MicroRNA 155 Contributes to Host Immunity against Leishmania donovani but Is Not Essential for Resolution of Infection

CD4⁺ T helper 1 (Th1) cells producing interferon gamma (IFN-γ) are critical for the resolution of visceral leishmaniasis (VL). MicroRNA 155 (miR155) promotes CD4⁺ Th1 responses and IFN-γ production by targeting suppressor of cytokine signaling-1 (SOCS1) and Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP-1) and therefore could play a role in the resolution of VL.

ABSTRACT

CD4⁺ T helper 1 (Th1) cells producing interferon gamma (IFN-γ) are critical for the resolution of visceral leishmaniasis (VL). MicroRNA 155 (miR155) promotes CD4⁺ Th1 responses and IFN-γ production by targeting suppressor of cytokine signaling-1 (SOCS1) and Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP-1) and therefore could play a role in the resolution of VL. To determine the role of miR155 in VL, we monitored the course of Leishmania donovani infection in miR155 knockout (miR155KO) and wild-type (WT) C57BL/6 mice. miR155KO mice displayed significantly higher liver and spleen parasite loads than WT controls and showed impaired hepatic granuloma formation. However, parasite growth eventually declined in miR155KO mice, suggesting the induction of a compensatory miR155-independent antileishmanial pathway. Leishmania antigen-stimulated splenocytes from miR155KO mice produced significantly lower levels of Th1-associated IFN-γ than controls. Interestingly, at later time points, levels of Th2-associated interleukin-4 (IL-4) and IL-10 were also lower in miR155KO splenocyte supernatants than in WT mice. On the other hand, miR155KO mice displayed significantly higher levels of IFN-γ, iNOS, and TNF-α gene transcripts in their livers than WT mice, indicating that distinct organ-specific antiparasitic mechanisms were involved in control of L. donovani infection in miR155KO mice. Throughout the course of infection, organs of miR155KO mice showed significantly more PDL1-expressing Ly6C^hi inflammatory monocytes than WT mice. Conversely, blockade of Ly6C^hi inflammatory monocyte recruitment in miR155KO mice significantly reduced parasitic loads, indicating that these cells contributed to disease susceptibility. In conclusion, we found that miR155 contributes to the control of L. donovani but is not essential for infection resolution.

INTRODUCTION

Leishmaniasis is a neglected tropical disease caused by more than 20 protozoan parasite species of the genus Leishmania. This disease afflicts 12 million people in over 80 countries, with an annual incidence of 2 million cases (1), and is considered by WHO to be one of the six priority parasitic diseases, second only to malaria (2). Visceral leishmaniasis (VL) is the most severe form of Leishmania infection, which is caused by Leishmania donovani or L. infantum (3). VL is a potentially life-threatening disease associated with systemic spread of parasites to the liver, spleen, and bone marrow, resulting in prolonged fever, anemia, weight loss, hepatosplenomegaly, and secondary bacterial infections (3, 4).

It is well documented that interleukin 12 (IL-12) and Th1-associated interferon gamma (IFN-γ) are indispensable for host resistance against VL and resolution of infection (5, 6). Furthermore, previous studies from our laboratory and others have shown that Th2-associated IL-4 and IL-13 are also required for formation of mature granuloma in the liver and effective clearance of the parasites (7, 8). Conversely, IL-4 or IL-4R deficiency has been shown to reduce therapeutic efficacy of anti-leishmanial chemotherapy. Collectively, these findings indicate that both Th1- and Th2-associated cytokines contribute to immunity against VL, unlike CL (cutaneous leishmaniasis), in which Th2 cytokines promote susceptibility.

