by activated hepatocytes

Lipopolysaccharide or LPS is an immunogen, derived from the cell

wall of the Gram-negative bacteria and is known to stimulate the

macrophage cells via activation of TLR4 receptor and p38/MAPK

pathway (Bode et al, 2012). LPS increases the expression of

proinﬂammatory cytokines by enhancing the NF-ĸβ–dependent

transcription and also by inac tivati ng the repressive miRNAs in

LPS-activated macrophages (Mazumder et al, 2013). In mammalian

liver, the LPS stimulation leads to activation of the tissue resident

macrophages, and thus, LPS increases liver inﬂammation (Rex

et al, 2019). Hepatocytes also respond to LPS and altered metabolic

function of the hepatocytes exposed to LPS has been documented

(Masaki et al, 2004; Momen-Heravi et al, 2015). miR-122 is the key

pro-inﬂammatory miRN A expressed in hepatocytes (Momen-

Heravi et al, 2015). We documented de creased miR-122 level in

mammalian liver cell H uh7 ex posed to LPS (Fig S1A and B). The

decreased miR-122 level was associated with enhanced phosp ho-

p38 MAPK level in LPS-treated Huh7 cells (Fig S1C). The decreased

cellular miR-122 was associated with increased miR-122 detected

in the EVs released by LPS-treated Huh7 cells (Fig S1D and E). What

consequence could this EV-associated miR-122 have on the tissue

resident macrophages infected with Leishmania?Uponinfection

of the host, the liver is the ﬁrst tissue where the Ld initiates the

infection process by targeting the tissue macrophage the Kupffer

cells (Beattie e t al, 2010). Hepatic miR-122, secreted as part of EVs

from hepatocytes, can interact with macrophages to transfer the

miRNA to the resident macrophages and could enhance the ex-

pression of pro-inﬂammatory cytokines (Momen-Heravi et al,

2015). Therefore, the Ld-infected macrophages must adopt strategies

to combat this activation process to protect themselves from getting

killed by the im munostimulatory effect of hepatocyte secreted

miR-122.

Leishmania prevent internalization of proinﬂammatory miR-122

to the infected cells

Ld is known to affect miRNA machineries of host cells to ensure its

proliferation (Ghosh et al, 2013; Chakrabarty & Bhattacharyy a, 2017).

Interestingly, like what happens in LPS-activated cells, expression

of miR-122 also caused an increase in the TNF-α mRNA and protein

levels in RAW264.7 macrophage cells (Fig 1A and C). Expression of

iNOS and NO were also getting increased with miR-122 expression in

macrophages (Fig 1C). Conversely, when Ld infection of RAW264.7

cells expr essing miR-122 was followed, we found increased

proinﬂammatory cytokine TNF-α and low anti-inﬂammatory cyto-

kine IL-10 mRNA levels upon miR-122 expression there (Fig 1D and

E). The infection level of respective cells was also found to be

reduced when the macrophages received the miR-122 positive EVs

before the infection ( Fig 1F and G). Thus, the hepatic miR-122 has an

immuno-protective role, and when transferred via EVs released by

hepatic

cells could prevent infection of neighbouring liver mac-

rophage cells—the ﬁrst target of invading Ld pathogen in the

mammalian host (Ghosh et al, 2013; Momen-Heravi et al, 2015;

Chakrabarty & Bhattacharyya, 2017 ). To survive, Ld must prevent

this EV-mediated miRNA transfer process to stop inﬂammatory

response in the host cells upon its interaction with miR-122 positive

EVs. We hypothesized that Ld could have hijacked the inﬂammatory

machinery of the host cell by preventing the miR-122–containing EV

transfer to infected macrophages, and thus could ensure survival

of the internalized pathogen. Consistent with the assumption, we

found an increased production of proinﬂammatory cytokines and

miR-155, the hallmark of inﬂammatory response in macrophage

cells upon treatment with miR-122 positive EVs. But in cells already

infected with Ld, the uptake of miR-122–containing EVs was found to

be signiﬁcantly compromised with concomitant reduction in miR-

155 or TNF-α production in the infection context (Fig 1H). miR-

122–containing EV treatment, however, do not have any major effect

on cellular NO and iNOS level (Fig 1I).

Leishmania inhibits entry of miR-122–containing EVs in the

infected Kupffer cells in mouse liver

EVs packed with miR-122 were generated from mouse liver cells

HePa1-6. The miR-122–containing EVs were used to treat the mouse

primary macrophage to score the effect of Ld infection on in-

ternalization of miRNA in infected mouse primary macrophages

(Fig 2 A). We documented a substa ntial reduction in EV-mediated

miRNA entry in infected cells (Fig 2B). To score the same in vivo, we

used mice infected with Ld and measured the effect of infection

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 2of23

Figure 1. Hepatic miR-122 acts as an immunostimulant for mammalian macrophage and Ld restricts miR-122 entry in infected macrophage.

(A) Diagrammatic depiction of experiments done with Leishmania donovani (Ld)–infected or uninfected RAW264.7 cells treated with miR-122–containing extracellular

vesicles (EVs). The macrophage cells were either infected or noninfected with Ld for 6 h after which both the groups were either treated with miR-122–containing EVs or

left untreated. (B) Western blot analysis of RAW264.7 cells to show the infection related up-regulation of Ucp2 and down-regulation of HRS protein expression in RAW264.7

cells (left panel). Graphical representation of densitometric analysis of Ucp2 Western blot (right panel )(n ≥ 3 independent experiments, unpaired t test, P = 0.2139).

Values for uninfected and untreated cells were set as unit. (C) Effect of miR-122 overexpression on cytokine expression and nitric oxide generation in RAW264.7 cells.

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 3of23

on EV-derived miR-122 entry in infected mouse liver macrophage

cells (Fig 2C). After infection of 30 d and 24 h of injection of miR-

122–containing EVs, the hepatocytes and Kupffer cells were

separated on a Percoll gradient and enrichment of each cell

population was monitored by following the expression of mRNAs

encoding the marker genes (Fig 2D). The levels of infection in both

control and EV-injected population were monitored by following

the kinetoplast DNA content (Fig 2E). In the u ninfected Kupffer

cells, there wa s internalization of miR-122 after treatment with

miR-122–containing EVs and a substantial increase in the ex-

pression of pro-inﬂammatory TNF-α mRNA in miR-122 EV-treated

cells was noted (Fig 2F and G). However, the Ld infection sub-

stantially reduces the entry of miR-122 EVs in infecte d mouse

Kupffer cells as both miR-122 and TNF-α expression was found to

be substantially low in infected state (Fig 2H and I).

Leishmania depolarizes mitochondria to prevent the entry of

miRNA-containing EVs

Ld is known to cause a robust change in endocytic pathway of the

host cells and alter components that are also found to be used for

endocytic entry or exit of miRNAs (Lievin-Le Moal & Loiseau, 2016;

Chakrabarty & Bhattacharyya, 2017). Proximity of endosomes with

Ld-containing parasitophorous vacuoles in infected macrophage

cells was noted (Fig S2A and B). The endosome maturation process

in Ld-infected cells is known to get affected (Scianimanico et al,

1999). We explored the status of Rab proteins in Ld-infected

macrophages and observed a decrease in endosomal protein

Hepatocyte growth factor-regulated tyrosine kinase substrate

(HRS) expression in infected cells (Fig 1B). However, there has

not been a major change in Rab5a or RILP expression with Ld in-

fection of RAW264.7 cells. Interestingly, treatment of cells with EVs

containing miR-122 has no signiﬁcant effect on endosome number or

Rab5a expression in infected RAW264.7 cells. The infection also had

no effect on Dynamin 2 expression and thus cannot account for the

reduced miR-122 entry that is known to be a dynamin 2 dependent

process in mammalian cells (Fig S2B and C;[Ghoshal et al, 2021]).

It is known that Ld targets the mitochondrial dynamics and

activity by inducing depolarization of mitochondria and reducing the

mitochondria–ER–endosome interaction in infected cells (Chakrabarty

& Bhattacharyya, 2017). Mitochondrial uncoupler protein Ucp2 gets up-

regulated upon Ld infection (Fig 1B) suggesting a depolarized mito-

chondrial state in infected cells (Chakrabarty & Bhattacharyya, 2017).

Does mitochondrial depolarization affect EV-entry? Mitochondria can

interact with several cellular compartments, like ER and endosomes

(Klecke r e t al, 2014; Todkar et al, 2 019). The Ucp2 is a mitochondrial

uncoupling protein causing defects in oxidative phosphorylation

and

ATP s ynthesis by changing membrane potential across mi-

tochondrial membranes. Hence, it was anticipated that uncou-

pling of the mitochondrial potential can affect the internalization

of EV-derived miRNAs in mammalian cells as it is known to affect

endogenous miRNA activity (Chakrabarty & Bhattacharyya, 2017).

FH-Ucp2 was expressed in the recipient HeLa cells and FH-

Ucp2–expressing cells were incubated with miR-122–containing

EVs (isolated from HeLa cells expressing pmiR-122). Ucp2 over-

expression caused a disruption in the mitochondrial structures as

observed microscopic ally (Fig 3 A). The low level of internalization

and consequently a lower repression activity of t he transferred

EV-derived miRNAs were observed in FH-Ucp2–expressing re-

cipient HeLa cells (Fig 3B–D). It has been reported earlier that Ld

infection or the loss of mitochondrial membrane potential by FH-

Ucp2 are accompanied by reduced juxtaposition of ER and mi-

tochondria (Chakrabarty & Bhattacharyya, 2 017)(Fig S3A and B).

Interestingly, oligomycin treatment that affects the ATP con-

centration alone and FCCP treatment that disrupts membrane

potential of m itochondria without much effect on cellula r ATP

content, do not have any notable inhibitory effect on EV-mediated

miR-122 entry in recipient cells (Fig S3C). These data suggest in-

volvement of altered mitochondrial dynamics and mitochondrial

interaction of subcellular organelles in FH-Ucp2–expressing cells to

The RAW264.7 macrophage cells were either kept as control, transfected with miR-122 expression plasmid pmiR-122 or with miR-146a expression plasmid pmiR-146a or

treated with LPS (as positive control) to determine the mRNA level of TNF-α (C, left panel, n = 3 independent experiments; P = 0.0361, 0.0084) and iNOS (C, middle right panel,

n = 3 independent experiments; P = 0.0562, 0.0002) from cellular RNA. Protein level of TNF-α (C, middle left panel, n = 3 independent experiments; unpaired t test, P =

0.0006) and nitric oxide level (right panel, n = 3 independent experiments; unpaired t test, P ≤ 0.0001, 0.0405, <0.0001) was also measured from culture supernatant.

Quantiﬁcation of TNF-α mRNA levels by qRT-PCR was done for the conditions described above. 18s RNA or GAPDH mRNA was used as endogenous control for qRT-PCR.

Values for control plasmid transfected untreated cells were considered as unit for qRT-PCR (control). (D) Effect of miR-122 expression on Ld infection. This panel shows

the schematic model for RAW264.7 cells transfected with miR-122 expression plasmid (pmiR-122) followed by L. donovani infection. (E) Comparison of the

proinﬂammatory TNF-α, and anti-inﬂammatory IL-10 cytokine expression in pmiR-122 transfected or pCIneo control vector transfected RAW264.7 cells followed by infection

with Ld. An increase in the proinﬂammatory cytokine TNF-α ( P = 0.0180) and a decrease in the anti-inﬂammatory cytokine IL-10 (P = 0.0422) were observed in the presence

of miR-122 (n = 3 independent experiments).18s rRNA was used as endogenous control. Values for pCIneo control transfected and infected RAW264.7 cells were

considered as unit. (F) Microscopic analysis of Ld infection in RAW264.7 cells treated or untreated with miR-122–containing EVs. Cells were then visualized under the

confocal microscope. The Leishmania protein GP63 imaged with indirect ﬂuorescence (red) and cells with internalized parasites were counted. Scale bar 8 μm. Marked

areas are zoomed for 5×. (G) Effect of miR-122–containing EVs on Ld infection. Graphical representation of the percent of RAW264.7 cells infected with L. donovani as

observed microscopically (n = 132, number of cells; P = 0.0038, unpaired t test). (H) Quantiﬁcation of miRNA levels upon Ld infection followed by EV treatment in RAW264.7

cells. Real-time PCR showed no increase in miR-122 levels in infected macrophages incubated with miR-122 positive EVs (top left panel

; P =

0.0215, 0.3211, 0.3808). miR-155

levels were also quantiﬁed under similar conditions (bottom left panel; P = 0.0024, 0.0736, 0.1368) (n = 4 independent experiments). U6 was used as endogenous control.

Relative levels of cytokine mRNA were also quantiﬁed. The proinﬂammatory cytokine, TNF-α (top right panel; P = 0.0142, <0.0001, 0.0013) and IL-1β (bottom right panel; P =

0.0438, <0.0001, 0.0366) did not increase in presence of the parasite followed by EV treatment (n = 4 independent experiments). 18s rRNA was used as endogenous control.

Values for uninfected cells were set as unit. (I) Quantiﬁcation of iNOS mRNA level and Nitric oxide (NO) generation after miR-122–containing EV treatment of RAW264.7

cells. Nitric oxide level was determined from culture supernatant of EV-treated RAW264.7 cells (upper panel, n = 3 independent experiments, unpaired t test; P = 0.3878).

qRT-PCR analysis was used to determine the relative level of iNOS mRNA in recipient RAW264.7 cells (lower panel, n = 3 independent experiments; P = 0.1275). GAPDH mRNA

was used as endogenous control. Values for control EV-treated cells were considered as unit for qRT-PCR. Data information: In all experimental data, error bars are

represented as mean ± SEM, ns, nonsigniﬁcant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, respectively. P-values were calculated by two-tailed paired t test in most of

the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.

Source data are available for this ﬁgure.

