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MicroRNA155 Plays a Critical Role in the Pathogenesis of Cutaneous Leishmania major Infection by Promoting a Th2 Response and Attenuating Dendritic Cell Activity

2021; Elsevier BV; Volume: 191; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2021.01.012

ISSN

1525-2191

Autores

Sanjay Varikuti, Chaitenya Verma, Gayathri Natarajan, Steve Oghumu, Abhay R. Satoskar,

Tópico(s)

Urticaria and Related Conditions

Resumo

Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4+ Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155−/−) mice. Infection was controlled significantly quicker in the miR155−/− mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155−/− mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ+CD8+ T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major−infected miR155−/− mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major−infected miR155−/− DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155−/− T cells was significantly enhanced in L. major−infected miR155−/− DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function. Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4+ Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155−/−) mice. Infection was controlled significantly quicker in the miR155−/− mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155−/− mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ+CD8+ T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major−infected miR155−/− mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major−infected miR155−/− DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155−/− T cells was significantly enhanced in L. major−infected miR155−/− DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function. Leishmania are obligate intracellular protozoans that infect phagocytes and cause a spectrum of clinical diseases such as cutaneous leishmaniasis (CL) and visceral leishmaniasis. Common in the tropical and subtropical regions, leishmaniasis affects over 1 billion people worldwide, with an incidence of up to 1 million cases per year.1Working to overcome the global impact of neglected tropical diseases - summary.Wkly Epidemiol Rec. 2011; 86: 113-120PubMed Google Scholar CL is the most common type of Leishmania infection, manifesting as localized skin lesions that can become chronic, leading to significant tissue destruction and disfigurement.2Scorza B.M. Carvalho E.M. Wilson M.E. Cutaneous manifestations of human and murine leishmaniasis.Int J Mol Sci. 2017; 18: 1296Crossref PubMed Scopus (86) Google Scholar,3Pearson R.D. Sousa A.Q. Clinical spectrum of leishmaniasis.Clin Infect Dis. 1996; 22: 1-13Crossref PubMed Scopus (403) Google Scholar It is well documented that the induction of a Th1 response and interferon (IFN)-γ are indispensable in the resolution of CL caused by Leishmania major,4Oghumu S. Stock J.C. Varikuti S. Dong R. Terrazas C. Edwards J.A. Rappleye C.A. Holovatyk A. Sharpe A. Satoskar A.R. Transgenic expression of CXCR3 on T cells enhances susceptibility to cutaneous Leishmania major infection by inhibiting monocyte maturation and promoting a Th2 response.Infect Immun. 2015; 83: 67-76Crossref PubMed Scopus (6) Google Scholar whereas disease progression is associated with the induction of a Th2 response and the production of cytokines such as IL-4 and IL-10.5Oghumu S. Lezama-Davila C.M. Isaac-Marquez A.P. Satoskar A.R. Role of chemokines in regulation of immunity against leishmaniasis.Exp Parasitol. 2010; 126: 389-396Crossref PubMed Scopus (68) Google Scholar Establishing a disease-resolving response in the host is largely dependent on the ability to mount an appropriate Th1 immune response.4Oghumu S. Stock J.C. Varikuti S. Dong R. Terrazas C. Edwards J.A. Rappleye C.A. Holovatyk A. Sharpe A. Satoskar A.R. Transgenic expression of CXCR3 on T cells enhances susceptibility to cutaneous Leishmania major infection by inhibiting monocyte maturation and promoting a Th2 response.Infect Immun. 