MicroRNAs (miRNAs) are a class of short single-stranded noncoding RNAs involved in gene regulation. They influence transcription of many protein-coding genes and mediate posttranscriptional repression (9). The regulatory properties of miRNAs make them important contributing factors to host responses, such as inflammation and development of immunity (10). MicroRNA 155 (miR155) is of particular interest, as it has been shown to regulate a wide array of genes, including chemokines, cytokines, and transcription factors (11). This microRNA is involved in both innate and adaptive immunity. Its expression is upregulated in many activated and mature cells of the immune system and can lead to the regulation of inflammatory cytokines and antigens responsible for immune responses (12, 13). Additionally, miR155 has been shown to promote efficient antigen presentation by dendritic cells (11) and to regulate members of the superfamilies of tumor necrosis factor (TNF) receptors and their ligands (14). After CD4⁺ T cell activation by antigen-presenting cells, miR155 was shown to promote Th1 and Th17 proinflammatory responses due to its inhibitory action on suppressor of cytokine signaling-1 (SOCS1) and Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP-1), known suppressors of cytokine signaling (15, 16). Along with CD4⁺ T cells, miR155 also plays a role in B-cell and CD8⁺ T cell proliferation and responses (12, 13). On the other hand, lack of miR155 leads to an increase in Th2 and a decrease in Th1 responses in vitro (11). Together, these findings indicate that miR155 could enhance or suppress proinflammatory immune responses that are critical for the resolution of L. donovani infection. To determine the role of miR155 in VL, we compared the progression of L. donovani infection and immune responses in miR155 gene-deficient mice with those of their age- and sex-matched wild-type (WT) counterparts. Our results show that miR155 plays a role in host defense against L. donovani but is not required for control of infection and resolution of VL.

RESULTS

miR155 deficiency increases susceptibility to L. donovani infection, which is associated with impairment of granuloma formation in the liver.

Following intravenous inoculation with 10⁷ L. donovani (LV82) amastigotes, the livers of miR155 knockout (miR155KO) mice showed significantly higher parasitic loads throughout the course of infection (15, 40, 60, 90, and 120 days postinfection [dpi]) than their WT counterparts (Fig. 1A). On the other hand, the spleens of miR155KO mice displayed very low parasitic loads, similar to those of WT mice, at 15 dpi. Spleen parasite burdens in WT mice peaked at 40 dpi and were significantly higher than those of miR155KO mice, in which parasite loads peaked at 60 dpi. At this time point and thereafter, spleens of miR155KO mice contained significantly more parasites than their WT counterparts. However, both groups eventually began to resolve the infection, as indicated by a decline in parasite burdens in both organs (Fig. 1A and B).

FIG 1.

Higher parasitic loads and lower levels of hepatic granulomas observed in miR155KO mice. WT and miR155KO mice were infected with L. donovani amastigotes. Mice were euthanized at 15, 40, 60, 90, and 120 dpi, and their organs were harvested. (A and B) Liver (A) and spleen (B) parasitic burdens of WT and miR155KO mice at the respective time intervals. Liver sections were stained with H&E, and granulomas were enumerated per 10 high-power fields and are indicated as means ± standard errors of the means (SEM). (C) Average numbers of developing (immature granuloma), mature, and parasitic free granulomas. Statistically significant differences are indicated between miR155KO and WT groups with regard to the total numbers of granulomas at each time point of infection. (D) Representative images of developing and mature granulomas (40×). Data represented are from 1 of the 3 independent experiments with n = 5 mice for each group/time point. *, P < 0.05; **, P = 0.01; ***, P = 0.001.

Hepatic granuloma formation is critical for parasite clearance in VL (17). We therefore assessed granulomas in the livers of L. donovani-infected WT and miR155KO mice. Histopathological analysis of livers from the infected mice revealed that miR155KO mice contained significantly fewer developing and mature granulomas than WT controls at 15, 40, and 60 dpi (Fig. 1C). However, the number of developing, mature, and parasitic free granulomas significantly increased in the livers of miR155KO mice at 120 dpi, which was associated with the decline in liver parasite loads (Fig. 1C and D). Collectively, these results suggest that miR155 plays an important role in host resistance against L. donovani infection and formation of hepatic granulomas, which is critical for parasitic clearance in VL.

Lack of miR155 impairs both Th1 and Th2 immune responses in the spleens of L. donovani-infected mice.

As miR155 has been shown to regulate T cell responses as well as Th1 versus Th2 cytokine balance by targeting SOCS1 and SHIP-1 (18), we analyzed T cell proliferation and production of Th1-associated IFN-γ and Th2-associated IL-4 and IL-10 by spleen cells harvested from WT and miR155KO mice at different time points after infection. Following in vitro stimulation with L. donovani antigen (LdAg), spleen cells from L. donovani-infected miR155KO mice displayed significantly reduced T cell proliferation responses at 40, 60, and 120 dpi compared to those of WT controls (Fig. 2E). Analysis of spleen cell supernatants showed that LdAg-stimulated spleen cells from miR155KO mice produced significantly lower levels of IFN-γ than those from similarly infected WT controls (Fig. 2A). At 40 dpi, culture supernatants from miR155KO mice contained significantly higher levels of TNF-α than WT controls, in which TNF-α production peaked at 90 dpi (Fig. 2B). LdAg-stimulated spleen cells from miR155KO mice produced significantly more IL-4 than WT mice at 15 dpi. However, IL-4 production by spleen cells from miR155KO mice declined significantly as infection progressed (Fig. 2C). Although miR155KO and WT mice showed analogous levels of IL-10 in the early stages of the infection (15 and 40 dpi), similar to IL-4, the levels of IL-10 also decreased in miR155KO mice compared to those in WT mice at later time points (60 and 90 dpi) (Fig. 2D). No significant levels of IL-12, IL-13, and IL-17 were observed (data not shown).