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 4of23

Figure 2. miRNA uptake is prevented in Ld -infected primary macrophages and Kupffer cells.

(A) A model to show the effect of Leishmania infection on uptake of miR-122–containing extracellular vesicles (EVs) in primary peritoneal macrophages. Macrophages

were either infected or noninfected with Ld followed by miR-122–containing EV treatment. The EVs were obtained from miR-122 expressing murine hepatic cell HePa 1–6.

(B) Relative levels of internalized miR-122 in infected or noninfected primary macrophages (n = 3 independent experiments; 0.0379, 0.0033, 0.2726, Unpaired t test P =

0.0227). U6 was used as endogenous control and values for uninfected control cells were considered as unit. (C) Schematic representation of Ld infection of mice

followed by miR-122–containing EV treatment. Mice were infected with Ld for 30 d followed by tail vein injection of miR-122–containing EVs. After 24 h of treatment, mice

were euthanized and Kupffer cells were isolated to quantify the miR-122 content. (D) Characterisation of isolated hepatocytes and Kupffer cells. Quantiﬁcation of

hepatocyte speciﬁc albumin mRNA in hepatocytes and Kupffer cells to show their low levels in Kupffer cell isolate (left panel; P = 0.143, n = 4 number of mice). Relative

levels of macrophage speciﬁc C-type Lectin Domain Family 4, Member F (Clec4f) mRNA between hepatocytes and Kupffer cells showing high levels in Kupffer cells (right

panel; P = 0.0378, n = 4 number of mice). GAPDH was used as endogenous control. Values for hepatocytes were considered as unit. (E) Comparison of normalized Ct values of

Kinetoplast DNA in infected or uninfected Kupffer cells isolated from animals untreated or treated with EVs containing miR-122 to determine the parasitic load in

Kupffer cells (n = 3 number of mice; P = 0.0009, 0.0005, unpaired t test). GAPDH was used as endogenous control. (F ) Mean Ct values of internalized miR-122 in

uninfected Kupffer cells either untreated or treated with miR-122–containing EVs (n = 3 number of mice; P = 0.00 49, unpaired t test). (G) Mea n Ct values of TNF-α

mRNA in uninfected Kupffer cells upon treatment with miR-122–EVs compared with untreated Kupffer cells (n ≥ 2; P = 0.0444, unpaired t test). (H) The internalization of

EV-derived miR-122 declined in the presence of Leishmania infection. Real-time PCR revealed the relative levels of miR-122 in uninfected or infected Kupffer cells isolated

from animals treated with miR-122–containing EVs (n = 3 number of mice; P = 0.0162). U6 was used as endogenous control and values for uninfected miR-122 EV-treated

cells were considered as unit. (I) Relative levels of proinﬂammatory cytokine TNF-α

in Leishmania infecte

d or uninfected miR-122–EV-treated animals’ Kupffer cells

(n = 3 number of mice; P < 0.0001). GAPDH mRNA was used as endogenous control and values for uninfected miR-122 EV-treated set were considered as unit. Data

information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsigniﬁcant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1, respectively. P-values

were calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise.

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 5of23

Figure 3. Mitochondrial depolarization and ER detethering by Ld prevents internalization and activity of extracellular vesicle (EV)-derived miRNAs in recipi ent cells.

(A) Structure of mitochondria (green) in Mito-GFP–expressing HeLa cells transfected with FH -Ucp2 (red) or control plasmid. Scale bar 5 μm. (B) Graphical representation

of the internalization of CD63-GFP–positive EVs in recipient HeLa cells transfected either with control or FH-Ucp2–expression plasmids (n = 18, number of cells; P < 0.0001,

unpaired t test). (C) Effect of FH-Ucp2 expression on uptake of miR-122. Relative level of internalized EV-derived miR-122 in recipient HeLa cells transfected either with

control or FH-Ucp2 expression plasmids (left panel; n = 3 independent experiments; P < 0.0001). U6 was used as endogenous control and values for control plasmid

transfected and miR-122 EV-treated cells was considered as unit. Right panel shows the Western blot for HA. β-Actin was used as loading control. (D) Luciferase assay to

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 6of23

cause the defect in cellular uptake of miRNA-containing EVs in

mammalian cells. Mfn2 is a protein responsible for mitochondrial

tethering with ER. Mfn2 knockout MEFs showed a decreased ER

tethering of mitochondria. To revalidate that the Ucp2 over-

expression mediated loss of ER–mitochondria contact is respon-

sible for the reduced EV-associated miRNA internalization and Ago2

association, EV-derived miR-122 levels were compared in Mfn2

knockout and wild-type MEFs. Importantly, as reported earlier, the

endogenous miR-16 level was found to be increased in Mfn2 de-

pleted cells (Fig 3E and F) that has been consistent with the pre-

vious ﬁndings on increased cellular miRNA content upon Ucp2

overexpression (Chakrabarty & Bhattacharyya, 2017). To reconﬁrm

the importance of Ucp2-lowering for cellular entry of EV-associated

miRNAs we used genipin, an inhibitor of Ucp2, to see its effect on

EV-mediated miR-122 entry. The miR-122 entry and associated

increase in T NF-α expressi on has been observed in infected

macrophage cells treated with Ucp2 inhibitor genipin ( Fig 3G).

Interestingly, ectopic expression of FH-Ucp2 has a marginal effect

on low endogenous levels of miR-122 expressed in RAW264.7 cells

compared to the changes in miR-122 levels in pmiR-122 transfected

RAW264.7 cells (Fig S3D). Conversely, there has been no detectable

change in the cellular ATP level or mitochondrial polarization status

or Ucp2 expression in RAW264.7 cells ectopically expressing miR-122

from pmiR-122 plasmid (Fig S3E–G). Overall, these data suggest Ld

infection–associated increase in Ucp2 that causes mitochondrial

detethering with ER and prevents the pro-inﬂammatory miR-122

entry via EVs into the infected macrophage to stop expression of

pro-inﬂammatory cytokines (Fig 3H).

Leishmania infection increases cellular and extracellular

miR-146a levels in infected cells

The hypothesis that the infected cells should release the EVs

enriched with factors to facilitate the propagation of infection

inspired us to examine the key protein components that are

speciﬁcally released by infected cells or their export is prevented in

the infection context. The EVs isolated from control and Ld-infected

cells were analysed for their protein content. Although upon in-

fection there has been a drop in expression of HRS or Rab27a

proteins that are known to have role in late endosome/MVB for-

mation and EV release (Fig 4A and B), we did not detect a signiﬁcant

change in EV diameter or marker protein levels in population

isolated from both control and Ld-infected RAW264.7 cells (Fig

4C–E). In the mass spectrometric analysis of released EVs that is

followed by candidate protein identiﬁcation, we have documented

several candidate proteins that showed exclusive presence in the

EVs isolated from control or Ld-infected cells. However, differential

expression of any important regulatory factors of cytokine ex-

pression, were not detected (Fig S4 and Tables S1–S3). Therefore, it

could be the small RNA population of the infected host that may

carry the required anti-inﬂammatory information to the neigh-

bouring cells as part of EVs. In infected cells, both miR-146a and

miR-155, two most important regulatory miRNAs of inﬂammation

pathways, were found to be up-regulated. Interestingly, among

them, only miR-146a were detected in EVs released by infected cells

and the content of miR-146a had increased several folds in EVs

from Ld-infected cells compared with EVs from control noninfected

cells (Fig 4F and G). Similar increase in miR-146a in EVs released by

infected primary macrophage was also detected (Fig 4H). The

miRNAs that are reported to be increased in Ld-infected cells

(Chakrabarty & Bhattacharyya, 2017), miR-21, miR-125b, or miR-16 all

were fou nd to be incr eased in the E Vs rel eased by infect ed ce lls

(Fi

g 4I).

Leishmania by targeting HuR ensures export of miR-146a but not

miR-155 from the infected cells

We report differential export of miR-146a from Ld- infected

RAW264.7 macrophage. But how miR-146a gets differe ntially

exported out of infected macrophage cells is a fascinating question.

Retention of miRNA and their targets with polysomes has been

found to be the reason for reduced miRNA export noted in

mammalian cells (Ghosh et al, 2015), whereas target RNA presence

positively inﬂuences the export process (Ghosh et al, 2021). We have

isolated the polysomes from control and infected cells and de-

tected increased retention of both miR-146a and miR-155 with

polysomes in the infected cells (Fig S5A and D). The increased

retention of IL-10 and MyD88 mRNAs, known to get expressed in

infected cells, were also detected more with polysomes isolated

from the infected cells but the miR-146a target TRAF6 mRNA and

TNF-α mRNA was found to be less with infected cell polysomes (Fig

S5B and C).

show the repression levels of RL-perf-miR-122 in recipient cells transfected with pCIneo control or FH-Ucp2–expression plasmids and treated with miR-122–containing

EVs. The repression levels were calculated as a ratio of ﬁreﬂy normalized RL-Control value to ﬁreﬂy normalized RL-perf-miR-122 value (n = 3 independent experiments;

0.0117, 0.0987, unpaired t test = 0.0013). (E) Relative level of internalized miR-122 (left panel; P = 0.0012) and endogenous miR-16 levels (right panel; P = 0.0316) in Mfn2

(Mitofusin2) wi ld-type and knockout MEF cells treated with miR-122–containing EVs. Quantiﬁcation was done by qRT-PCR and U6 levels were used as normalizing control

(n = 3 independent experiments). Values for Mfn2 wild-type cells were set as unit. (F) FH-Ago2 associated miR-122 levels in Mfn2 wild-type and knockout MEF cells

(transfected with FH-Ago2) co-cu ltured with miR-122 expressing HeLa cells. Immunoprecipitated Ago2 levels were used for normalization of associated miR-122 (n = 3

independent experiments; P = 0.0077). Values for Mfn2 wild-type cells was considered as unit. (G) Rescue of EV-miR-122 internalization in Leishmania donovani infected

RAW264.7 cells in presence of genipin, the inhibitor of Ucp2. RAW264.7 cells infected with Ld were treated with genipin (100 μM after 4 h of infection) followed by addition of

miR-122 positive EVs. Relative levels of miR-122 (top panel; P = 0.0311) and TNF-α mRNA (bottom panel; P = 0.0968) increased in infected RAW264.7 cells treated with genipin

(n = 3 independent experiments). U6 levels were used as control for normalization of miRNA and 18s rRNA was used normalizing levels for mRNA. Values for infected and

genipin untreated set was consider ed as unit. (H) Diagrammatic representation of the EV-mediated crosstalk between hepatocytes and macrophages. Hepatocytes release

miR-122–containing EVs which can be transferred to macrophag es that causes production of proinﬂammatory cytokines and can prevent Ld infection (red arrow).

Inversely, Ld-infected macrophages are unable to take up the miR-122–containing EVs due to up-regulation of Ucp2 protein in cells and are tuned to have high

production of anti-inﬂammatory cytokines for sustained parasitic infection (green arrow). Data information: In all experimental data, error bars are represented as mean ±

SEM, ns, nonsigniﬁcant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, respectively. P-values were calculated by two-tailed paired t test in most of the experiments unless

mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.

Source data are available for this ﬁgure.

Leishmania hijacks host miRNA machinery Ganguly et al.

https://doi.org/10.26508/lsa.202101229 vol

5 | no 6 | e202101229 7of23

Figure 4. Leishmania donovani (Ld) infection triggers extracellular export of miRNAs from infected macrophage cells.

(A) Schematic representation of the experiment. After 24 h of Ld infection, the host macrophage and extracellular vesicles (EVs) isolated from culture supernatant were

analysed for cellular and EV-associated protein and RNA analysis. (B) Ld infection altered cellular protein levels. Western blot images showed protein le vels after 24 h of

infection of macrophages. β-Actin was used as loading control. (C, D, E) Characterisation and quantiﬁcation of EVs released from control and Ld-infected macrophages.

Nanoparticle Tracking Analysis (NTA) of EVs released from control (C, left panel) and infected cell (C, right panel). (D) Western blot images showed EV marker proteins in

control and infected cell released EVs. (E) Number and size of EVs released by control and infected cells were quantiﬁed by NTA analysis (E, left panel, P = 0.0008 and right

panel, P = 0.0677, n = 3 independent experiments, respectively, unpaired t test). (F, G) miR-146a level increases and the miRNA gets exported from Ld-infected macrophage

cells. qRT-PCR based relative quantiﬁcation of cellular miR-155 (F, left panel, P = 0.0817, n = 3 independent experiments) and miR-146a (F, right panel, P = 0.0146, n = 4

independent experiments) after 24-h infection of RAW264.7 cells. U6 was used as endogenous control. miRNA levels of infected cells were normalized against noninfected

controls. qRT-PCR data represented the relative level of miR-146a and miR-155 in EVs from RAW264.7 cells after infection. EV marker protein Alix was used for normalization

of EV-associated miR-146a level (G, P = 0.0229, n = 4 independent experiments). Values for uninfected cell–derived EVs were set as unit. (H) Ld infection triggers the

extracellular export of miRNAs from mouse peritoneal macrophages. miR-146a level was also estimated in EV-derived from mouse peritoneal macrophage(H,upper

panel, P = 0.0006, n = 5 independent experiments, unpaired t test) and Western blot data also showed EV marker proteins (H, lower panel). (I) Average Ct value of miR-21 (I,

left panel, P = 0.0011, n = 3 independent experiments, unpaired t test), miR-125b (I, middle panel, P = 0.2654, n = 3 independent experiments, unpaired t test), and miR-16 (I,

right panel, P = 0.0794, n = 3 independent experiments, unpaired t test) in EVs released from Ld-infected and noninfected RAW264.7 cells. Data information: In all

experimental data, error bars are represented as mean ± SEM, ns, nonsigniﬁcant,

*P < 0.05, **P < 0.01, ***P < 0.001, respectively. P-values were calculated by two-tailed

paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.