2015; 83: 67-76Crossref PubMed Scopus (6) Google Scholar Crucial in this response is the stimulation and activation of DCs that direct T-cell proliferation and differentiation toward IFN-γ–producing Th1 cells.6Kawakami Y. Inagaki N. Salek-Ardakani S. Kitaura J. Tanaka H. Nagao K. Kawakami Y. Xiao W. Nagai H. Croft M. Kawakami T. Regulation of dendritic cell maturation and function by Bruton's tyrosine kinase via IL-10 and Stat3.Proc Natl Acad Sci U S A. 2006; 103: 153-158Crossref PubMed Scopus (78) Google Scholar,7Pulendran B. Smith J.L. Caspary G. Brasel K. Pettit D. Maraskovsky E. Maliszewski C.R. Distinct dendritic cell subsets differentially regulate the class of immune response in vivo.Proc Natl Acad Sci U S A. 1999; 96: 1036-1041Crossref PubMed Scopus (871) Google Scholar In addition to activating of phagocytic cells, IFN-γ induces the production of reactive nitrogen species, specifically nitric oxide (NO), leading to enhanced parasite clearance.4Oghumu S. Stock J.C. Varikuti S. Dong R. Terrazas C. Edwards J.A. Rappleye C.A. Holovatyk A. Sharpe A. Satoskar A.R. Transgenic expression of CXCR3 on T cells enhances susceptibility to cutaneous Leishmania major infection by inhibiting monocyte maturation and promoting a Th2 response.Infect Immun. 2015; 83: 67-76Crossref PubMed Scopus (6) Google Scholar miR155 is a recognized regulator of immune cell function and immune response. miR155 enhances macrophage and DC activation and induces inflammatory response,8Du F. Yu F. Wang Y. Hui Y. Carnevale K. Fu M. Lu H. Fan D. MicroRNA-155 deficiency results in decreased macrophage inflammation and attenuated atherogenesis in apolipoprotein E-deficient mice.Arterioscler Thromb Vasc Biol. 2014; 34: 759-767Crossref PubMed Scopus (147) Google Scholar,9Qin Y. Wang Q. Zhou Y. Duan Y. Gao Q. Inhibition of IFN-gamma-induced nitric oxide dependent antimycobacterial activity by miR-155 and C/EBPbeta.Int J Mol Sci. 2016; 17: 535Crossref PubMed Scopus (21) Google Scholar and up-regulation of miR155 in CD4+ T cells promotes preferential Th1 differentiation and IFN-γ production10Chen Y. Li L. Lu Y. Su Q. Sun Y. Liu Y. Yang D. Upregulation of miR-155 in CD4(+) T cells promoted Th1 bias in patients with unstable angina.J Cell Physiol. 2015; 230: 2498-2509Crossref PubMed Scopus (13) Google Scholar by suppressing the expression of suppressor of cytokine signaling (SOCS)-1.11Trotta R. Chen L. Ciarlariello D. Josyula S. Mao C. Costinean S. Yu L. Butchar J.P. Tridandapani S. Croce C.M. Caligiuri M.A. miR-155 regulates IFN-gamma production in natural killer cells.Blood. 2012; 119: 3478-3485Crossref PubMed Scopus (157) Google Scholar, 12Cheng Y.Q. Ren J.P. Zhao J. Wang J.M. Zhou Y. Li G.Y. Moorman J.P. Yao Z.Q. MicroRNA-155 regulates interferon-gamma production in natural killer cells via Tim-3 signalling in chronic hepatitis C virus infection.Immunology. 2015; 145: 485-497Crossref PubMed Scopus (55) Google Scholar, 13Rao R. Rieder S.A. Nagarkatti P. Nagarkatti M. Staphylococcal enterotoxin B-induced microRNA-155 targets SOCS1 to promote acute inflammatory lung injury.Infect Immun. 2014; 82: 2971-2979Crossref PubMed Scopus (63) Google Scholar, 14Hu J.Y. Zhang J. Ma J.Z. Liang X.Y. Chen G.Y. Lu R. Du G.F. Zhou G. MicroRNA-155-IFN-gamma feedback loop in CD4(+)T cells of erosive type oral lichen planus.Sci Rep. 2015; 5: 16935Crossref PubMed Scopus (30) Google Scholar Conversely, miR155 gene–deficient mice exhibit diminished levels of Th1/Th17 cells, macrophages, and DCs.15Singh U.P. Murphy A.E. Enos R.T. Shamran H.A. Singh N.P. Guan H. Hegde V.L. Fan D. Price R.L. Taub D.D. Mishra M.K. Nagarkatti M. Nagarkatti P.S. miR-155 deficiency protects mice from experimental colitis by reducing T helper type 1/type 17 responses.Immunology. 2014; 143: 478-489Crossref PubMed Scopus (85) Google Scholar miR155 has also been shown to play a role in regulating effector Th2 response.16Malmhall C. Alawieh S. Lu Y. Sjostrand M. Bossios A. Eldh M. Radinger M. MicroRNA-155 is essential for T(H)2-mediated allergen-induced eosinophilic inflammation in the lung.J Allergy Clin Immunol. 2014; 133: 1429-1438e1-7Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 17Okoye I.S. Czieso S. Ktistaki E. Roderick K. Coomes S.M. Pelly V.S. Kannan Y. Perez-Lloret J. Zhao J.L. Baltimore D. Langhorne J. Wilson M.S. Transcriptomics identified a critical role for Th2 cell-intrinsic miR-155 in mediating allergy and antihelminth immunity.Proc Natl Acad Sci U S A. 2014; 111: E3081-E3090Crossref PubMed Scopus (89) Google Scholar, 18Knolle M.D. Chin S.B. Rana B.M.J. Englezakis A. Nakagawa R. Fallon P.G. Git A. McKenzie A.N.J. MicroRNA-155 protects group 2 innate lymphoid cells from apoptosis to promote type-2 immunity.Front Immunol. 