FIG 2.

miR155 deficiency impairs both Th1 and Th2 immune responses. Spleens of WT- and miR155KO-infected mice were harvested at the respective time intervals; single-cell suspensions were prepared and stimulated with LdAg for 72 h. Production of cytokines in the splenocyte culture supernatants was measured by sandwich ELISA. (A) IFN-γ. (B) TNF-α. (C) IL-4. (D) IL-10. (E) T cell proliferation measured by alamarBlue reduction method at 15, 40, 60, 90, and 120 dpi. Data represented are from 1 of the 3 independent experiments with n = 5 mice for each group/time interval. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

miR155 deficiency is associated with induction of distinct organ-specific immune responses following L. donovani infection.

Since miR155KO mice harbored significantly more parasites in their livers and spleens than WT controls, we analyzed gene transcript levels of pro- and anti-inflammatory cytokines, chemokines, and miR155-targeted SHIP-1 and SOCS1 in the organs of L. donovani-infected WT and miR155KO mice at different time points. Interestingly, at 60, 90, and 120 dpi, livers of miR155KO mice expressed higher transcript levels of IFN-γ (Fig. 3A), iNOS (Fig. 3B), and TNF-α (Fig. 3C) than WT counterparts. In contrast, spleens of miR155KO mice contained significantly smaller amounts of IFN-γ mRNA transcripts than WT mice throughout the course of infection (Fig. 3G), which correlated with lower IFN-γ production in these mice. miR155KO mice expressed higher TNF-α mRNA levels at 40 dpi, reflecting high TNF-α levels in spleen cell supernatants, but no significant differences were noted in TNF-α transcript levels between the groups at other time points (Fig. 3I). Interestingly, despite lower IFN-γ production and no differences in TNF-α level, miR155KO mice expressed significantly more iNOS in their spleens at 90 and 120 dpi (Fig. 3H), suggesting upregulation of iNOS via TNF-α/IFN-γ-independent pathways in this organ. No significant differences were noted in the transcript levels of IL-10, IL-12, and IL-17 (data not shown) in the spleen and liver between WT and miR155KO mice, but CXCL-10 (C-X-C motif chemokine 10) (Fig. 3F and L) was significantly elevated in both the livers and spleens of miR155KO mice at chronic stages of the infection.

FIG 3.

Livers and spleens of miR155KO-infected mice exhibit distinct host immune mechanisms. Livers and spleens of WT and miR155KO mice were harvested at respective time intervals. Gene expression analysis was done by RT-PCR. (A to F) Gene expression of IFN-γ (A), iNOS (B), TNF-α (C), SHIP-1 (D), SOCS1 (E), and CXCL10 (F) from the livers. (G to L) Gene expression of IFN-γ (G), iNOS (H), TNF-α (I), SHIP-1 (J), SOCS1 (K), and CXCL10 (L) from the spleens. Data are represented as fold induction in response to liver and spleen from uninfected naive mice from 1 of the 3 independent experiments with n = 5 mice for each group/time interval. Data were analyzed by using unpaired t test. *, P < 0.05; **, P < 0.01.

At days 40 to 120 dpi, the livers of miR155KO mice showed increased expression of both SHIP-1 (Fig. 3D) and SOCS1 (Fig. 3E), which are known to be direct targets of miR155. Consistent with this finding, the spleens of miR155KO mice also showed increased transcripts of SHIP-1 (Fig. 3J) and an increased trend of SOCS1 levels (Fig. 3K) at days 40 to 120 dpi. Together, these findings indicate that distinct immune mechanisms are involved in resolution of L. donovani infection in the liver and spleen of miR155KO mice.