Source data are available for this ﬁgure.

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 8of23

The polysome retention data thus could not clearly explain why

there has been a preference of miR-146a for export from Ld-

infected cells. miRNAs accumulate at endosomes before they get

packed and exported out (Mukherjee et al, 2016). We had isolated

the endosomes and ER fractions from control and infected cells to

document preferential enrichment of miR-146a over miR-155 at the

endosomal fraction that can explain its enhanced export observed

in infected macrophage cells (Fig S5E–G). HuR is known for its role

in export of miRNAs in mammalian liver and macrophage cells

(Mukherjee et al, 2016). HuR drives miRNA accumulation with

endosomes before the export but HuR level is known to get reduced

in Ld-infected cells (Goswami et al, 2020)(Fig S5H). The HuR level

increases after LPS treatment that is associated with enhanced

export of miR-155 from LPS-activated macrophage cells (Goswami

et al, 2020), it is possible that low HuR in Ld-infected cells may cause

reduced export of miR-155. Does binding of HuR makes miR-155

available in endosomal fraction and for export? Supporting the

notion, we have documented strong interaction of HuR with miR-

155 in LPS-treated cells (Fig S5I and J). Similarly export of miR-155

from HA-HuR expressing cells has also been noted (Fig S5K ).

EV-containing miR-146a down-regulates miR-122 maturation in

hepatocytes

The infected cells prevent the entry of miR-122–containing EVs

thereby restricting the activation of the Ld-infected cells by miR-122

to prevent the death of the internalized parasites owing to en-

hanced proin ﬂammatory response observed in miR-122 recipient

macrophages (Chen et al, 2011). However, miR-122 in hepatic EVs

could also get transferred to a na

ıve neighbouring macrophage not

infected with Ld. How the parasite prevents the activation of resident

neighbouring na

ıve macrophages to prevent the proinﬂammatory

cytokine accumulation in the infection microenvironment? It is likely

that Ld must have adopted a mechanism to reduce the miR-122–

containing EV release by neighbouring hepatocytes in the infection

niche to prevent miR-122–mediated activation of na

ıve noninfected

macrophage cells. Can Ld doit through cross talk with hepatocytes

via the EVs released by infected cells? We have noted miR-146a

as the predomina nt anti-inﬂammatory miRNA in the EVs se-

creted by the infected macrophage (Fu et al, 2017). Does miR-

146a reduce the miR-122 level in the hepatocytes?

We have isolated the EVs from infected macrophage for treating

human hepatoma cell Huh7. We have documented an increase in

miR-146a content of recipient hepatic cells with a reduction in

mature miR-122 level there (Fig 5A and B). The decrease of mature

miR-122 was accompanied by an increase in pre-miR-122 level and

with an increase in endogenous control miR-16 level in the he-

patocyte. Thus, the miR-146a–mediated lowering of hepatic miR-122

is a miRNA speciﬁc effect. To conﬁrm that the effect of miR-146a on

mature miR-122 level is miR-146a speciﬁc, we have isolated the EV

from RAW264.7 cells ectopically expressing miR-146a and used the

isolated EVs for the treatment of the hepatocytes (Fig 5C). The EVs

isolated from control and miR-146a expression plasmid transfected

cells were analysed by NTA to document no major change in size

and number of the EVs under miR-146a overexpression condition

(Fig S6A). Increased miR-146a content in EVs released from pmiR-

146a–expressing cells was also noted (Fig S6B) and cellular and EV

fractions were Western blotted for EVs and cytosolic markers to

conﬁrm the purity of the fractions (Fig S6C). The miR-146a–

containing EV treatment enhanced cellular miR-146a level in re-

cipient hepatocytes with concomitant decrease in mature miR-122

and increase in pre-miR-122 levels observed there (Fig 5D). To

substantiate the idea of miR-146a–induced reduction in miR-122

levels in hepatocytes, we have expressed miR-146a in hepatocytes

and documented decreased miR-122 level associated with in-

creased pre–miR-122 and miR-146a levels in Huh7 cells (Fig 5E).

WehavenotedareductioninDicer1leveluponLd-infected cell

EV treatment of hepatocyt es. However, mi R-146a–c ontai ning EV

treatment or miR-146a expression in hepatocytes, did not alter

Dicer1 level (Fig 5F–H). Thus, altered pre–miR-122 processing by

Dicer1 in miR-146a enriched hepatocyte could not be explained

by the unchanged Dicer1 level. The increased pre–miR-122 however

got associated with the Dicer1 to a lesser extent in Huh7 cells

expressing pmiR-146a where total pre–miR-122 level increases (Fig

5J and K). Therefore, there must be a mechanism that excludes the

pre–miR-122 association with Dicer1 for its subsequent processing

to the mature form. The miR-146a expression, however, enhances

the P-ERK1/2 level and a decrease in P-p38 level (Fig 5I), suggesting

a possible lowering of transcriptional events for pre-miR-122 known

to be linked with P-p38 level (Basu & Bhattacharyya, 2014).

Infected cell secreted EVs downgrade inﬂammatory response in

neighbouring macrophage and induce macrophage polarization

Although lowering of miR-122 levels in hepatocytes could be one of

the mechanisms to reduce the proinﬂammatory response in the

ıve macrophage, there must be additional mechanisms to restrict

the production of inﬂammatory cytokines in the neighbouring na

ıve

macrophage cells adjacent to the infected macrophages. To score

the effect of infected cell EVs on na

ıve macrophage cells, we treated

ıve RAW264.7 cells with EVs isolated from Ld-infected RAW264.7

cells (Fig 6A). Interestingly, when the na

ıve RAW264.7 cells were

pretreated with Ld

-infected cell–derived

EVs, we documented a

lesser responsiveness and relatively low proinﬂammatory cytokine

production in the treated cells when exposed to pro-inﬂammatory

agents such as LPS. Remarkably, the microvesicles (MVs) isolated

from the infected cells increase the production of cytokines in

recipient cells on LPS exposure (Fig 6B).

To score the effect of infected cell EVs on polarization of na-

ive macrophage, we treated the RAW264.7 macrophage with Ld-

infected EVs (Fig 6C) and measured the amount of internalized miRNA

and found an increase in miR-146a content. This was accompanied

by a reduction in iNOS expression (Fig 6D). With EV treatment, we

noted no change in Dicer1 level and a decrease in HuR expression

with increased levels of P-p38 and P-ERK1/2 levels (Fig 6E). These

were accompanied by an increase in IL-10 expression and decrease

in expression of proinﬂammatory cytokine IL-1β (Fig 6F). To con-

clude on casualty of EV-associated miR-146a with changed cytokine

expression observed in Ld-infected cell–derived EV-treated cells,

we used EVs released by pmiR-146a–expressing RAW264.7 cells and

treated na

ıve RAW264.7 cells with miR-146a–containing EVs. We

noted an increase in IL-10 and a decrease in IL-1β as well as iNOS

mRNAs after miR-146a–containing EV treatment (Fig 6G and H).

Increased expression of miR-146a has also found to be associated

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 9of23

Figure 5. Extracellular vesicle (EV)-mediated transfer of Ld-infected cell derived miR-146a reduces miR-122 level in hepatic cells.

(A, B) Effect of control and Ld-infected cell–derived EVs on na

ıve hepatocytes. (A) A schematic representation of the experiment (A). (B) Relative levels of miR-146a (B, left panel,

P = 0.0329, n = 3 independent experiments), miR-122 (B, middle left panel, P = 0.0364, n = 4 independent experiments), Pre-miR-122 (B, middle right panel, P =0.0993,n=3

independent experiments), miR-16 (B, right panel,unpairedt test P = 0.2074, n = 3 independent experiments) were measured by qRT-PCR in recipient hepatic Huh7 cells after 24 h of

control and infected cell–derived EV treatment. U6 was used as endogenous control for miRNA normalization and for Pre-miR-122 level, GAPDH was used as endogenous control.

Values for control EV-treated cells were used for normalization of infected cell–EV-treated cells. (C, D) Effect of pmiR-146 a–expressing macrophage derived-EV treatment on

recipient hepatocytes. (C) A schematic representation of the experiment has been described in panel. (D) Relative levels of miR-146a (D, left panel, P = 0.0213, n = 3 independent

experiments), miR-122 (D, middle panel, P = 0.0036, n = 3 independent experiments) and Pre-miR-122 (D, right panel, P = 0.0307, n = 3 independent experiments) were measured by

qRT-PCR in recipient Huh7 cells after pCIneo and pmiR-146a transfected RAW264.7 cell–derived EV treatment for 24 h. U6 was used as endogenous control for cellular miRNA level

normalization. Values for pCIneo-transfected cell–derived EV-treated cell were set as unit. For Pre-miR-122 level, GAPDH was used as endogenous control. (E) Effect of pmiR-146a

overexpression on miR-122 level in hepatocytes. Relative levels of miR-146a (E, left panel, P = 0.2251, n = 3 independent experiments), miR-122 (E, middle panel, P = 0.0003, n = 4

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 10 of 23

with decreased iNOS mRNA and NO levels in RAW264.7 cells (Fig 1C)

associated with a decrease in TNF-α protein level (Fig 1C) and cellular

IL-1β protein expression (Fig S6D), respectively. We expressed miR-

146a in na

ıve and Ld-infected RAW264.7 cells and have noted a

decrease in IL-1β and TNF-α expression and increase in IL-10 ex-

pression (Fig 6I). Interestingly, inhibition of miR-146a by anti-miR-

146a reduced the expression of IL-10 both in uninfected and

Ld-infected cell–derived EV-treated RAW264.7 cells, whereas the ac-

tivation of miR-146a–expressing cells with LPS have a induced effect

on anti-inﬂammatory cytokine IL-10 mRNA level (Fig 6J and K). This

data suggests an M2 polarization of macrophage expressing miR-

146a and that become refractory to immuno-stimulation by LPS (Fig

6I–K). To further reafﬁrm the contribution of Ld-derived factors

associated with EVs-released by infected cells on na

ıve macro-

phage cell polarization and IL-10 production, we treated RAW264.7

cells with Ld-derived EVs and lipophosphogylcan (LPG) isolated

from the Ld membrane. We have noted no important change in the

expression of major signalling component in Ld-derived EV-treated

cells (Fig S7A, B, and F). As reported earlier, we had noted decreased

TNF-α expression in both sets of treated cells, whereas IL-1β only

got reduced in presence of Ld-derived EVs but not with Ld LPG (Fig S7C

and E,[de Carvalho et al, 2019]). The IL-10 level decreased in both Ld-

derived EVs or LPG treatment and therefore, these Leishmania-derived

factors cannot be the major player for the observed increase in IL-10 in

neighbouring macrophage cells (Fi g S7 C and G). iNOS mRNA level,

however, decreases with Ld-derived EV treatment (Fig S7B), whereas

the Ld-derived EV, LPG, or soluble antigens of Leishmania (SLA) has an

insigniﬁcant effect on endogenous level of miR-122 in RAW264.7 cells

(FigS7D,E,andG). On contrary, the effect of

Ld-derive

d EVs, LPG, or SLA

also has an inhibitory effect on endogenous miR-146a levels in he-

patocytes as observed in Huh7 cells (FigS7D,E,andG). Taken together,

thedatasuggestthattheeffectof miR-146a rather than the Ld-derived

factors, as part of infected cell secreted EVs, has the potential to

change the IL-10 level in recipient na

ıve macrophage cells and is

essential for m acrophage M2 po larizati on. Thus, we conclude that

the EV-associated miR-146a of Ld-infected cell EVs, have an anti-

inﬂammatory effect on na

ıve macrophage cells and is associated

with an increase in IL-10 production in EV-treated cells, whereas the

LPS responsiveness as well as pro-inﬂammatory cytokine expression

level remain reduced in miR-146a–enriched RAW264.7 cells.

HuR is required for miR-146a–mediated IL-10 induction

From the data discussed so far, it has become more likely that miR-

146a control the IL-10 level in macrophage cells. How does miR-146a

ensure a high IL-10 expression in treated macrophage to polarize

them to have an anti-inﬂammatory response pathway—a prelude to

the infection niche establishment? HuR is an important post-

transcriptional gene regulator in mammalian macrophage and is

essential for activation and expression of proinﬂammatory cyto-

kines in macrophage exposed to LPS. Activated macrophage also

showed increased expression of HuR with LPS treatment time

(Goswami et al, 2020). Interestingly, expression of HA-HuR also

enhances the expression of pro-inﬂammatory cytokines in un-

treated macrophages and could get the cells to the activated state

(Fig 7A and B, Goswami et al, 2020). Therefore, to have the strong

anti-inﬂammatory effect of miR-146a, the HuR-mediated proin-

ﬂammatory effect should be neutralized or countered by the

transferred miR-146a from the infected cells. Leishmania could

down-regulate HuR in mammalian macrophage cells (Fig S5H).

However, it is possible that the extracellular miR-146a released by

the infected cells could bring down the effect of HuR in noninfected

cells present in the infection niche to ensure an overall anti-

inﬂammatory pathway prevalent in all i mmune cells present

there. miR-146a–mediated down-regulation of HuR has been re-

ported previously (Cheng et al, 2013). Does miR-146a counter the

action of the HuR to stop inﬂammatory responses in macrophage

cells? In miR-146a–containing EV-treated cells, we have noted that

the expression of HuR could not enhance the IL-1β or TNF-α ex-

pression in RAW264.7 cells. Surprisingly, an increase in IL-10 ex-

pression in HuR expressing cells was detected when treated with

miR-146a–containing EVs (Fig 7C and D). Inversely, siHuR treatment

causes a reduction in IL-10 expression in cells treated with miR-

146a–containing EVs (Fig 7E and F). However, miR-146a mimic

decreases HuR levels as well as HuR induced increase in TNF-α and

IL-1β expression, whereas IL-10 level increases in presence of HA-

HuR when miR-146a is also expressed in RAW264.7 cells (Fig

7G–K).