2018; 9: 2232Crossref PubMed Scopus (12) Google Scholar Collectively, these findings suggest that miR155 regulates both Th1 and Th2 responses, which control the outcome of CL caused by L. major. Therefore, the role of miR155 in immunity to L. major using miR155−/− mice was investigated in the present study. The findings show that miR155 is not required for the induction of a Th1 response and IFN-γ in L. major infection. Rather, miR155 plays a disease-exacerbating role in CL by attenuating DC function and Th1 response and promoting Th2 response. Female C57BL/6 wild-type (WT) mice (catalog number 000664) and miR155−/− mice (catalog number 07745) aged 6 to 8 weeks were purchased from The Jackson Laboratory (Bar Harbor, ME). L. major LV39 parasites were obtained from the lesions of previously infected animal stock, and cultured in M199 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 26°C to obtain stationary-phase metacyclic promastigotes. All experimental mice were infected by inoculation of 2 × 106 stationary-phase metacyclic promastigotes into the footpad. Disease progression was monitored by measurement of the footpad lesion growth and assessment of parasite loads as described previously.4Oghumu S. Stock J.C. Varikuti S. Dong R. Terrazas C. Edwards J.A. Rappleye C.A. Holovatyk A. Sharpe A. Satoskar A.R. Transgenic expression of CXCR3 on T cells enhances susceptibility to cutaneous Leishmania major infection by inhibiting monocyte maturation and promoting a Th2 response.Infect Immun. 2015; 83: 67-76Crossref PubMed Scopus (6) Google Scholar Footpad lesion progression was monitored by using a dial-gauge micrometer (Mitutoyo, Tokyo, Japan). All experimental protocols were approved by the Institutional Animal Care and Use Committee, The Ohio State University (Columbus, OH). The draining lymph nodes were harvested from infected WT and miR155−/− mice at weeks 4, 8, and 14 post-infection (PI), and single-cell suspensions were prepared. Cells were plated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin, and stimulated with 20μg/mL of freeze–thaw L. major antigen for 72 hours. T-cell proliferation and cytokine production were quantified by Alamar Blue (purchased from Thermo Fisher Scientific, Waltham, MA) and cytokine enzyme-linked immunosorbent assay methods as described previously.19Varikuti S. Oghumu S. Saljoughian N. Pioso M.S. Sedmak B.E. Khamesipour A. Satoskar A.R. Topical treatment with nanoliposomal amphotericin B reduces early lesion growth but fails to induce cure in an experimental model of cutaneous leishmaniasis caused by Leishmania mexicana.Acta Trop. 2017; 173: 102-108Crossref PubMed Scopus (13) Google Scholar Cells were isolated from the draining lymph nodes of infected WT and miR155−/− mice, and single-cell suspensions were prepared and stained with respective stain cocktails. Anti-mouse CD3, CD4, CD8, natural killer (NK) 1.1, CD11c, CD11b, major histocompatibility complex (MHC)-II, and Ly6C (purchased from BioLegend, San Diego, CA) were used for analyzing the T-cell and DC populations. For intracellular staining, lymph node cells were stimulated with a cell-activation cocktail (purchased from BioLegend) for 4 hours, stained with cell-surface antibodies anti-mouse CD3, CD4, CD8, and NK1.1, and permeabilized and stained with anti-mouse IFN-γ, tumor necrosis factor (TNF)-α, IL-10, and IL-4 antibodies purchased from BioLegend. Samples were acquired by BD LSRFortessa (BD Biosciences, San Jose, CA) and data were analyzed by FlowJo software version 10 (Tree Star, Inc., Ashland, OR). Bone marrow–derived DCs (BMDCs) from WT and miR155−/− mice were prepared as described previously.20Natarajan G. Oghumu S. Terrazas C. Varikuti S. Byrd J.C. Satoskar A.R. A Tec kinase BTK inhibitor ibrutinib promotes maturation and activation of dendritic cells.Oncoimmunology. 2016; 5: e1151592Crossref PubMed Scopus (13) Google Scholar BMDCs were infected with L. major promastigote parasites at 1:5 (cell/parasite) ratio and stimulated with 1 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO) or 2 ng/mL of recombinant IFN-γ (BD Biosciences). After 24 hours, culture supernatants were collected and analyzed for the production of cytokines IL-12 and IL-10 by enzyme-linked immunosorbent assay, and NO by Greiss assay, as previously described.21Natarajan G. Terrazas C. Oghumu S. Varikuti S. Dubovsky J.A. Byrd J.C. Satoskar A.R. Ibrutinib enhances IL-17 response by modulating the function of bone marrow derived dendritic cells.Oncoimmunology. 