Lack of miR155 leads to the impairment of CD4⁺ and CD8⁺ T cell recruitment in the livers and spleens of L. donovani-infected mice.

It is well known that miR155 deficiency leads to decreased trafficking of CD4⁺ and CD8⁺ T cells in various viral infections, like coronavirus (19) and herpes simplex encephalitis (20), as well as under other conditions, like acute graft-versus-host disease (21). It also has been shown that miR155 is highly expressed on regulatory T cells and is a direct target for Foxp3 (22). To investigate the effect of miR155 deficiency in T cell subset recruitment, we analyzed the livers and spleens of L. donovani-infected WT and miR155KO mice temporally by flow cytometry. At all time points, livers and spleens of miR155KO mice contained significantly fewer CD4⁺ T cells than their WT counterparts (Fig. 4A and D, respectively). Additionally, miR155KO mice also showed significant impairment of CD8⁺ T cell recruitment to the livers at 40, 60, 90, and 120 dpi (Fig. 4B) and to the spleens at 60, 90, and 120 dpi (Fig. 4E). Our data also revealed that there was no significant difference in Foxp3 gene expression in liver and spleen of miR155KO-infected mice and that of the WT counterparts (Fig. 4C and F). No significant differences were noted in NK cell recruitment to organs between the groups (data not shown). Together, these results suggest that miR155 plays a role in mediating recruitment of CD4⁺ and CD8⁺ T cells to the visceral organs during L. donovani infection.

FIG 4.

miR155 deficiency impairs CD4⁺ and CD8⁺ T cell recruitment into the visceral organs. WT- and miR155KO-infected mice were euthanized at the respective time intervals and analyzed for lymphoid and myeloid cell populations. (A to C) Percentage of CD4⁺ T cells (A), percentage of CD8⁺ T cells in the total lymphocytes (B), and FOXP3 gene expression (C) from the livers. (D to F) Percentage of CD4⁺ T cells (D), percentage of CD8⁺ T cells in the total lymphocytes (C), and FOXP3 gene expression (F) from the spleens. Data represented are from 1 of the 3 independent experiments with n = 5 mice for each group/time interval. *, P  < 0.05; **, P < 0.01; ***, P < 0.001.

L. donovani-infected miR155KO mice show accumulation of immunosuppressive PDL-1^hi Ly6C^hi inflammatory monocytes in their organs.

A recent study from our group has established the detrimental role of CD11b⁺ Ly6C^hi cells (Ly6C^hi inflammatory monocytes) in L. donovani infection (23). In the present study, flow cytometric analysis of phagocyte populations in the organs revealed that miR155KO mice contained significantly higher numbers of CD11b⁺ Ly6C^hi cells in their livers (Fig. 5A and B) throughout the course of infection than their WT counterparts. Similarly, the spleens of miR155KO mice also showed increased numbers of inflammatory monocytes at 60, 90, and 120 dpi (Fig. 5C and D) compared to those of the spleens of WT mice. It is known that PD-L1 expression by the myeloid cell population plays an important role in immune regulation in various infectious diseases as well as in several cancer models (24–26). Furthermore, CD11b⁺ Ly6C^hi cells isolated from the organs of miR155KO-infected mice expressed significantly higher PD-L1 mRNA transcript levels than their WT counterparts (Fig. 6A). Since PDL-1 has been implicated in suppressing T cell response in VL, in vitro T cell-monocyte coculture studies were performed to compare T cell-immunosuppressive activities of WT versus miR155 Ly6C^hi inflammatory monocytes. These studies showed that CD11b⁺ Ly6C^hi cells from miR155KO mice were significantly more effective in suppressing proliferation of naive T cells upon anti-CD3 activation than CD11b⁺ Ly6C^hi cells isolated from WT mice (Fig. 6B). These data indicate that miR155 deficiency is associated with increased recruitment of Ly6C^hi inflammatory monocytes, and these cells could contribute to high parasitic burdens in miR155KO mice by suppressing T cell responses (Fig. 1A and B).

FIG 5.