How does HuR ensure the high IL-10 level? It is known that miR-21

negatively regulates IL-10 (Wang et al, 2017) and HuR is also known

to interact and sponge out miR-21 to inactivate it in mammalian

cells (Poria et al, 2016). We have noted reduction in miR-21 level in

HA-HuR–expressing cells, whereas siHuR treatment prevents miR-

21 lowering. We have also noted increased miR-21 export in HuR-

expressing cells that suggests HuR-mediated export of miR-21 that

helps the IL-10 expression (Fig 7L and M). Thus, HuR by up-

regulating IL-10 level also ensures the strong anti-inﬂammatory

polarization in miR-146a–containing EV-treated cells. Interestingly,

HuR eventually gets decreased in cells expressing high levels of

miR-146a to possibly balance the HuR-mediated stabilization of

cytokine mRNAs such as TNF-α and IL-1β possibly to restrict excess

independent experiments) and Pre-miR-122 (E, right panel , P = 0.0279, n = 4 independent experiments) were measured by qRT-PCR in Huh7 cells after pCIneo and pmiR-146a

expression in hepatocytes. U6 was used as endogenous control for miRNA normalization. pCIneo transfected cells was used for normalization of pmiR-146a transfected cells. For

Pre-miR-122 level, GAPDH was used as endogenous control. (F, G, H) Effect of EV treatment on Dicer1 level in na

ıve hepatocyt es. (F) Level of Dicer1 was measured by Western blot in

control and Ld-infected cell–EV-treated Huh7 cells. β-Actin was used as loading control (F). (G) Dicer1 level was also measured in Huh7 treated with EVs derived from pCIneo and

pmiR-146a transfected RAW264.7 cells. β-actin was used as loading control. (H) Effect of pmiR-146a expression on Dicer1 in Huh7 hepatocytes was also determined. β-Actin was used

as loading control. (I) Effect of pmiR-146a overexpression on signalling component proteins in hepatocytes. Levels of HuR, P-ERK1/2, P-p38 were determined by Western blot

analysis in pCIneo and pmiR-146a transfected Huh7 cells. β-Actin was used as loading control. (J, K) Effect of NHA-Dicer1 expression on Dicer1 associated precursor miRNA level in

cells expressing pmiR-146a. (J) Average Ct value of NHA-Dicer1 associated Pre-miR-146a (J, left panel, P = 0.0005, n = 3 independent experiments, unpaired t test) and Pre-miR-122 level

(J, right panel, P = 0.2630, n = 3 independent experiments, unpaired t test) were determined by Real-time PCR after HA-Dicer1 was immunoprecipitated from pCIneo and pmiR-146a

transfected Huh7. (K) NHA-Dicer1wasimmunoprecipitatedusinganti-HAantibodyandwasdetected in immunoprecipitated and input samples using anti-HA antibody. HC, heavy

chain. Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsigniﬁcant, *P <0.05,**P < 0.01, ***P < 0.001, respectively. P-values were calculated

by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.

Source data are available for this ﬁgure.

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 11 of 23

Figure 6. Extracellular vesicle (EV)–mediated transfer of miR-146a from infected macrophage cells promotes anti-inﬂammatory response in na

ıve recipient macrophag es.

(A, B) Effect of EV treatment on LPS induced activation of recipient macrophages. (A) A schematic representation of the experiment has been shown in the left. (B) Relative

cellular levels of TNF-α (B, left panel, P = 0.0224, n = 6 independent experiments), IL-1β (B, middle left panel, P = 0.0280, n = 5 independent experiments) mRNAs were measured

by qRT-PCR from Infected cell–EV-treated macrophages after LPS treatment. TNF-α and IL-1β (B, middle right, P = 0.2487, n = 6 independent experiments, unpaired t test and

right panel, P = 0.1133, n = 6 independent experiments, unpaired t test, respectively) were measured by qRT-PCR from infected cell derived Microvesicle (MV)–treated

macrophages followed by 4 h of LPS (1 ng/ml) treatment. 18s rRNA was used as endogenous control. Values in the noninfected cell EV treated set were used asunits.

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 12 of 23

anti-inﬂammatory response in miR-146a expressing cells. There-

fore, it is the balanced expression of HuR and miR-146a that de-

termine the polarization status of the macrophage. In miR-146a

compromised situation, expression of HuR promotes M1 polariza-

tion (Goswami et al, 2020), while HuR-mediated miR-21 export

contributes in increased IL-10 expression. High IL-10, in the pres-

ence of miR-146a, possibly down-regulates HuR protein level to

ensure the subdued expression of proinﬂammatory cytokines and

expression of IL-10 itself as a feedback effect (Rajasingh et al, 2006).

miR-146a acts as a balancer of IL-10 production and Ld infection

Does miR-146a uptake via EV affect the Ld infection process? We

have measured the number of parasites internalized in the treated

macrophage after Ld-infected cell EVs or miR-146a–containing EVs

treatment. We have performed quantiﬁcation and noted reduced

entry of Ld after the miR-146a positive or infected cell EV treatment

(Fig 8A–D). Lower expression level of Leishmania amastigote–

speciﬁc gene amastin also conﬁrmed reduced levels of Ld entry

after the miR-146a–containing EV or infected cell EV treatment of

RAW264.7 cells (Fig 8E –G ). miR-146a expression in RAW264.7 cells

also reduces the Ld infection process. We have used single cell

infection level examination to conclude on preferential reduction

of Ld internalization in cells expressing miR-146a (Fig 8H). Infection

level may be down-regulated due t o a problem i n signalling

pathways or downstream factors. We have noted an increased

P-p38 level in miR-146a–expressing macrophage that signiﬁes the

existence of a possible counteractive pathway that usually gets

reduced in cells infected with Ld where a p38/MAPK down-

regulation has been noted (Fig 8M). The decreased Ld infection

could have also been attributed by a possible reduction in the

endocytosis process itself. However, in a latex bead internalization

assay, we did not document any reduction in latex bead inter-

nalization after miR-146a–containing EV or infected cell EV treat-

ment of RAW264.7 cells (Fig 8I–L).

Discussion

EVs are known for their role in intercellular communication. They

play an important role in immune response during any pathological

condition and also help to establish tumour microenvironment

(Thery et al, 2002). In disease condition, EVs either can help in

progression of the disease or in providing protection against the

disease. There are ma ny studies where it has been repor ted that

exosomes from Mycobacterium infected cells can trigger proin-

ﬂammatory response in noninfected cells (Bhatnagar et al, 2007).

Leishmania parasitesreleaseexosomesasavehicletotransport

proteins to the host cell for immunosuppressive action (Silverman

et al, 2010). Leishmania protein G P63 was found to be t ransported

to neighbouring hepatocytes via secreted exosomes that target

Dicer1, a pr ocessor of pre-miRNAs, to down-regulate expression of

liver speciﬁc miR-122 (Ghosh et al, 2013). Leishmania parasite

secretes exosomes within sandﬂy midgut and thes e exosomes are

part of the sandﬂy inocula during bite which helps in patho-

genesis via overproduction of IL-17a in target cells (Atayde et al,

2015). Giosefﬁ et al (2020), in recent time, have reported Leish-

mania-infected EVs as a carrier of large number of Leishmanial

and host proteins (Giosefﬁ et al, 2020). Among the proteins

exported out, they have identi ﬁed that a Leishmania homolog of

mammalian angiogenesis promoting factor, vasohibin is found to

induce endothelial cells to release angioge nesis promoting fac-

tors and thereby helping in promotion of lesion v ascularization

during infection.

miRNAs, being the regulator of expression of several cytokines,

are important players in determining the fate of immune cells and

in particular have a major role in buffering the immune activation

processes by regulating the expression of several cytokines directly

or indirectly (Lindsay, 2008; Testa et al, 2017). miR-146a and miR-155

are the two most important players that are explored for their

potential role as immune checkpoint regulators in the mammalian

system. Whereas miR-155 is a pro-inﬂammatory miRNA, miR-146a

(C,D,E,F)Effect of infected cell–derived EV treatment on recipient macrophages. (C) A schematic representation of the experiment has been shown (C). (D) Relative levels of

miR-146a and iNOS mRNA in recipient macrophage after 24 h of EV treatment were measured by qRT-PCR and miRNA and mRNA levels in noninfected cell EV-treated cell were

set as unit. U6 and GAPDH was used as endogenous control for miRNA and mRNA, respectively (D, left panel P = 0.0184, n = 4 independent experiments and right panel,

P = 0.0035, n = 5 independent experiments, respectively). (E) Levels of Dicer1, HuR, P-ERK1/2, and P-p38 were measured by Western blot analysis in infected cell–derived

EV-treated RAW264.7 cells. β-Actin was used as loading control. (F) Relative levels of IL-1β (F, left panel, P = 0.0967, n = 3 independent experiments) and IL-10 (F, right panel,

P = 0.0357, n = 3 independent experiments) were measured by qRT-PCR in recipient RAW264.7 cells after 24 h of EV treatment. GAPDH was used as endogenous control and

values for noninfected cell EV-treated cells were set as unit. (G, H) Effect of EV-mediated transfer of miR-146a on recipient macrophage. (G) A schematic diagram has been

given. (H) Relative levels of cytokine mRNAs; IL-1β,IL-10andiNOS(H,upper panel, P = 0.2131, n = 3 independent experiments, H, middle panel, P = 0.0406, n = 4 independent

experiments and H, lower panel, P = 0.0662, n = 3 independent experiments, respectively). GAPDH was used as endogenous control and values for pCIneo control EV-treated

cells were set as units. (I) Effect of Ld infection on cytokine mRNA levels in pmiR-146a overexpressed macrophages. A schematic diagram of the experiment has been shown

(I, upper panel). Relative cytokine mRNA level of TNF-α (I, left panel, P =0.0138,P =0.1740,P = 0.7287, n = 3 independent experiments), IL-1β (I, middle panel, P =0.0183,P =0.0684,

P = 0.4727, n = 3 independent experiments), and IL-10 (I, rightpanel, P =0.1491,P =0.2417,P = 0.7457, n = 3 independent experiments) were measured by qRT-PCR from pCIneo and

pmiR-146a transfected RAW264.7 cells after 24 h of Ld infection or no infection. GAPDH was used as endogenous control and values for pCIneo control cells were set as units

(I, unpaired t test was performed for comparison between pmiR-146a–

expressing noninfected control and infected set). (J

) Effect of control and infected cell derived-EV

treatment on recipient macrophages transfected with control or miR-146a inhibitor oligos. A schematic diagram has been given (J, left panel). Relative mRNA level of IL-10 was

measured by qRT-PCR from miR-146a inhibitor transfected and EV-treated RAW264.7 cells (J, right panel, paired t test, P = 0.0149,0.3208, unpaired t test was performed for

comparison between miR-146a inhibitor transfected control EV and infected cell EV-treated sets, P = 0.0456, n = 3 independent experiments, respectively). GAPDH was used as

endogenous control and values for negative control inhibitor transfected cells were set as units. (K) Effect of LPS treatment on pmiR-146a–expressing macrophages. Relative

level of IL-10 was measured by qRT-PCR from control or pmiR-146a–expressing cells with or without LPS exposure (1 ng/ml) (K. paired t test, P = 0.0984, 0.0921, unpaired t test

was performed for comparison between LPS-treated sets, P = 0.0132, n = 3 independent experiments). GAPDH was used as endogenous control and values for untreated control

cells were set as units. Data information: In all experimental data, error bars are represented as mean ± SEM, ns, nonsigniﬁcant, *P <0.05,**P < 0.01, respectively. P-values were

calculated by two-tailed paired t test in most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the

respective Western blots.

Source data are available for this ﬁgure.

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 13 of 23

Figure 7. Crosstalk between miRNA-exporter HuR and miR-146a determines the fate of cytokine response in macrophage cells.

(A) LPS treatment (100 ng/ml) for different time points in RAW264.7 cells. The cell lysates were immunoblotted for HuR. β-Actin was used as loading control. (B) Real-

time PCR was performed for proinﬂammatory cytokine mRNAs (TNF-α and IL-1β) in pCIneo and pHA-HuR cells co-transfected with pre-miR control mimic. GAPDH was

used for normalization (n = 5 independent experiments; P-values = 0.2048, 0.0467, respectively). Va lues observed in pCIneo transfected cells were considered as units for

normalization of values obtained with pHA-HuR transfected cells. (C, D) Extracellular vesicles (EVs) isolated from pmiR-146a transfected RAW264.7 cells were treated to

recipient RAW264.7 cells expressing pCIneo or pHA-HuR. (C) Recipient cells treated with pmiR-146a–expressing cell EVs were immunoblotted for HA and HuR. β-Actin was

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 14 of 23

acts oppositely to balance the immune activation process (Testa

et al, 2017). In the infection context, the pathogens need to target

the miRNA pathways to control the inﬂammation level. The Ld

parasite has shown previously to control expression of miRNAs

in a negative manner in infected cells. By down-regulating HRS

and depolarizing mitochondria in the host, Ld affects the miRNA

recycling process to enhance the miRNA content of the infected

cells. However, the accumulated miRNAs are largely non-functional

as they were found to be accumulated with endosomal fraction and

they fail to recycle for new round of target repression on ER attached

polysomes (Chakrabarty & Bhattacharyya, 2017; Bose et al, 2020). The

miR-146a that also increases with Ld infection gets exported to

neighbouring cells. The miR-122 is another miRNA in hepatocytes that

Ld needs to control to ensure lower cholesterol biogenesis in the

liver cells essential for the parasite survival. Ld, by targeting Dicer1 in

hepatocytes, control the expression of miR-122 in infected liver tissue

(Ghosh et al, 2013; Chakrabarty & Bhattacharyya, 2017). Our results

show that miR-146a, transferred from infected macrophage cells,

also has a negative role to play on miR-122 expression in mouse liver.