2016; 5: e1057385Crossref PubMed Scopus (24) Google Scholar Briefly, 96-well plates were coated with respective capture antibodies (2 μg/mL; purchased from BioLegend) overnight at 4°C, and plates were washed with 0.05% Tween 20 in phosphate-buffered saline and blocked with 10% fetal bovine serum in phosphate-buffered saline for 2 hours. Plates were washed and incubated with respective recombinant mouse cytokine standards (BD Biosciences) or culture supernatants overnight at 4°C. Respective biotinylated antibodies (1 μg/mL; purchased from BioLegend) were added for 1 hour at room temperature, and plates were washed and added with streptavidin–alkaline phosphatase (BioLegend) for 30 minutes at room temperature. Plates were washed, and 1 mg/mL p-nitrophenyl phosphate (purchased from Thermo Fisher) in glycine buffer was added to each well, and absorbance was recorded at 405 nm. Cytokine concentrations were calculated by Softmax Pro software version 7.1 (Molecular Devices LLC, San Jose, CA). Briefly, T cells were enriched from the splenocytes of WT and miR155−/− mice by nylon wool column and then stimulated with plate-bound anti-CD3 for 2 hours in vitro as described previously.21Natarajan G. Terrazas C. Oghumu S. Varikuti S. Dubovsky J.A. Byrd J.C. Satoskar A.R. Ibrutinib enhances IL-17 response by modulating the function of bone marrow derived dendritic cells.Oncoimmunology. 2016; 5: e1057385Crossref PubMed Scopus (24) Google Scholar Activated T cells were washed, stained with 5 μmol/L carboxyfluorescein succinimidyl ester (Thermo Fisher), and co-cultured with L. major–infected and 1 μg/mL LPS (Sigma-Aldrich) stimulated BMDCs (1:4 ratio) isolated from WT and miR155−/− mice. After 72 hours, T-cell proliferation was measured by flow cytometry and represented as the proliferative index (calculates the total number of divisions by the number of cells that have undergone division) by using FlowJo software version 10 as described previously.22De Biasi S. Meschiari M. Gibellini L. Bellinazzi C. Borella R. Fidanza L. Gozzi L. Iannone A. Lo Tartaro D. Mattioli M. Paolini A. Menozzi M. Milic J. Franceschi G. Fantini R. Tonelli R. Sita M. Sarti M. Trenti T. Brugioni L. Cicchetti L. Facchinetti F. Pietrangelo A. Clini E. Girardis M. Guaraldi G. Mussini C. Cossarizza A. Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with COVID-19 pneumonia.Nat Commun. 2020; 11: 3434Crossref PubMed Scopus (313) Google Scholar,23Boasso A. Hardy A.W. Anderson S.A. Dolan M.J. Shearer G.M. HIV-induced type I interferon and tryptophan catabolism drive T cell dysfunction despite phenotypic activation.PLoS One. 2008; 3: e2961Crossref PubMed Scopus (96) Google Scholar Cytokine secretion from the culture supernatants was determined by cytokine enzyme-linked immunosorbent assay as described previously.21Natarajan G. Terrazas C. Oghumu S. Varikuti S. Dubovsky J.A. Byrd J.C. Satoskar A.R. Ibrutinib enhances IL-17 response by modulating the function of bone marrow derived dendritic cells.Oncoimmunology. 2016; 5: e1057385Crossref PubMed Scopus (24) Google Scholar Statistical analysis was conducted using GraphPad software version 8.4.3 (GraphPad Software, San Diego, CA). Statistical significance was determined by the unpaired t-test for independent univariate data set and one-way analysis of variance with Bonferroni correction for multiple comparisons. P values of <0.05 were considered statistically significant. After footpad infection with 2 × 106 L. major (LV39 strain), stationary-phase promastigotes lesion growth was comparable for the first 5 weeks in both WT and miR155−/− C57BL/6 mice (Figure 1A). However, as the infection progressed, lesions resolved significantly faster in the L. major–infected miR155−/− mice compared to those in the WT controls. At week 6 post-infection (PI) and thereafter, the lesion sizes were significantly smaller in the L. major-infected miR155−/− mice compared to those in their WT counterparts (Figure 1A). No differences were noted in the footpad parasite loads between the WT and miR155−/− mice at week 4 PI; however, at weeks 8 and 14 PI, lesions contained significantly fewer parasites in the miR155−/− mice compared to those in the WT mice (Figure 1B). At 4, 8, and 14 weeks PI, L. major–infected miR155−/− and WT mice were euthanized and in vitro T-cell proliferation and cytokine production by L. major antigen–stimulated draining lymph node cells were analyzed as described previously.19Varikuti S. Oghumu S. Saljoughian N. Pioso M.S. Sedmak B.E. Khamesipour A. Satoskar A.R. Topical treatment with nanoliposomal amphotericin B reduces early lesion growth but fails to induce cure in an experimental model of cutaneous leishmaniasis caused by Leishmania mexicana.Acta Trop. 2017; 173: 102-108Crossref PubMed Scopus (13) Google Scholar At weeks 8 and 14, L. major antigen–stimulated lymph node cells from miR155−/− mice displayed higher proliferation compared to those from their WT counterparts (Figure 2A). At these time points, lymph node culture