Accumulation of inflammatory monocytes correlates with the increased parasitic loads. WT- and miR155KO-infected mice were euthanized at the respective time intervals; accumulation of inflammatory monocytes in the livers and spleens was analyzed by flow cytometry. (A) Percentage of LY6C^hi cells out of the total CD11b gated cells (CD11b⁺ Ly6C^hi cells, i.e., inflammatory monocytes) in the livers of WT- and miR155KO-infected mice at 15, 40, 60, 90, and 120 dpi. (B) Bar graph of percentage of inflammatory monocytes in total liver cells at respective time intervals. (C) Percentage of LY6C^hi cells out of the total CD11b gated cells in the spleens of WT- and miR155KO-infected mice at 15, 40, 60, 90, and 120 dpi. (D) Bar graph of percentage of inflammatory monocytes in total splenocytes at respective time intervals. Data represented are from 1 of the 3 independent experiments, with n = 5 mice for each group/time interval. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6.

Inflammatory monocytes from miR155KO mice express PD-L1, and their blockade leads to reduced parasitic loads. (A) CD11b⁺ Ly6C^hi cells were sorted from spleens of WT- and miR155KO-infected mice at 60 dpi, and the levels of PD-L1 gene expression were analyzed by RT-PCR. (B) CD11b⁺ Ly6C^hi cells were sorted from the spleens of WT- and miR155KO-infected mice and then cocultured with CFSE-stained T cells isolated from the naive WT mice at a 1:1 ratio for 72 h. Proliferation of anti-CD3-stimulated T cells incubated with either sorted WT or miR155KO CD11b⁺ Ly6C^hi cells was measured by flow cytometry, and data are represented as the mean fluorescent intensity. (C and D) WT- and miR155KO-infected mice were treated with CCR2-specific antagonist once daily from 15 dpi to 40 dpi, and parasitic loads were calculated in the livers (C) and spleens (D) at 40 dpi. Data represented are from 1 of the 2 independent experiments. *, P < 0.05; **, P < 0.01.

Blockade of Ly6C^hi inflammatory monocyte recruitment to liver and spleen reduces organ parasite loads in miR155KO mice.

We next determined whether increased numbers of Ly6C^hi inflammatory monocytes in the organs of miR155KO mice contribute to higher parasite loads in miR155KO mice. L. donovani-infected miR155KO mice were treated with phosphate-buffered saline (control) or the chemokine receptor type 2 (CCR2) antagonist RS-504393 from 15 dpi to 40 dpi to reduce accumulation of Ly6C^hi inflammatory monocytes in the organs as described previously (23), and parasite loads in the organs were compared at 40 dpi. Our results showed that RS-504393 treatment effectively reduced the parasitic loads in both the livers and spleens of miR155KO mice (Fig. 6C and D). These observations suggest that miR155 deficiency leads to the accumulation of Ly6C^hi inflammatory monocytes, which are suppressive toward T cells, and that these cells contribute to increased susceptibility of miR155KO mice to L. donovani.

DISCUSSION

Immunological responses required for immunity against L. donovani infection are complex and involve the induction of IFN-γ- and IL-4-associated signaling pathways. Although miR155 has been shown to regulate the development and activity of Th1, Th17, and CD8⁺ T cells (11, 12), its specific role in immunity against experimental VL caused by L. donovani has yet to be characterized. Our results establish that miR155 contributes to host immunity against VL via modulation of IFN-γ-associated Th1 and IL-4-mediated Th2 immune responses (27–29).

Our findings revealed that lack of miR155 resulted in a significant increase in parasitic burdens in the livers and spleens of L. donovani-infected mice. Additionally, miR155KO mice exhibited increased hepatomegaly and splenomegaly and impaired granuloma formation compared to those of WT mice; these are the most prominent symptoms of VL. miR155KO-infected mice expressed lower numbers of both developing and mature granulomas than WT-infected mice at early stages of infection, which is indicative of immediate-early protection mediated by miR155. At later time intervals miR155KO-infected mice exhibited equal (90 dpi) and higher (120 dpi) numbers of granulomas than WT-infected mice, which explains the persistence of infection in miR155KO and resolution of infection in WT mice. This was associated with the inability of miR155KO mice to mount an efficient Th1 immune response, as shown by the reduced IFN-γ and TNF-α production by antigen-restimulated splenocytes. These results were not surprising, as miR155 is known to be involved in mechanisms that lead to IFN-γ production via the targeting of SOCS1 (16). Interestingly, we observed a previously uncharacterized role for miR155 in the generation of optimal Th2 immune responses against L. donovani infection. These results demonstrate the importance of miR155 in regulating both Th1 and Th2 immune responses during L. donovani infection.