Reciprocally, miR-122 can get transferred from activated or stressed

liver cells to resident macrophage to get them activated (Ghosh et al,

2015; Mukherjee et al, 2016). The parasite uses a secondary mech-

anism of lowering the miR-122 in liver cells to ensure reduced trans-

mission of the proinﬂammatory miR-122 to macrophages to prevent the

expression of inﬂammatory cytokines there. EV-mediated crosstalk

between the macrophages and hepatocytes fosters an environment of

cell-to-cell communication to take place. However, in the liver, it is

known that the EVs released from LPS-treated THP-1 macrophages

could stimulate the proliferation of hepatic stellate cells, whereras

miR-103-3p present in the EVs isolated f rom macrophages could

get transferred to the hepatic stellate cells to promote its growth

(Chen et al, 2020).

Mitochondrial activity is found to be a determining factor for

intercellular transfer of miRNAs. Does miR-122 affect mitochondrial

function? miR-122 is known to regulate mitochondrial metabolic

function. Cationic amino acid transporter gene CAT-1 level de-

creases, whereras PPARGC1A (PGC-1α) and succinate dehydroge-

nase subunit A and B level increases in HCC cells expressing

miR-122. PGC-1α is the regulator of mitochondrial biogenesis and it

is also involved in energy metabolism. Succinate dehydrogenase

(SDH) is the enzyme complex located in inner mitochondrial

membrane and is involved in both TCA cycle and electron transport

chain. PGC-1α and succinate dehydrogenase both are found to be

the putative secondary targets of miR-122 (Burchard et al, 2010).

Mitochondrial depolarization as well as ATP content are found to be

non-responsive to miR-122 levels in macrophage cells.

How mitochondria affects the EV entry in the recipient cells? We

have found how the mitochond ria-ER tethering and mitochondria–

endosome interaction play a key role in miRNA internalization process

which also affect miRNA turnover in mammalian cells (Chakrabarty &

Bhattacharyya, 2017). Any factor that can inﬂuence these interactions

will also modify organellar dynamics in mammalian cells to eventually

affect the EV-internalization. Increased expression of Ucp2 due to Ld

infection or exogeneous expression of FH-Ucp2 is associated with

mitochondrial depolarization and changed mitochond rial dynamics

that resulted in defective miRNA compartmentalization. Importantly

Mfn2 loss in Ld-infectedcellsorMfn2nullconditioninMfn2−/− MEF

cells are also associated with miRNA compartme ntalization defect

(Chakrabarty & Bhattacharyya, 2017). Therefore, microRNA compart-

mentalization or defective EV uptake possibly directly gets affected

by the change in mitochondrial dynamics rather than changed

membrane potential or ATP level. p-Triﬂouromethoxyphenylhydrazone

(FCCP) is an uncoupler of mitochond

rial membrane potential but does

not affect cellular ATP content whereas oligomycin inhibit F0F1-ATPase

used as loading control. (D) Real-time PCR was performed for cytokine mRNAs (TNF-α, IL-1β, and IL-10) in pCIneo and pHA-HuR transfected recipient cells treated with

pmiR-146a–EVs. GAPDH was used for normalization (n = 3 independent experiments; P-values: 0.3952, 0.0134, and 0.0352, respectively). Values obtained with pCIneo

transfected and EV-treated cells were considered as units. (E, F) EVs were isolated from pmiR-146a transfected RAW264.7 cells and used to treat the recipient RAW264.7

cells depleted of HuR (siHuR transfected). (E) Recipient cells treated with pmiR-146a–EVs were immunoblotted for HuR to check for proper silencing and β-Actin was

used as loading control (E). (F) Real-time PCR was performed for cytokine mRNAs (TNF-α, IL-1β and IL-10) in siCon or siHuR-containing recipient cells treated with pmiR-

146a–EVs, GAPDH was used for normalization (n = 3 independent experiments; P-values: 0.0004, 0.0015, 0.2641, respectively) (F). In this particular Experiment, siCon

transfected and EV-treated cells was used for normalization of values noted in siHuR-containing cells. (G, H, I, J, K) Effect of pHA-HuR expression in RAW264.7 cells

transfected with pre-miR control mimic or pre-miR-146a mimic. Transfection was performed either wit h pCIneo (control plasmid) or pHA-HuR. (G) Immunoblotting of the

cell lysates for HuR using β-Actin as loading control (G). (H) Immunoblotting of the respective cell lysates for HA to check for proper overexpression of HA-HuR, β-Actin

was used as loading control. Real-time PCR was performed for TNF-α, where GAPDH has served as control. (I) Values obtained for pCIneo and pre-miR control mimic co-

transfected sets were taken as units (n ≥ 5 independent experiments; P-values: 0.2048, 0.8587, 0.8620, respectively). (J) Real-time PCR was performed for IL-1β, GAPDH

served as control. Values obtained for pCIneo and pre-miR control mimic co-transfected sets were taken as units (n ≥ 5 independent experiments; P-values: 0.0467,

0.3453, 0.0375, respectively). (K) Real-time PCR was performed for IL-10, where GAPDH has served as control. Values obtained for pCIneo and pre-miR control mimic co-

transfected sets were taken as units (n ≥ 4 independent experiments; P-values: 0.0239, 0.2710, 0.0267, respectively). (L, M) Relative level of miR-21 in HA-HuR expressing

RAW264.7 cells. Real-time PCR for miR-21 level in RAW264.7 cells expressing pHA-HuR and transfected with pre-miR-146a mimic. Values obtained for pCIneo and pre-miR

mimic co-transfected sets were taken as units. U6 snRNA served as control (n = 3 independent experiments; P-value = 0.0015) (L, left panel). Real-time PCR for miR-21

level in recipient RAW264.7 cells depleted of HuR which were treate d with miR-146a–containing EVs. Values obtained for siCon and miR-146a–containing EV-treated sets

were taken as units. U6 snRNA served as control (n = 3 independent experiments; P-value = 0.5259) (L, middle panel). Real-time PCR of EV-associated miR-21 level from EVs

of RAW264.7 cells expressing or not expressing pHA-HuR. EV marker protein Flotillin-1 was used for normalization of miR-21 level (n = 3 independent experiments; P-

value = 0.4313) (L, right panel). Values obtained for pCIneo transfected cell–derived EVs was considered as unit. (M) Immunoblotting of the cell lysates with HA using β-Actin

as loading control in pCIneo or pHA-HuR transfected RAW264.7 cells.

(N) The

proposed model depicts the role of EV-associated miRNAs in Ld infection. The left half shows

that Ld infection triggers EV-mediated secretion of miR-146a from macrophage which when transfers to na

ıve macrophage polarizes it toward M2 phenotype because of

its anti-inﬂammatory role whereas the right half represents what happens when miR-146a get delivered to na

ıve hepatocyte via EV. miR-146a reduces miR-122 level in

hepatocytes. If not restricted, miR-22 as part of hepatocyte secreted EVs polarizes naive macrophage to M1 phenotype because of its proinﬂammatory role. Data

information: In all experimental data, error bars are represented as mean ± SEM,*P < 0.05; **P < 0.01; ***P < 0.001. P-values were calculated by two-tailed paired t test in

most of the experiments unless mentioned otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.

Source data are available for this ﬁgure.

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 15 of 23

Figure 8. Effect of extracellular vesicle (EV)–associated miR-146a treatment on Ld infection.

(A, B, C, D) Infected cell–derived EV treatment decreases Ld infection in recipient cells. (A) Parasites were labelled with CFSE dye and infection was given for 6 h after 24 h

of control and infected cell–derived EV treatment (A, upper and lower panel, respectively). Parasite infection was determined by counting the CFSE positive structures

inside cells. Scale bar 10 μm. (B) Percentage of infected cells was calculated for both control cell EV and infected cell EV-treated RAW264.7 cells (B, P = 0.0064, unpaired

t test, n ≥ 16 number of ﬁelds). (C) Parasites were labelled with CFSE dye and infection was given for 6 h after 24 h of pCIneo and pmiR-146a–transfected cell–derived EV

treatment. Percentage of infected cells was calculated for both pCIneo and pmiR-146a transfected cell–derived EV-treated cells (C, P = 0.0208, unpaired t test, n ≥ 18

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 16 of 23

and prevents ATP production without affecting mitochondrial mem-

brane potential (Chakrabarty & Bhattacharyya, 2017). We found oli-

gomycin treatment could not inhibit EV-mediated internalization of

miR-122 and there was no change in miR-122 uptake in 3-h FCCP

treated cells. Thus, it is not the mitochondrial depolarization or ATP

level, but is the change in mitochondrial dynamics and its interaction

with endosomes or ER in Mfn2 KO or FH-Ucp2–expressing or Ld-

infected cells (Chakrabarty & Bhattacharyya, 2017) that probably

hampers endocytosis of miR-122–containing EVs in recipient cells.

MicroRNAs are the small noncoding RNAs that are involved in

post-transcriptional and translational regulation of various genes.

Thus, altered expression of these miRNAs is one of the main

reasons for various pathogenic conditions (Bartel, 2018). Earlier

reports suggested up-regulation of miR-146a/b during Salmonella

infection (Ordas et al, 2013). Up-regulation of miR-155 and miR-

146a/b and down-regulation of miR-20a, miR-191, and miR-378 in

Mycobacterium avium–infected condition were also reported

(O’ Conne ll et al, 2012; Curtale et al, 2019 ). miR-155 favours the

pro-inﬂammatory environment, whereas miR-146a favours the

anti-inﬂammatory environment (Lindsay, 2008). Recently miR-

146a–mediated M2 polarization during L. donovani (Ld) infection

has been reported. The investigation revealed that L. donovani (Ld)

infection up-regulates miR-146a expression which favoured par-

asite survivability in hosts by maintaining the anti-inﬂammatory

environment. Inhibition of miR-146a lowered parasite burden in

infected Balb/C mice as well as anti-inﬂammatory environment (M2

phenotype). Interestingly, this up-regulation of miR-146a during Ld

infection is regulated by Super Enhancer components like BET

bromodomain 4 (BRD4) and P300 (Das et al, 2021).

The polarization of macrophage is contributed by several factors

including speciﬁc activation of signalling component that favours

either the TLR4-p38/MAPK-NF-kB–mediated activation of proin-

ﬂammatory cytokine expression or it may be an inhibitory circuit

involving ERK1/2–dependent inactivation of the proinﬂammatory

cytokines with con comitant increase in expression of anti-

inﬂammatory cytokine like IL-10 (Mukherjee et al, 2021). The par -

asite s eems to use the infected cell EVs packed with miR-146a to

transmit the anti-i nﬂammatory signals to the na ive neighbouring

macrophage to polarize them to the M2 stage. It is not known

whether the polarization is reversible, but subsequent treatment

of miR-146a overexpressed cell s with LPS suggest the refractory

nature of the recipient macrophage to inﬂammatory response.

Thus, miR-146a–containing EVs could be considered as a good

immune modulator and use of that could be important to control

the inﬂammation in different physiological contexts like in pre-

vention of viral infection related cytokine storm (Ar isan et al, 2020

)

inﬂammation associated with tumour (Rupaimoole et al, 2016).

The cross communication of miR-146a–containing EVs to other

noninfected tissue is another important aspect. Do EVs with miR-

146a get transferred via the bloodstream to the spleen and do they

have any effect on subsequent establishment of infection and

T-cell polarization observed in the spleen of infected animals?

These are important questions for futur e exploration.

In our experimental system, the effect of Leishmanial secretory

proteins on the M2 state of macrophages seems to be marginal as

the cytokine pattern of recipient macrophages treated with Ld

promastigote culture–derived EVs showed decreased level of IL-10

and IL-1β as well. Thus, these results clearly explain that the ob-

served M2 state of the na

ıve macrophages treated with infected

cell–derived EVs is due to EV-mediated transfer of miR-146a and

possibly not by Leishmania speciﬁc factors. The results obtained

from the SLA or LPG or parasite-derived EV-treated macrophages

and hepatocytes suggest Leishmania derived factors are not pri-

marily responsible also for miR-122 and miR-146a up-regulation in

respective cell types (Fig 7N).

Although miR-122 can up-regulate IL-1β mRNA, we could not

detect an increase in mature IL-1β by ELISA in miR-122 expressing

cells. Thus, the transcriptional surge of IL-1β mRNA due to miR-122

expression is not associated with increase detectable in the protein

level. It is possible that the excess IL-1β mRNA produced in the

presence of miR-122 may get transferred to other immune cells

where they may elicit a proinﬂammatory response. Otherwise, the

inﬂammatory response observed in miR-122 expressing cells is

manifested primarily by TNF-α rather than IL-1β.

HuR is a RNA binding protein with immense functional diversity.