supernatants from the miR155−/− mice contained significantly more IL-12 (Figure 2B) and IFN-γ (Figure 2C) but significantly less Th2-associated IL-4 (Figure 2D), IL-10 (Figure 2E), and IL-13 (Figure 2F) compared to those from the WT mice. No significant differences were noted between the groups in IL-17A levels at weeks 4 and 8 of PI, but miR155−/− mice expressed significantly less IL-17A at week 14 PI (data not shown). Given that L. major–infected miR155−/− mice showed higher levels of IFN-γ in their lymph node culture supernatants, CD4+ and CD8+ T cells and NK cells in the draining lymph nodes of L. major–infected miR155−/− and WT mice were analyzed by flow cytometry at 4, 8, and 14 weeks PI. At all time points, the draining lymph nodes of miR155−/− mice contained fewer CD4+ T cells (Figure 3A) but significantly higher concentrations of CD8+ T cells (Figure 3B) and NK cells (Figure 3C) compared to WT mice. Intracellular flow-cytometry analysis revealed that the draining lymph nodes of L. major–infected miR155−/− mice contained significantly higher concentrations IFN-γ+CD8+ T cells and NK cells and lower concentrations of IL-10+CD8+ T cells and NK cells compared to WT mice (Figures 3, D–H). Analysis of DC populations in the lymph nodes revealed that although there were no noticeable differences in the concentrations of cDC1 (CD8+ cDC) and cDC2 (CD11b+ cDC) cells, total DCs (CD11c+MHCII+) from infected miR155−/− mice expressed significantly higher levels of MHCII and CD80 compared to WT DCs (Figure 3, I and J). To analyze the functional responses of miR155−/− DCs with L. major infection, L. major–infected bone marrow–derived DCs from miR155−/− and WT mice were stimulated with 1 μg/mL LPS or 2 ng/mL recombinant IFN-γ, and the production of pro- and anti-inflammatory cytokines and NO was analyzed. The percentage of miR155−/− BMDCs infected with L. major was significantly lower compared to that in WT BMDCs (Figure 4A). After stimulation with LPS or recombinant IFN-γ, L. major–infected BMDCs from miR155−/− mice produced significantly higher levels of IL-12 (Figure 4B) and NO (Figure 4C) compared to L. major–infected BMDCs from WT mice. Conversely, the level of IL-10 in culture supernatant from miR155−/− BMDCs was significantly lower compared to that in the WT BMDCs (Figure 4D). Next, the effect of L. major–infected miR155−/− DCs on proliferation and cytokine production by anti-CD3–activated T cells was determined using DC–T cell co-culture assays. No significant differences were noted in the proliferation of anti-CD3–activated miR155−/− T cells co-cultured with L. major–infected miR155−/− BMDCs and anti-CD3–activated WT T cells co-cultured with L. major–infected WT BMDCs (Figure 4E). Similarly, no significant differences were noted in the proliferation response between WT T cells cultured with L. major–infected miR155−/− BMDCs and miR155−/− T cells cultured with L. major–infected WT BMDCs. Interestingly, miR155−/− T cells co-cultured with either miR155−/− BMDCs or WT BMDCs produced significantly more IFN-γ compared to WT T cells co-cultured under the same conditions (Figure 3F). The novel findings in this study were that miR155−/− mice displayed impairment of a Th2 response, mounted an enhanced Th1-like response, and resolved their infection significantly faster than did the WT controls. Furthermore, LPS-stimulated L. major–infected miR155-deficient DCs produced significantly more IL-12 and NO compared to their WT counterparts and significantly enhanced IFN-γ production from activated T cells. These results indicate that miR155 plays a crucial role in mediating susceptibility to CL caused by L. major by promoting Th2 response, suppressing protective Th1 response, and modulating DC function. The results from the present study differ from previous findings that miR155 contributed to host immunity in visceral leishmaniasis caused by L. donovani but was not required for resolution of infection.24Varikuti S. Natarajan G. Volpedo G. Singh B. Hamza O. Messick G.V. Guerau-de-Arellano M. Papenfuss T.L. Oghumu S. Satoskar A.R. MicroRNA 155 contributes to host immunity against Leishmania donovani but is not essential for resolution of infection.Infect Immun. 2019; 87: e00307Crossref PubMed Scopus (7) Google Scholar This was not surprising as it is becoming increasingly evident that different immune mechanisms control the outcome of L. major versus L. donovani infection.25Cummings H.E. Tuladhar R. Satoskar A.R. Cytokines and their STATs in cutaneous and visceral leishmaniasis.J Biomed Biotechnol. 