Our analysis of parasite burdens and associated immune responses in the liver and spleen of L. donovani-infected miR155KO mice indicates that the mechanisms of miR155-mediated immunity against this parasite differ in these two organs. Coupled with the distinct immune cell profiles that exist within these organs, infection rates of L. donovani are known to be different in the livers and spleens of infected mice (7, 30), and these differences could account for the organ-specific immune responses observed at different time points. For example, in contrast with the spleen, livers of miR155KO mice showed higher gene expression of IFN-γ and TNF-α at 60, 90, and 120 dpi than WT mice. This suggests that the mechanisms behind IFN-γ production in the liver occur via miR155-independent mechanisms. Further analysis of miR155 targets involved in IFN-γ regulation showed an increased gene expression of SHIP-1 and SOCS1 in both the livers and spleens of L. donovani-infected miR155KO mice. SHIP-1 and SOCS1 are negative regulators of IFN-γ production (31, 32). Therefore, the increased levels of IFN-γ combined with higher SHIP-1 and SOCS1 levels in infected miR155KO livers suggest that the production of IFN-γ in the liver during L. donovani infection is mediated by miR155-independent mechanisms.

Consistent with the increased gene expression of IFN-γ in the liver of miR155KO mice, we observed increased expression of iNOS at chronic stages of the infection. It is well known that IFN-γ and nitric oxide play an important role in protective Th1 immunity during VL (23, 33, 34). Surprisingly, the spleens of miR155KO-infected mice also expressed higher iNOS levels at 90 and 120 dpi than WT mice despite the decrease in IFN-γ production. This could be due to the increased TNF-α expression in miR155KO mice, which can provide an additional mechanism of macrophage activation, resulting in nitric oxide production.

Recent studies from our group show that Ly6C^hi inflammatory monocytes migrate from bone marrow to the liver and spleen, via the CCR2-dependent pathway, and contribute to susceptibility during L. donovani infection (23, 35). Our analysis of the effects of miR155 on cellular immune responses showed an increased accumulation of Ly6C^hi inflammatory monocytes throughout the course of the infection in miR155KO mice. Interestingly, infected organs of miR155KO mice expressed higher levels of mRNA transcripts of CXCL10 and CCL2 (chemokine [C-C motif] ligand 2, a monocyte chemoattractant protein), and an increasing trend of CCR2 at chronic stages of VL infection was observed (see Fig. S1A to D in the supplemental material). These cytokines have been shown to play an important role in monocyte migration and recruitment to the site of inflammation in leishmaniasis (36, 37) and other diseases, such as HIV (38), rheumatoid arthritis, and atherosclerosis (39, 40). Not surprisingly, chemical inhibition of CCR2 resulted in a reduction of parasitic burdens in L. donovani-infected miR155KO mice. These results demonstrate that miR155 inhibits L. donovani infection partly via reduction of CCR2-mediated inflammatory monocyte infiltration.

In conclusion, we demonstrate the contribution of miR155 to host immunity against experimental VL. Our findings revealed that miR155 plays an essential role in the induction of both Th1 and Th2 protective immune responses, as well as the suppression of inflammatory monocyte infiltration and activity during L. donovani infection. It should be noted that miR155 was not essential to the resolution of L. donovani infection in this model. However, although miR155KO mice ultimately were able to control the infection, they took twice as long as their WT counterparts to clear the parasites. This substantial delay suggests that miR155 plays a crucial role in the development of protective immunity during the early stages of VL infection. miR155 can therefore be explored as a target for enhancing immunity against VL.

MATERIALS AND METHODS

Mouse strains.

miR155KO C57BL/6 and WT C57BL/6 mice were purchased from the Jackson Laboratory (miR155KO stock number 07745, BL/6 stock number 000664; Bar Harbor, ME, USA). All mice were maintained and bred at The Ohio State University animal facility by following approved animal protocols and University Laboratory Animal Resources (ULAR) regulations. All experiments were performed using age-matched 6- to 8-week-old female mice.

Parasites and infections.

Leishmania donovani parasites (LV82 strain) were obtained from the spleen of previously infected golden Syrian hamsters. The spleens of L. donovani-infected hamsters were harvested and mashed, and the amastigotes were counted. All of the experimental mice were intravenously injected with 10⁷ LV82 amastigotes via the tail vein.

Parasite burdens.