HuR is known to stabilize the mRNAs with AU-rich elements in their

39 UTR and thus have a role in the inﬂammation process as most of

the proinﬂammatory cytokines such as TNF-α or IL-1 β bear the AU-

rich elements in their 39 UTR (Srikantan & Gorospe, 2012). In this

report, we have detected a non-canonical role of HuR in stabili-

zation of IL-10 mRNA. The anti-inﬂammatory IL-10 mRNA is regu-

lated negatively by miR-21 (De Melo et al, 2021), the suppression of

number of ﬁelds). (D) Parasites were labelled with CFSE dye and infection was given for 24 h to pCIneo and pmiR-146a–transfected cell. Percentage of infected cells was

calculated for both pCIneo and pmiR-146a transfected cells (P = 0.0401, unpaired t test, n ≥ 16 number of ﬁelds). (E) RAW264.7 cells were infected with Ld for 24 h and

infection was measured in terms of amastin mRNA level by qRT-PCR (E, P = 0.2213, n = 4 independent experiments). (F) Relative level of amastin mRNA was determined by

qRT-PCR from control and infected cell–der ived EV-treated cells followed by 6 h infection (F, P = 0.0205, n = 4 independent experiments). (G) Relative level of amastin

mRNA from pCIneo and pmiR-146a transfected cells after 24 h of infection was determined by qRT-PCR (G, P = 0.0160, n = 4 independent experiments). GAPDH was used as

endogenous control. Values for uninfected cell or control EV-treated cell or pCIneo transfected cells were considered as units. (H) RAW264.7 cells were co-transfected

with DsRed and pmiR-146a and was infected with Ld for 24 h and infection level was visualized at individual cells (lower panel) and quantiﬁed (upper panel). Percent of

infection in pmiR-146a/DsRed expressing versus non-transfected Ld-infected cells were measured. Scale bar 10 μm. (n = 38, Number of ﬁelds). (I, J, K, L) Latex bead

phagocytosis after EV treatment. Red ﬂuorescent bead (Fluorescent Red; Sigma-Aldrich) was diluted in medium and added to EV-treated RAW264.7 cells at 1:10 (cell:bead)

ratio for 6 h. Red ﬂuorescent bead was visualized by a confocal microscope. (I, J) Phalloidin 488 was used for staining the cytoskeleton of cells (I). Scale bar 10 μm.

Percentage of beads phagocytosed in EV-treated macrophages was plotted and compared between control cell EV-treated and Ld-infected cell EV-treated cells (J,

unpaired t test, P = 0.0347, Number of ﬁelds, n ≥ 13 number of ﬁelds). (K, L) In panel (K) visualization of beads phagocytosed in EV-treated macrophages was monitored in

pCIneo transfected control cell EV-treated and pmiR-146a– expressing cell EV-treated cells. The quantiﬁcation was completed and plotted in panel L (unpaired t test, P =

0.6761, Number of ﬁelds, n ≥ 13 number of ﬁelds). Scale bar = 10 μm. (M) Cellular levels of the major signalling pathway components in pmiR-146a and pCIneo expres sion

conditions were determined by Western blot. β-Actin was used as loading control. Data information: In all experimental data, error bars are represented as mean ± SEM,

ns, nonsigniﬁcant, *, ** represent P-value of <0.05, <0.01, respectively. P-values were calculated by two-tailed paired t test in most of the experiments unless mentioned

otherwise. Positions of molecular weight markers are marked and shown with the respective Western blots.

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 17 of 23

miR-21 activity by HuR has been reported previously (Poria et al,

2016). In the context of miR-146a induced expression of IL-10, HuR

acts synergistically to remove miR-21 from the cell to boost the anti-

inﬂammatory IL-10 production in macrophages. Interestingly, in

the context of an anti-inﬂammatory environment the miRNA

antagonistic role of HuR predominate to ensure expression of

miRNA-repressed cytokine mRNAs, whereas in the proinﬂammatory

context, the mRNA ’s stabilizer role of HuR is important to ensure

proinﬂammatory cytokine expression there.

Materials and Methods

Cell culture, peritoneal macrophage isolation, and parasite

infections

Human hepatic Huh7, mouse hepatic HePa1-6, and Mfn2 wild-type

and knockout MEFs cells (WT/Mfn2

−/−

) were cultured in Dulbecco’s

Modiﬁed Eagle’sMedium(Gibco-BRL)and 10% FBS (heat-

inactivated foetal bovine serum). In case of RAW264.7 macro-

phage cells, RPMI-1640 medium (Gibco) with 2 mM L-glutamine and

0.5% β-mercaptoethanol along with 10% FBS was used. Primary

murine peritoneal macrophages were isolated from BALB/c mice

subjected to intraperitoneal injection of 1 ml of 4% starch solution.

Cold 1× PBS was used to lavage or wash the peritoneal cavity to

isolate the peritoneal macrophages the following day which were

then pelleted and plated. These cells were also cultured in RPMI-

1640. EV isolation of Peritoneal Exudate Cells (PECs) was done from

a 60 mm plate. All experiments with PEC were carried out after 48 h

of isolation (Goswami et al, 2020 ). For macrophage or Huh7 acti-

vation, LPS dose of 1 or 100 or 500 ng/ml was used for different time

points depending on the experiments. RAW264.7 and Huh7 cells

were stimulated with LPS from Escherichia coli O111:B4 (Calbio-

chem). For EV-free growth medium, 10% EV-depleted FBS (made by

ultracentrifugation of FBS at 1,000,00g for 4 h) was added to DMEM

or RPMI-1640 for respective cell types.

Animal experiments

BALB/c male mice (4–6 wk) were divided into four groups (three

animals each) of either uninfected or infected and treated or

untreated with EVs. Two groups (six animals) were infected with

1×10

promastigotes by intracardiac puncture and maintained for

30 d. On the 30

day, EVs were injected into six animals (three

infected and three uninfected with parasites), whereas 1× PBS was

injected in the remaining six animals (three infected and three

uninfected). For EV isolation, HePa1-6 cells were transfected with

miR-122–expressing plasmid and ~5 × 10

EVs (measured by Nano-

particle Tracker Nanosight NS300) isolated from these transfected

cells were suspended in 1× PBS and injected into the tail vein. The

animals were euthanized on the 31

day for Kupffer cell isolation.

All the experiments were carried out in accordance with the

National Regulatory Guidelines issued by the Committee for the

Purpose of Supervision of Experiments on Animals, Ministry of

Environment and Forest, Govt. of India. The animal experiments

were performed in agreement with the Institutional Animal Ethics

Committee. The BALB/c mice were kept under controlled conditions

(temperature 23 ± 2°C, 12 h/12 h light/dark cycle) in individually

ventilated cages.

Kupffer cell isolation was done from control and experimental

group of mice. The animals were anaesthetized, and livers were

perfused with HBSS via the hepatic portal vein with an incision in

the inferior vena cava until they turned bloodless and then

digested with a Liver Digest Medium. Livers were then excised,

minced, and ﬁltered through a 70-μm cell strainer. The resultant

single cell suspension was centrifuged at 50g for 5 min to pellet

and store the hepatocytes. The supernatant was then collected and

loaded onto a Percoll gradient of 25% and 50%. It was centrifuged at

1,200g for 30 min at 4°C without brakes. The interphase containing

the Kupffer cells was collected and washed twice with PBS and the

pellet was stored. The Kupffer cell pellet and hepatocyte pellet were

subjected to RNA isolation by TriZol reagent.

For detection of purity of isolation, the Kupffer cells of all the four

groups were compared with the respective hepatocytes by quan-

tiﬁcation of mRNA levels of C-type Lectin Domain Family 4, Member

F (Clec4f), a Kupffer Cell marker, and hepatocyte marker, Albumin.

The kinetoplast DNA levels were also quanti ﬁ ed in the four groups

to detect the level of leishmanial infection.

Parasite culture and infection to macrophage cell line

L. donovani (Ld) strain AG83 (MAOM/IN/83/AG83) was obtained

from a Indian Kala-azar patient and was maintained in golden

hamsters (Chakrabarty & Bhattacharyya, 2017). Amastigotes were

obtained from infected hamster spleen and transformed into

promastigotes. Promastigotes were maintained in M199 medium

(Gibco) supplemented with 10% FBS (Gibco) and 1% Pen-Strep

(Gibco) in 22°C. RAW264.7 cells or PEC were infected with station-

ary phase Ld promastigotes of second to fourth passage at a ratio of

1:10 (cell: Ld) for 6, 16, or 24 h depending upon the experiment

(Goswami et al, 2020).

Plasmid constructs and transfection

pCIneo, precursor miR-146a–overexpressing plasmid (pmiR-146a),

precursor miR-122–overexpressing plasmid (pmiR-122), and HA-HuR

plasmid(pHA-HuR) were transfected using Fugene HD (Promega) for

RAW264.7 cells as described previously (Goswami et al, 2020). For

Huh7 cells, all transfections of plasmids were performed using

Lipofectamine 2000 reagent (Life Technologies) according to the

manufacturer’s protocol. For miRNA inhibitor (anti-miR-146a) ex-

periments, 30 pmol per well (30 nM) was transfected using RNA imax

reagent in RAW264.7 cells according to manufacturer’s protocol.

Transfection of Negative control inhibitor was used as controls. For

co-transfection of plasmid and pre-miR mimic, Lipofectamine 2000

(Invitrogen) was used. 50 pmoles of pre-miR mimic were trans-

fected per well of a 12 well plate (50 nM). siRNA transfection was

performed using RNAi max (Invitrogen) following the manufac-

turer’s instructions. RAW264.7 cells were transfected with 50 pmoles

of siRNA per well of a 12-well plate (50 nM). siRL was used as siCon

for the experiments. EV treatment was given to the recipient cells

48 h after siRNA transfection and the cells were harvested 24 h after

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 18 of 23

EV treatment. Donor RAW264.7 cells were transfected with 2 μg

pmiR-146a plasmid per well of a six-well plate using Fugene HD.

EV isolation and characterization

RAW264.7 cells were plated in a 90 mm plate and given infection

with stationary phase parasite at about 80% conﬂuency and kept

for 6 h. After 6 h of infection, the medium was discarded and cells

were washed with PBS to remove extracellular unattached para-

sites and cells were replenished with fresh RPMI-1640 medium

supplemented with 10% EV depleted FBS and 1% Pen-Strep for 20 h.

Total infection time was 24 h before EV isolation. After 24 h, the

supernatant was collected for EV isolation. Brieﬂy, the supernatant

ﬁrst centrifuged at 3,000g for 15 min at 4°C followed by 30 min

centrifugation at 10,000g at 4°C. For all EV isolation experiments,

supernatant was passed through a 0.22-μm ﬁlter unit followed by

ultracentrifugati on on a 30% sucrose cushion at 1,00,000g for 70 min.

The supernatant was discarded leaving a medium layer of interface-

containing EVs behind, which was then washed with 1× PBS at 1,00,000g

for 70 min again. The pellet then was resuspended in PBS for NTA or 1×

passive lysis buffer (PLB) for RNA or protein precipitation (Promega).

For EV-associated RNA and protein, the pellet was resuspended in PLB.

One-third was used for total RNA isolation using TriZzol LS reagent

(Invitrogen) following the manufacturer’s protocol. Two-thirds of the

PLB sample was used for protein precipitation using methanol pre-

cipitation. In brief, for 200 μlofPLBsample,640μl of sterile water,

480 μl of methanol (Merck), and 160 μl of chloroform (Sigma-

Aldrich) w ere added and vortexed well followed by centrifugation

at 20,000g for 5 min at room temperature. Then the supernatant

was discarded and 300 μl o f methanol was added. The mixture

was vortexed well and again centr ifuged for 30 min at 20,000g 4°C.

The supernatant was discarded. The pellet was air drie d for 1 0 min

and then dissolved in 1× SDS Dye and heated at 95°C before run on

aSDS–PAGE.

For experiments with EV treatment, culture supernatant ﬁrst

centrifuged at 3,000g for 15 min at 4°C followed by 30 min cen-

trifugation at 10,000g and ﬁltered pass through 0.22 μm following

ultracentrifugation at 1,00,000g for 70 min. The pellet was then

resuspended in RPMI-1640 medium supplemented with 10% EV

depleted FBS and 1% Pen-strep for further use.

For nanoparticle tracking analysis (NTA), the control and infected

RAW264.7 cell–derived EV pellet was resuspended in 300 μlof

sterile PBS and resuspended well. Then, diluted 10-folds in PBS and

1 ml of diluted sample was used for NTA (Nanosight NS300).

EV treatment of recipient cells

After EV isolation, the pellet was resuspended in fresh RPMI-1640

supplemented with 10% EV–depleted FBS and 1% Pen-Strep fol-

lowed by ﬁltration through a 0.22-μ

m ﬁlter

unit. The EVs were then

added to the recipient macrophage or hepatic cell line for 24 h.

Post-LPS treatment was given at a dose of 1 ng/ml for 4 h after 20 h

of EV treatment to macrophages. For treatment, EVs isolated from

8×10

cells (one 90 mm plate) were added to 2.4 × 10

recipient cells

(per well of a 12 well plate). For estimation of parasite infection after

EV treatment, parasites were added to EV-treated cells for 6 h.

Optiprep density gradient ultracentrifugation

For cell fractionation using 3–30% iodixanol gradient (Optiprep

gradient) 1.6 × 10

cells were used as described earlier (Mukherjee

et al, 2016). Optiprep (Sigma-Aldrich) solution was used to prepare

3–30% gradient. In brief, the cell pellet was homogenized in a

buffer with glass Dounce homogenizer. The lysate was centri-

fuged at 1,00 0g twice and the supernatant was loaded on the

gradient and centrifuged at 36,000 rp m in a Beckman-Coulter

SW60Ti rotor for 5 h. Approximately 400 μl of 10 fractions were

collected for RNA and protein.

Total RNA isolation and quantiﬁcation of miRNA and mRNA

Total RNA extract from experimental samples using TriZol reagent

(Invitrogen) for cell and TriZol LS (Invitrogen) for Optiprep/

subcellular fractio n according t o manufacturer ’sprotocol.All

miRNA and mRNA quantiﬁcations were performed as described

previously ( Basu & Bhattacharyya, 2014; Mukherjee et al, 2016). In

brief, miRNA estimation was performed with 100 ng of RNA for cellular

and EV samples using speciﬁc primers for human miR-146a, mouse

miR-155, human miR-122, human miR-21, human miR-16, miR-125b,

and U6 snRNA was used as endogenous control. For the assay, one-

third of the reverse transcription mix was subjected to PCR ampli-

ﬁcation with TaqMan Universal Master Mix No AmpErase (Applied

Biosystem) and respective TaqMan assay reagents for target miRNAs.

Samples were analysed in triplicates for each biological replicates.

The comparative C

method which involved normalization by U6

snRNA was used for quantiﬁcation (Mukherjee et al, 2016).