2010; 2010: 294389Crossref PubMed Scopus (41) Google Scholar,26Kedzierski L. Evans K.J. Immune responses during cutaneous and visceral leishmaniasis.Parasitology. 2014; 141: 1-19Crossref PubMed Scopus (31) Google Scholar For example, Th2-associated IL-4 and IL-13 are factors of susceptibility to CL, but they are required for the induction of a protective immune response during visceral leishmaniasis.27Stager S. Alexander J. Carter K.C. Brombacher F. Kaye P.M. Both interleukin-4 (IL-4) and IL-4 receptor alpha signaling contribute to the development of hepatic granulomas with optimal antileishmanial activity.Infect Immun. 2003; 71: 4804-4807Crossref PubMed Scopus (112) Google Scholar,28McFarlane E. Carter K.C. McKenzie A.N. Kaye P.M. Brombacher F. Alexander J. Endogenous IL-13 plays a crucial role in liver granuloma maturation during Leishmania donovani infection, independent of IL-4Ralpha-responsive macrophages and neutrophils.J Infect Dis. 2011; 204: 36-43Crossref PubMed Scopus (33) Google Scholar Similarly, it has been shown that IL-17 contributes to immunity to L. major29Lopez Kostka S. Dinges S. Griewank K. Iwakura Y. Udey M.C. von Stebut E. IL-17 promotes progression of cutaneous leishmaniasis in susceptible mice.J Immunol. 2009; 182: 3039-3046Crossref PubMed Scopus (160) Google Scholar,30Gonzalez-Lombana C. Gimblet C. Bacellar O. Oliveira W.W. Passos S. Carvalho L.P. Goldschmidt M. Carvalho E.M. Scott P. IL-17 mediates immunopathology in the absence of IL-10 following Leishmania major infection.PLoS Pathog. 2013; 9: e1003243Crossref PubMed Scopus (107) Google Scholar but that it mediates susceptibility to L. donovani.31Terrazas C. Varikuti S. Kimble J. Moretti E. Boyaka P.N. Satoskar A.R. IL-17A promotes susceptibility during experimental visceral leishmaniasis caused by Leishmania donovani.FASEB J. 2016; 30: 1135-1143Crossref PubMed Scopus (39) Google Scholar Taken together, these findings also suggest that miR155 plays a distinct role in regulating immunity against cutaneous L. major versus visceral L. donovani infection. Previous studies have reported that miR155 promotes Th1 response and IFN-γ production,32O'Connell R.M. Kahn D. Gibson W.S. Round J.L. Scholz R.L. Chaudhuri A.A. Kahn M.E. Rao D.S. Baltimore D. MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development.Immunity. 2010; 33: 607-619Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar,33Goncalves-Alves E. Saferding V. Schliehe C. Benson R. Kurowska-Stolarska M. Brunner J.S. Puchner A. Podesser B.K. Smolen J.S. Redlich K. Bonelli M. Brewer J. Bergthaler A. Steiner G. Bluml S. MicroRNA-155 controls T helper cell activation during viral infection.Front Immunol. 2019; 10: 1367Crossref PubMed Scopus (13) Google Scholar which are indispensable in the control of cutaneous L. major infection. On the other hand, others have shown that inhibitors of miR155 reduces IL-5 and IL-13 expression in Th2 cells16Malmhall C. Alawieh S. Lu Y. Sjostrand M. Bossios A. Eldh M. Radinger M. MicroRNA-155 is essential for T(H)2-mediated allergen-induced eosinophilic inflammation in the lung.J Allergy Clin Immunol. 2014; 133: 1429-1438e1-7Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar,17Okoye I.S. Czieso S. Ktistaki E. Roderick K. Coomes S.M. Pelly V.S. Kannan Y. Perez-Lloret J. Zhao J.L. Baltimore D. Langhorne J. Wilson M.S. Transcriptomics identified a critical role for Th2 cell-intrinsic miR-155 in mediating allergy and antihelminth immunity.Proc Natl Acad Sci U S A. 2014; 111: E3081-E3090Crossref PubMed Scopus (89) Google Scholar,34Lin R.T. Liu J.Q. Lu H.Y. Chen Y.M. Guan L. Liu Z.G. Liu Z.J. Yang P.C. Micro RNA-155 plays a critical role in the initiation of food allergen-related inflammation in the intestine.Oncotarget. 2017; 8: 67497-67505Crossref PubMed Scopus (3) Google Scholar and that miR155 protects group 2 innate lymphoid cells to promote a Th2 response.18Knolle M.D. Chin S.B. Rana B.M.J. Englezakis A. Nakagawa R. Fallon P.G. Git A. McKenzie A.N.J. MicroRNA-155 protects group 2 innate lymphoid cells from apoptosis to promote type-2 immunity.Front Immunol. 2018; 9: 2232Crossref PubMed Scopus (12) Google Scholar In the present study, antigen-stimulated lymph node cells from miR155−/− mice produced significantly more IL-12 and IFN-γ but less Th2-associated IL-4, IL-10, and IL-13 compared to cells from WT mice during resolution of the infection. miR155 is also a driver of a Th17 response,35Escobar T.M. Kanellopoulou C. Kugler D.G. Kilaru G. Nguyen C.K. Nagarajan V. Bhairavabhotla R.K. Northrup D. Zahr R. Burr P. Liu X. Zhao K. Sher A. Jankovic D. Zhu J. Muljo S.A. miR-155 activates cytokine gene expression in Th17 cells by regulating the DNA-binding protein Jarid2 to relieve polycomb-mediated repression.Immunity. 