All experimental mice were euthanized at 15, 40, 60, 90, and 120 days postinfection (dpi). Spleens and livers were harvested and used to make smear impressions on glass slides. The slides were then fixed with methanol for 10 min, air dried, and stained with Giemsa stain for 30 min (purchased from Sigma-Aldrich, St. Louis, MO). The number of amastigotes per 1,000 nucleated cells was calculated using a microscope. The parasitic loads were represented by Leishman-Donavan units (LDU) using the following formula: LDU equals the number of L. donovani amastigotes per 1,000 nucleated cells times tissue weight (in grams).

Histopathology.

The spleens and livers of infected mice were sectioned at 5 μm and processed with the hematoxylin and eosin (H&E) staining procedure. The number and type of granulomas were determined by a certified pathologist using the following method: each sample was scored as (i) no cellular response, (ii) developing granuloma (initial influx of lymphocytes and monocytes, amastigotes present), (iii) mature granuloma (“functional” parasitic free granuloma), (iv) parasitic free granuloma (involuting epithelioid granuloma devoid of amastigotes), and (v) parasitic free tissue without granulomas. All scores were calculated using microscopy (10× and 40× lenses).

Cytokine ELISA and T cell proliferation.

Splenocytes were harvested and plated at a concentration of 5 × 10⁶ cells/ml in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% HEPES. Cells were stimulated for 72 h with 20 μg/ml L. donovani antigen (LdAg) prepared by freeze-thawing L. donovani promastigote cultures. Supernatants were collected, and production of IFN-γ, TNF-α, IL-4, IL-10, IL-12, and IL-13 cytokines was analyzed by sandwich enzyme-linked immunosorbent assay (ELISA). All the capture and detection antibodies were purchased from BioLegend (San Diego, CA, USA), and all the recombinant mouse cytokine standards were purchased from BD Biosciences (San Jose, CA, USA). Cytokine production was quantified by measuring the absorbance using a SpectraMax microplate reader and Softmax Pro software (Molecular Devices LLC, Sunnyvale, CA, USA). T cell proliferation of L. donovani antigen-stimulated splenocytes was measured at 72 h by using the alamarBlue reduction method. Ten percent alamarBlue (ThermoFisher Scientific, Waltham, MA) was added to the cells at 60 h, and absorbance was measured 10 h later at 570 and 600 nm using a SpectraMax microplate reader and Softmax Pro software (Molecular Devices LLC, Sunnyvale, CA, USA).

RT-PCR analysis.

Total RNA was extracted from spleens and livers of experimental mice by the TRIzol extraction method (Life Technologies, Carlsbad, CA). Complementary DNAs (cDNAs) were prepared with iScript reverse transcriptase, and reverse transcription-PCRs (RT-PCRs) were performed using IQ SYBR green super mix and a CFX 96 RT-PCR cycler (Bio-Rad, Hercules, CA, USA). Primers were selected from the Primer Bank website (http://pga.mgh.harvard.edu/primerbank). Data obtained were normalized by using the housekeeping gene β-actin and are presented as the fold induction over the level of uninfected WT mice.

In vitro coculture studies.

Spleens and livers were harvested from L. donovani-infected WT and miR155KO mice at 40 dpi. Single-cell suspensions were prepared and stained with CD11b, Ly6C, Ly6G, and F4/80 antibody cocktail. CD11b⁺ Ly6C^hi cells were sorted from both spleens and livers by using a FACS Aria (BD FACS Aria high-speed cell sorter). T cells were isolated from naive WT mice by the nylon wool column method. T cells were stimulated with anti-CD3 antibodies for 2 h and then stained with carboxyfluorescein succinimidyl ester (CFSE). Sorted CD11b⁺ Ly6C^hi cells were cocultured with CFSE-stained T cells at a 1:1 ratio for 72 h, and T cell proliferation was analyzed by flow cytometry.

Flow cytometry.

Single-cell suspensions were prepared from the spleens and livers of experimental mice at all respective time intervals. Briefly, spleens and livers were mashed, red blood cells were lysed with ACK lysis buffer, and single-cell suspensions were prepared. Normal mouse serum was used to block FC receptors, and cells were stained with the following antibodies: CD3, CD4, CD8, NK1.1, CD11b, F480, Ly6G, and Ly6C (BioLegend, San Diego, CA). Cells were acquired through a fluorescence-activated cell sorter (BD Biosciences, San Jose, CA, USA), and analysis was performed with FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Statistical analysis.

Student’s unpaired t test was used to determine statistical significance of differences among the groups. A P value of <0.05 was considered significant.