For mRNA, 200 ng of total RNA was used for estimation based on

SYBR Green based real-time cDNA estimation using speciﬁc primer

for target genes. The comparative C

method which involved nor-

malization by GAPDH or 18s rRNA was used for relative quantiﬁ-

cation of mRNA (Goswami et al, 2020). The details of Primers, miRNA

assays are provided as part of Tables S4 and S5. Information on

expression plasmids, siRNAs, miRNA-mimic and miRNA inhibitors

are part of Table S6.

Western blot

Western blot analyses were performed as described elsewhere

(Mazumder et al, 2013). Imaging of the blots was performed using an

UVP BioImager 600 system equipped with VisionWorksLife Science

software (UVP) V6.80. ImageJ software was used for quantiﬁcation.

Details of all antibodies used for Wes tern blot and immuno ﬂuo-

rescence experiments are summarized in Tab le S 7.

Immunoﬂuorescence

For internalization, Ld parasites were stained with 1 μ M carboxy-

ﬂuorescein succinimidyl ester (CFSE) dye (green) for 30 min at 22°C

followed by PBS wash thrice and then resuspended in medium

before adding to the cell. Cells were ﬁxed with 4% PFA for 20 min

after three PBS wash. Nuclei were stained with DAPI. For calculating

the percentage of infected cells, a minimum number of 100 cells

were counted. Cells were observed under the Zeiss LSM800 con-

focal microscope.

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 19 of 23

Polysome isolation

For polysome isolation ~8 × 10

cells were used as starting material.

After 24 h of infection, cells were scraped in PBS and pelleted down

at 600g for 5 min at 4°C. The cell pellet was collected and incubated

in 1× lysis buffer (10 mM Hepes, 25 mM KCL, 5 mM MgCl

, 1 mM DTT, 5

mM VRC, 1× PMSF, cycloheximide 100 μg/ml, 1% Triton X-100, and 1%

sodium deoxycholate) for 15 min at 4°C followed by centrifugation

at 3,000g for 10 min at 4°C. Then the supernatant was collected and

centrifuged at 20,000g for 10 min at 4°C. The supernatant was

collected and loaded on 30% sucrose cushion in gradient buffer

and centrifuged at 31,200 rpm for 1 h in 4°C in a SW61Ti rotor.

After centrifugation the non-polysome fractions were collected

andtherestofthesolutionwasmixedfollowedbycentrifugation

at 31,200 rpm for 30 min at 4°C in a SW61Ti rotor. T he pelleted

polysome was suspended in polysome buffer (10 mM Hepes,

25 mM KCL, 5 m M MgCl

, 1 mM DTT, 5 mM VRC, a nd 1× PMS F) and

kept for RNA isolation and Western blot (Ghoshal et al, 2021).

LPG extraction and treatment

LPG isolation was performed as mentioned earlier (Goswami et al,

2020). In brief, 2 × 10

stationary phase parasites (AG83) were used

for LPG extraction. The pellet was resuspended in 2 ml of Chlo-

roform: Methanol: Water mixture (1:2:0.5 vol/vol) followed by vor-

texing and sonication for 10 s thrice at 30 s interval and incubated in

mutarotator at room temperature for 2 h. Then the lysate was

centrifuged at 4,000g for 30 min at 4°C. The pellet was used for

extraction in 9% 1-Butanol in water. After vortex and sonication as

before, pellet was incubated for 1 h at room temperature in

mutarotator. Second extraction was done with the pellet in 9%

1-Butanol in water again after centrifugation at 4,000g for 30 min at

4°C. The supernatants of both extractions were pooled for ly-

ophilization. The lyophilized LPG was resuspended in 1 ml sterile

PBS before cell culture treatment. For treatment, 1:50 (cell: LPG)

dose was used for different time points.

SLA preparation

SLA was prepared from the stationary-phase promastigotes (~10

cells) as described earlier (Basu et al, 2005). Brieﬂy, stationary

phase parasites were pelleted down at 3,000g for 10 min followed

by repeated cycles of freezing (−70°C) and thawing (37°C) followed

by 5 min incubation on ice. These cells were then further completely

lysed by sonication thrice for 30 s and centrifuged for 10,000g for

30 min at 4°C. The supernatant was used for protein estimation

using the Bradford assay. Macrophages or Huh7 were treated with

10 μg/ml SLA for 24 h.

Mass spectrometry

For proteomics sample preparation, exosomes from two 90 mm

were pooled and protein was precipitated with 100% chilled ace-

tone in −20°C overnight. The protein precipitated was then washed

with 80% chilled acetone followed by another wash with 40%

chilled acetone. The pellet was then air dried for 5–10 min to

remove excess acetone at room temperature and resuspended in

50 mM ammonium bicarbonate (AmBic) followed by sonication

thrice. 85 mM DTT was then added, and the sample was heated at

60°C for 1 h. Sample was then incubated in dark with 55 mM

Iodoacetamide (IAA) at RT for 30 min. Trypsin (1 μg/μl) digestion was

carried out to the sample for overnight at 37°C. The reaction was

stopped with 100% formic acid (5% of total volume) and snap frozen

until lyophilization. Lyophilized sample was used for proteomics

and run in Orbitrap analyzer.

Latex bead phagocytosis assay

Red ﬂuorescent lab elled Latex bead (Fluorescent Red; Sigma-

Aldrich) was diluted in RPMI-1640 medium and added to mac-

rophages at a ratio of 1:10 (cell:bead) for 6 h. Cells were ﬁxe d w ith

4% PFA as described earlier and mount in DAPI. Cell cytoskeleton

was s tained with Phalloidin 488. Red latex bead phagocytosis was

imaged in Zeiss LSM800 confocal microscope.

Cytokine measurement by ELISA

Protein level of TNF-α was measured from culture s upernatant

of RAW264.7 macrophages using sandwich ELISA Kit (BD Phar-

mingen) as per the manufacturer ’ s protocol. Protein l evel of IL-

1β was m easured from RAW264.7 cell lysate (1 mg/ml) instead of

cell culture supernatant using sandwich ELISA Kit (BD Phar-

mingen) as per the manufacturer’sprotocol.Cytokinelevelwere

determined by measuring the OD at 450 nm using a microtiter

plate reader.

Measurements of NO in cell culture supernatant

Nitric oxide (NO) was measured from RAW264.7 cell supernatant

using G reiss reagent. In brief, equal volume of sample was in-

cubated with eq ual volume of 1% sulphanilam ide for 15 min in

dark at room temperature followed by equal volume of 0.1%

NEDD (N-1-napththylethylenediamine dihydrochloride) was added

and incubated until colour developed. NO level was deter-

mined by measuring the OD at 550 nm using a microtiter plate

reader.

Flow cytometry analysis for mitochondrial depolarization

Approximately 0.4 × 10

RAW264.7 cells were scraped and washed in

PBS. Then the cells were incubated in 100 nM MitoTracker Red for

30 min in 37° C followed by washing with PBS to remove excess stain

and ﬁnally analysed in BD FACS Fortessa.

Cellular ATP-level measurement

Amount of ATP was determined using the ATP bioluminescent assay

Kit (Sigma-Aldrich)

. Approximately 1.5 × 10

RAW264.7 cells were

scraped and washed in PBS. Brieﬂy, the cells were lysed and the

equal volume of lysate was added to equal volume of ATP assay mix

solution followed by rapid mixing and the amount of light produced

was determined by luminometer.

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 20 of 23

Immunoprecipitation

Immunoprecipitation was performed as described earlier with

minor modiﬁcations (Goswami et al, 2020). Brieﬂy, protein G agarose

beads (Life Technologies) was used for exogeneous HA protein or

any other endogenous protein. Beads were washed twice with 1× IP

buffer (20 mM TRIS–HCL, pH 7.5, 150 mM KCL, 5 mM MgCl

, and 1 mM

DTT 1× EDTA free protease inhibitor cocktail) at 2,000g for 2 min at

4°C. Beads were then blocked with 5% BSA in lysis buffer for 1 h at

4°C in mutarotator followed by washing with IP buffer as mentioned

previously. Afte r blocking, requi red amount of antibody was

added to t he bead for a nother 4 h incubation at 4°C before lysate

addition. The ﬁnal dilution of antibody was 1:100. Cells w ere

lysedinalysisbuffer(1×IPbufferwith0.5%TritonX-100and0.5%

of sodium deoxycholate) for 30 min in 4°C and sonicated thrice

for 10 s interval and 30 s incu bation in ice. Then l ysate was

collected after centrifugation at 16,000g for 10 min at 4°C and

was added to the bead-antibody mixture for overnight immu-

noprecipitation to occur at 4°C. Af ter the reaction, the lysate–

bead–antibody mixture was washed with 1× IP buffer thrice and

was resuspended i n 400 μl I P buffer. Equal volume was kept for

protein and RNA. For RNA, TriZol LS was added thrice of the

volume and for protein 5× SDS dye was added so that the ﬁnal

concentration would be 1×.

Statistical analysis

All graphs and statistical signiﬁcance were calculated using

GraphPad Prism 5.00 and 8.00 version (GraphPad). Experiments

were carried out for minimum three times to get P-value, unless

otherwise mentioned. Two-tailed paired and unpaired t tests were

used for determination of P-values. The sample size was chosen by

convenience and no exclusion criteria were used.

Ethics statement

Balb/C mice and Syrian golden hamsters w ere obtained from

CSIR-Indian Institute of Chemical Biology animal house facility.

All experiments were performed according to the national

regulatory guidelines stated by the Committee for the Purpose of

Supervision of Experiments on Animals, Ministry of Environment

and Forest, Govt. of India. All animal experiments were per-

formed with prior approval of the Institutional Animal Ethics

Committee.

Data Availability

All data are available in the supplementary Tables (Tables S1–S3).

This study includes no data deposited in external repositories.

Supplementary Information

Supplementary Information is available at https://doi.org/10.26508/lsa.

202101229.

Acknowledgements

We acknowledge Witold Filipowicz for different plasmid constructs. We also

acknowledge the support of Malini Sen, Shilpak Chatterjee , and Syamal Roy

for different reagents used in ELISA and ATP measurement assays. SN

Bhattacharyya and K Mukherjee acknowledge Council of Scientiﬁc and In-

dustrial Research (CSIR)-Indian Institute of Chemical Biology (IICB) for

infrastructural support. The work is supported by The Swarnajayanti Fel-

lowship (DST/SJF/LSA-03/2014-15) and CSIR funded Niche Creating Project

RNAMOD (MLP-139), Leishmaniasis (MLP-136), and the Indo-Swiss Bilateral

Project Grant from Department of Biotechnology, Govt of India (BT/IN/

Swiss/53/SNB/2018-19). K Mukherjee and SN Bhattacharyya both received

support from CSIR. SN Bhattacharyya also acknowledges support of National

Bioscience Award Fund from Department of Biotechnology, Government of

India. S Ganguly received support from University Grant Commission, India. B

Ghoshal, I Banerji, S Chakraborty, S Bhattacharjee, and A Goswami received

their support from CSIR, India. The funders had no role in study design,

data collection and analysis, decision to publish, or preparation of the

manuscript.

Author Contributions

S Ganguly: data curation, formal analysis, validation, investigation,

and methodology.

B Ghoshal: formal analysis, valida tion, investiga tion, and methodology.

I Banerji: data curation, formal analysis, validation, investigation,

and methodology.

S Bhattacharjee: validation and investigation.

S Chakraborty: validation and investigation.

A Goswami: validation and investigation.

K Mukherjee: conceptualization, formal analysis, supervision, vi-

sualization, methodology, and writing—original draft, review, and

editing.

SN Bhattacharyya: conceptualization, data curation, supervision,

visualization, methodology, project administration, and writing—original

draft, review, and editing.

Conﬂict of Interest Statement

The authors declare that they have no conﬂict of interest.

References

Arisan ED, Dart A, Grant GH, Arisan S, Cuhadaroglu S, Lange S, Uysal-Onganer

P (2020) The prediction of mirnas in sars-cov-2 genomes: Hsa-mir

databases identify 7 key mirs linked to host responses and virus

pathogenicity-related kegg pathways signiﬁcant for comorbidities.

Viruses 12: 614. doi:10.3390/v12060614

Atayde VD, Aslan H, Townsend S, Hassani K, Kamhawi S, Olivier M (2015)

Exosome secretion by the parasitic protozoan leishmania within the

sand ﬂy midgut. Cell Rep 13: 957–967. doi:10.1016/j.celrep.2015.09.058

Bartel DP (2018) Metazoan micrornas. Cell 173: 20–51. doi:10.1016/

j.cell.2018.03.006

Basu R, Bhaumik S, Basu JM, Naskar K, De T, Roy S (2005) Kinetoplastid

membrane protein-11 DNA vaccination induces complete protection

against both pentavalent antimonial-sensitive and -resistant strains

of leishmania donovani that correlates with inducible nitric oxide

synthase activity and il-4 generation: Evidence for mixed th1- and th2-

like responses in visceral leishma niasis. J Immunol 174: 7160–7171.

doi:10.4049/jimmunol.174.11.7160

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 21 of 23

Basu S, Bhattacharyya SN (2014) Insulin-like growth factor-1 prevents mir-122

production in neighbouring cells to curtail its intercellular transfer to

ensure proliferation of human hepatoma cells. Nucleic Acids Res 42:

7170–7185. doi:10.1093/nar/gku346

Beattie L, Peltan A, Maroof A, Kirby A, Brown N, Coles M, Smith DF, Kaye PM

(2010) Dynamic imaging of expe rimental leishmania donovani-

induced hepatic granulomas detects kupffer cell-restricted antigen

presentation to antigen-speciﬁc cd8 t cells. PLoS Pathog 6: e1000805.

doi:10.1371/journal.ppat.1000805

Bhatnagar S, Shinagawa K, Castellino FJ, Schorey JS (2007) Exosomes released

from macrophages infected with intracellular pathogens stimulate a

proinﬂammatory response in vitro and in vivo. Blood 110: 3234–3244.

doi:10.1182/blood-2007-03-079152

Bode JG, Ehlting C, H

aussinger D (2012) The macrophage response towards

lps and its control through the p38(mapk)-stat3 axis. Cell Signal 24:

1185–1194. doi:10.1016/j.cellsig.2012.01.018

Bose M, Chatterjee S, Chakrabarty Y, Barman B, Bhattacharyya SN (2020)

Retrograde trafﬁcking of argonaute 2 acts as a rate-limiting step for

de novo mirnp formation on endoplasmic reticulum-attached

polysomes in mammalian cells. Life Sci Alliance 3: e201800161.

doi:10.26508/lsa.201800161

Burchard J, Zhang C, Liu AM, Poon RT, Lee NP, Wong KF, Sham PC, Lam BY,

Ferguson MD, Tokiwa G, et al (2010) Microrna-122 as a regulator of

mitochondrial metabolic gene network in hepatocellular carcinoma.