2014; 40: 865-879Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar which is associated with immune pathogenesis and disease progression in L. major infection. In one study, IL-17−/− mice developed smaller lesions after L. major infection associated with reduced recruitment of neutrophils into the lesions.29Lopez Kostka S. Dinges S. Griewank K. Iwakura Y. Udey M.C. von Stebut E. IL-17 promotes progression of cutaneous leishmaniasis in susceptible mice.J Immunol. 2009; 182: 3039-3046Crossref PubMed Scopus (160) Google Scholar Another study reported that IL-17A contributed to immunopathology associated with the lesion in CL.30Gonzalez-Lombana C. Gimblet C. Bacellar O. Oliveira W.W. Passos S. Carvalho L.P. Goldschmidt M. Carvalho E.M. Scott P. IL-17 mediates immunopathology in the absence of IL-10 following Leishmania major infection.PLoS Pathog. 2013; 9: e1003243Crossref PubMed Scopus (107) Google Scholar In agreement with this, the levels of IL-17A in the current study were also significantly lower in the lymph node culture supernatants from miR155−/− mice compared to those in WT controls at week 14 PI. Taken together, these findings suggest that miR155 promotes susceptibility to cutaneous L. major infection by promoting Th2 response and that increased production of IL-12 and IFN-γ in miR155−/− mice could be due to the attenuation of Th2 response. NK cells and CD8+ T cells also contribute to host resistance against cutaneous L. major infection. miR155 is a positive regulator of IFN-γ production in NK cells.11Trotta R. Chen L. Ciarlariello D. Josyula S. Mao C. Costinean S. Yu L. Butchar J.P. Tridandapani S. Croce C.M. Caligiuri M.A. miR-155 regulates IFN-gamma production in natural killer cells.Blood. 2012; 119: 3478-3485Crossref PubMed Scopus (157) Google Scholar On the other hand, Sullivan et al36Sullivan R.P. Fogel L.A. Leong J.W. Schneider S.E. Wong R. Romee R. Thai T.H. Sexl V. Matkovich S.J. Dorn 2nd, G.W. French A.R. Fehniger T.A. MicroRNA-155 tunes both the threshold and extent of NK cell activation via targeting of multiple signaling pathways.J Immunol. 2013; 191: 5904-5913Crossref PubMed Scopus (46) Google Scholar found that miR155−/− NK cells exhibited increased IFN-γ production during cytomegalovirus infection, suggesting that miR155 deficiency during NK cell development results in lowering of activation threshold for NK cells, resulting in more NK cells expressing IFN-γ during infection. In the present study, the lymph nodes of L. major–infected miR155−/− mice contained significantly higher concentrations of IFN-γ–producing NK cells but fewer IL-10+ NK cells compared to the lymph nodes of WT mice. Several studies have documented that miR155 is required for effector CD8+ T-cell response against viruses and cancers.37Dudda J.C. Salaun B. Ji Y. Palmer D.C. Monnot G.C. Merck E. Boudousquie C. Utzschneider D.T. Escobar T.M. Perret R. Muljo S.A. Hebeisen M. Rufer N. Zehn D. Donda A. Restifo N.P. Held W. Gattinoni L. Romero P. MicroRNA-155 is required for effector CD8+ T cell responses to virus infection and cancer.Immunity. 2013; 38: 742-753Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 38Ji Y. Fioravanti J. Zhu W. Wang H. Wu T. Hu J. Lacey N.E. Gautam S. Le Gall J.B. Yang X. Hocker J.D. Escobar T.M. He S. Dell'Orso S. Hawk N.V. Kapoor V. Telford W.G. Di Croce L. Muljo S.A. Zhang Y. Sartorelli V. Gattinoni L. miR-155 harnesses Phf19 to potentiate cancer immunotherapy through epigenetic reprogramming of CD8(+) T cell fate.Nat Commun. 2019; 10: 2157Crossref PubMed Scopus (33) Google Scholar, 39Lind E.F. Ohashi P.S. Mir-155, a central modulator of T-cell responses.Eur J Immunol. 2014; 44: 11-15Crossref PubMed Scopus (52) Google Scholar Therefore, the finding that the lymph nodes of L. major–infected miR155−/− mice contain higher concentrations of IFN-γ–producing CD8+ T cells compared to the lymph nodes of their WT counterparts is unexpected. One possible explanation of this finding is that miR155-deficient CD8+ T cells also have a lower activation threshold similar to miR155−/− NK cells, resulting in higher concentrations of CD8+ T cells producing IFN-γ. Collectively, these results show that miR155 deficiency leads to an augmentation of IFN-γ in NK and CD8+ T cells that may at least in part contribute to faster resolution of L. major infection in miR155−/− mice. It is well documented that miR155 regulates maturation and migration of DCs and activation of T cells by DCs during cancer and viral infections.37Dudda J.C. Salaun B. Ji Y. Palmer D.C. Monnot G.C. Merck E. Boudousquie C. Utzschneider D.T. Escobar T.M. Perret R. Muljo S.A. Hebeisen M. Rufer N. Zehn D. Donda A. Restifo N.P. Held W. Gattinoni L. Romero P. MicroRNA-155 is required for effector CD8+ T cell responses to virus infection and cancer.Immunity. 