Mol Syst Biol 6: 402. doi:10.1038/msb.2010.58

Chakrabarty Y, Bhattacharyya SN (2017) Leishmania donovani restricts

mitochondrial dynami cs to enhance mirnp stability and target rna

repression in host macrophages. Mol Biol Cell 28: 2091–2105.

doi:10.1091/mbc.E16-06-0388

Chen L, Yao X, Yao H, Ji Q, Ding G, Liu X (2020) Exosomal mir-103-3p from lps-

activated thp-1 macrophage contributes to the activation of hepatic

stellate cells. FASEB J 34: 5178–5192. doi:10.1096/fj.201902307RRR

Chen Y, Shen A, Rider PJ, Yu Y, Wu K, Mu Y, Hao Q, Liu Y, Gong H, Zhu Y, et al

(2011) A liver-speciﬁc microrna binds to a highly conserved rna

sequence of hepatitis b virus and negatively regulates viral gene

expression and replication. FASEB J 25: 4511–4521. doi:10.1096/fj.11-

187781

Cheng HS, Sivachandran N, Lau A, Boudreau E, Zhao JL, Baltimore D, Delgado-

Olguin P, Cybulsky MI, Fish JE (2013) Microrna-146 represses

endothelial activation by inhibiting pro-inﬂammatory pathways.

EMBO Mol Med 5: 1017–1034. doi:10.1002/emmm.201202318

Curtale G, Rubino M, Locati M (2019) Micrornas as molecular switches in

macrophage activation. Front Immunol 10: 799. doi:10.3389/

ﬁmmu.2019.00799

Das S, Mukherjee S, Ali N (2021) Super enhancer-mediated transcription of

mir146a-5p drives m2 polarization during leishmania donovani

infection. PLoS Pathog 17: e1009343. doi:10.1371/journal.ppat.1009343

de Carvalho RVH, Andrade WA, Lima-Junior DS, Dilucca M, de Oliveira CV,

Wang K, Nogueira PM, Rugani JN, Soares RP, Beverley SM, et al (2019)

Leishmania lipophosphoglycan triggers caspase-11 and the non-

canonical activation of the nlrp3 inﬂ

ammasome. Cell

Rep 26:

429–437.e5. doi:10.1016/j.celrep.2018.12.047

De Melo P, Pineros Alvarez AR, Ye X, Blackman A, Alves-Filho JC, Medeiros AI,

Rathmell J, Pua H, Serezani CH (2021) Macrophage-derived microrna-

21 drives overwhelming glycolytic and inﬂammatory response during

sepsis via repression of the pge2/il-10 axis. J Immunol 207: 902–912.

doi:10.4049/jimmunol.2001251

Fern

andez-Messina L, Guti

errez-V

azquez C, Rivas-Garc

ıaE,S

anchez-Madrid

F, de la Fuente H (2015) Immunomodulatory role of micrornas

transferred by extracellular vesicles. Biol Cell 107: 61–77. doi:10.1111/

boc.201400081

Fu Y, Zhang L, Zhang F, Tang T, Zhou Q, Feng C, Jin Y, Wu Z (2017) Exosome-

mediated mir-146a transfer suppresses type i interferon response

and facilitates ev71 infection. PLoS Pathog 13: e1006611. doi:10.1371/

journal.ppat.1006611

Ghosh J, Bose M, Roy S, Bhattacharyya SN (2013) Leishmania donovani targets

dicer1 to downregulate mir-122, lower serum cholesterol, and

facilitate murine liver infection. Cell Host Microbe 13: 277–288.

doi:10.1016/j.chom.2013.02.005

Ghosh S, Bose M, Ray A, Bhattacharyya SN (2015) Polysome arrest restricts

mirna turnover by preventing exosomal export of mirna in growth-

retarded mammalian cells. Mol Biol Cell 26: 1072–1083. doi:10.1091/

mbc.E14-11-1521

Ghosh S, Mukherjee K, Chakrabarty Y, Chatterjee S, Ghoshal B, Bhattacharyya

SN (2021) Gw182 proteins restrict extracellular vesicle-mediated

export of micrornas in mamma lian cancer cells. Mol Cell Biol 41:

e00483-20. doi:10.1128/MCB.00483-20

Ghoshal B, B ertrand E, Bhattacharyya SN (2021) Non-canonical argonaute

loading of extracellular vesicle-derived exogenous single-stranded

miRNA in recipient cells. J Cell Sci 134: jcs253914. doi:10.1242/jcs.253914

Giosefﬁ A, Hamerly T, Van K, Zhang N, Dinglasan RR, Yates PA, Kima PE (2020)

Leishmania-infected macrophages release extracellular vesicles that

can promote lesion development. Life Sci Alliance 3: e202000742.

doi:10.26508/lsa.202000742

Goswami A, Mukherjee K, Mazumder A, Ganguly S, Mukherjee I, Chakrabarti S,

Roy S, Sundar S, Chattopadhyay K, Bhattacharyya SN (2020) Microrna

exporter hur clears the internalized pathogens by promoting pro-

inﬂammatory response in infected macrophages. EMBO Mol Med 12:

e11011. doi:10.15252/emmm.201911011

Kalluri R, LeBleu VS (2020) The biology, function, and biomedical applications

of exosomes. Science 367: 6478. doi:10.1126/science.aau6977

Klecker T, B

ockler S, Westermann B (2014) Making connections:

Interorganelle contacts orchestrate mitochondrial behavior. Trends

Cell Biol 24: 537–545. doi:10.1016/j.tcb.2014.04.004

Lenk EJ, Redekop WK, Luyendijk M, Fitzpatrick C, Niessen L, Stolk WA, Tediosi F,

Rijnsburger AJ, Bakker R, Hontelez JAC, et al (2018) Socioeconomic

beneﬁt to individuals of achieving 2020 targets for four neglected

tropical diseases controlled/eliminated by innovative and intensiﬁed

disease management: Human african trypanosomiasis, leprosy,

visceral leishmaniasi s, chagas disease. PLoS Negl Trop Dis 12:

e0006250. doi:10.1371/journal.pntd.0006250

evin-Le Moal V, Loiseau PM (2016) Leishmania hijacking of the macrophage

intracellular compartments. FEBS J 283: 598–607. doi:10.1111/

febs.13601

Lindsay MA (2008) Micrornas and the immune response. Trends Immunol 29:

343–351. doi:10.1016/j.it.2008.04.004

Masaki T, Chiba S, Tatsukawa H, Yasuda T, Noguchi H, Seike M, Yoshimatsu H

(2004) Adiponectin protects lps-induced liver injury through

modulation of tnf-alpha in kk-ay obese mice. Hepatology 40: 177–184.

doi:10.1002/hep.20282

Mazumder A, Bose M, Chakraborty A, Chakrabarti S, Bhattacharyya SN (2013) A

transient reversal of mirna-mediated repression controls

macrophage activation. EMBO Rep 14: 1008–1016. doi:10.1038/

embor.2013.149

Momen-Heravi F, Bala S, Kodys K, Szabo G (2015) Exosomes derived from

alcohol-treated hepatocytes horizontally transfer liver speciﬁc

mirna-122 and sensitize monocytes to lps. Sci Rep 5: 9991. doi:10.1038/

srep09991

Mukherjee B, Mukherjee K, Nanda P, Mukhopadhayay R, Ravichandiran V,

Bhattacharyya SN, Roy S (2021) Probing the molecular mechanism of

aggressive infection by antimony resistant leishmania donovani.

Cytokine 145: 155245. doi:10.1016/j.cyto.2020.155245

Mukherjee B, Mukhopadhyay R, Bannerjee B, Chowdhury S, Mukherjee S,

Naskar K, Allam US, Chakravortty D, Sundar S, Dujardin JC, et al (2013)

Antimony-resistant but not antimony-sensitive leishmania donovani

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 22 of 23

up-regulates host il-10 to overexpress multidrug-resistant protein 1.

Proc Natl Acad Sci U S A 110: E575 –E582. doi:10.1073/pnas.1213839110

Mukherjee K, Ghoshal B, Ghosh S, Chakrabarty Y, Shwetha S, Das S,

Bhattacharyya SN (2016) Reversible hur-microrna binding controls

extracellular export of mir-122 and augments stress response. EMBO

Rep 17: 1184–1203. doi:10.15252/embr.201541930

O’Connell RM, Rao DS, Baltimore D (2012) Microrna regulation of

inﬂammatory responses. Annu Rev Immunol 30: 295–312. doi:10.1146/

annurev-immunol-020711-075013

Ordas A, Kanwal Z, Lindenberg V, Rougeot J, Mink M, Spaink HP, Meijer AH

(2013) Microrna-146 function in the innate immune transcriptome

response of zebraﬁsh embryos to salmonella typhimurium infection.

BMC Genomics 14: 696. doi:10.1186/1471-2164-14-696

Poria DK, Guha A, Nandi I, Ray PS (2016) Rna-binding protein hur sequesters

microrna-21 to prevent translation repression of proinﬂammatory

tumor suppressor gene programmed cell death 4. Oncogene 35:

1703–1715. doi:10.1038/onc.2015.235

Rajasingh J, Bord E, Luedemann C, Asai J, Hamada H, Thorne T, Qin G,

Goukassian D, Zhu Y, Losordo DW, et al (2006) Il-10-induced tnf-alpha

mrna destabilization is mediated via il-10 suppression of p38 map

kinase activation and inhibition of hur expression. FASEB J 20:

2112–2114. doi:10.1096/fj.06-6084fje

Regev-Rudzki N, Wilson DW, Carvalho TG, Sisquella X, Coleman BM, Rug M,

Bursac D, Angrisano F, Gee M, Hill AF, et al (2013) Cell-cell

communication between malaria-infected red blood cells via

exosome-like vesicles. Cell 153: 1120–1133. doi:10.1016/j.cell.2013.04.029

RexJ,LutzA,FalettiLE,AlbrechtU,ThomasM,BodeJG,BornerC,SawodnyO,

Merfort I (2019) IL-1β and TNFα different ially inﬂuence NF-κB activity and

FasL-induced apoptosis in primary murine hepatocytes during LPS-

induced inﬂammation. Front Physiol 10: 117. doi:10.3389/fphys.2019.00117

Rupaimoole R, Calin GA, Lopez-B erestein G, Sood AK (2016) Mirna

deregulation in cancer cells and the tumor microenvironment. Cancer

Discov 6: 235–246. doi:10.1158/2159-8290.CD-15-0893

Scianimanico S, Desrosiers M, Dermi ne JF, M

eresse S, Descoteaux A,

Desjardins M (1999) Impaired recruitment of the small gtpase rab7

correlates with the inhibition of phagosome maturation by

leishmania donovani promastigotes. Cell Microbiol 1: 19–32.

doi:10.1046/j.1462-5822.1999.00002.x

Silverman JM, Clos J, de’Oliveira CC, Shirvani O, Fang Y, Wang C, Foster LJ,

Reiner NE (2010) An exosome-based secretion pathway is responsible

for protein export from leishmania and communication with

macrophages. J Cell Sci

123: 842–852. doi:10.1242/jcs.056465

Srikantan S, Gorospe M (2012) Hur function in disease. Front Biosci

(Landmark Ed) 17: 189–205. doi:10.2741/3921

Sunter J, Gull K (2017) Shape, form, function and leishmania pathogenicity:

From textbook descriptions to biological understanding. Open Biol 7:

170165. doi:10.1098/rsob.170165

Testa U, Pelosi E, Castelli G, Labbaye C (2017) Mir-146 and mir-155: Two key

modulators of immune response and tumor development. Noncoding

RNA 3: 22. doi:10.3390/ncrna3030022

ery C, Zitvogel L, Amigorena S (2002) Exosomes: Composition, biogenesis

and function. Nat Rev Immunol 2: 569–579. doi:10.1038/nri855

Todkar K, Chikhi L, Germain M (2019) Mitochondrial interaction with the

endosomal compartment in endocytosis and mitochondrial

transfer. Mitochondrion 49: 284–288. doi :10.1016/

j.mito.2019.05.003

Walker DM, Oghumu S, Gupta G, McGwire BS, Drew ME, Satoskar AR (2014)

Mechanisms of cellular invasion by intracellular parasites. Cell Mol

Life Sci 71: 1245–1263. doi:10.1007/s00018-013-1491-1x

Wang H, Xu W, Shao Q, Ding Q (2017) Mir-21 silencing ameliorates

experimental autoimmune encephalomyelitis by promoting the

differentiation of il-10-producing b cells. Oncotarget 8: 94069–94079.

doi:10.18632/oncotarget.21578

License: This article is available under a Creative

Commons License (Attribution 4.0 International, as

described at https://creativecommons.org/

licenses/by/4.0/).

Leishmania hijacks host miRNA machinery Ganguly et al. https://doi.org/10.26508/lsa.202101229 vol 5 | no 6 | e202101229 23 of 23