2013; 38: 742-753Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar,40Hodge J. Wang F. Wang J. Liu Q. Saaoud F. Wang Y. Singh U.P. Chen H. Luo M. Ai W. Fan D. Overexpression of microRNA-155 enhances the efficacy of dendritic cell vaccine against breast cancer.Oncoimmunology. 2020; 9: 1724761Crossref PubMed Scopus (7) Google Scholar Published studies have shown that the expression of co-stimulatory molecules and cytokines IL-1, IL-6, IL-12, and TNF-α is impaired in miR155-deficient DCs.41Wang J. Iwanowycz S. Yu F. Jia X. Leng S. Wang Y. Li W. Huang S. Ai W. Fan D. microRNA-155 deficiency impairs dendritic cell function in breast cancer.Oncoimmunology. 2016; 5: e1232223Crossref PubMed Scopus (27) Google Scholar,42Lind E.F. Millar D.G. Dissanayake D. Savage J.C. Grimshaw N.K. Kerr W.G. Ohashi P.S. miR-155 upregulation in dendritic cells is sufficient to break tolerance in vivo by negatively regulating SHIP1.J Immunol. 2015; 195: 4632-4640Crossref PubMed Scopus (38) Google Scholar In contrast, in the present study, the total DCs (CD11c+MHCII+) from infected miR155−/− mice expressed significantly higher levels of MHCII and CD80 compared to WT DCs, and no noticeable differences were noted in cDC1 (CD8+ cDC) and cDC2 (CD11b+ cDC) populations between the groups. Furthermore, L. major–infected BMDCs from miR155−/− mice displayed lower infection rates and produced significantly more IL-12 and NO after stimulation with LPS or recombinant IFN-γ compared to WT DC controls. Conversely, levels of IL-10 in culture supernatants from miR155−/− BMDCs were significantly lower compared to the WT BMDCs. Although the exact biological basis for the difference between previously published findings and the results in the present study remains to be determined, it should be noted that Leishmania parasites including L. major have been shown to suppress IL-12 and NO production in DCs.43Moll H. Scharner A. Kampgen E. Increased interleukin 4 (IL-4) receptor expression and IL-4-induced decrease in IL-12 production by Langerhans cells infected with Leishmania major.Infect Immun. 2002; 70: 1627-1630Crossref PubMed Scopus (16) Google Scholar, 44Liu D. Uzonna J.E. The early interaction of Leishmania with macrophages and dendritic cells and its influence on the host immune response.Front Cell Infect Microbiol. 2012; 2: 83Crossref PubMed Scopus (188) Google Scholar, 45Jayakumar A. Widenmaier R. Ma X. McDowell M.A. Transcriptional inhibition of interleukin-12 promoter activity in Leishmania spp.-infected macrophages.J Parasitol. 2008; 94: 84-93Crossref PubMed Scopus (17) Google Scholar Therefore, it is possible that loss of Leishmania-induced suppression in the infected miR155−/− DCs due to lower infection is at least in part responsible for increased IL-12 and NO production in these cells. It is also important to note that CD80 expressed at high levels can provide an adequate co-stimulatory signal to induce the production of cytokines such as IFN-γ even in the absence of CD86.46Thomas I.J. Petrich de Marquesini L.G. Ravanan R. Smith R.M. Guerder S. Flavell R.A. Wraith D.C. Wen L. Wong F.S. CD86 has sustained costimulatory effects on CD8 T cells.J Immunol. 2007; 179: 5936-5946Crossref PubMed Scopus (14) Google Scholar These data indicate that miR155−/− DCs infected with L. major display an enhanced activation profile and up-regulate IL-12 production, which could be involved in the enhancement of Th1-associated IFN-γ production observed in L. major–infected miR155−/− mice. Additionally, reduced IL-10 production in miR155−/− DCs could at least in part contribute to increased host resistance of miR155−/− mice to L. major. Indeed, DC–T cell co-culture studies revealed that L. major–infected and LPS-activated miR155−/− DCs significantly enhanced IFN-γ production by anti-CD3–activated miR155−/− T cells. Interestingly, anti-CD3–activated miR155−/− T cells co-incubated with LPS-activated WT DCs also produced more IFN-γ than did controls, suggesting that T cell–intrinsic miR155-dependent mechanisms could be involved in suppressing IFN-γ production in these cells during L. major infection. In conclusion, the results from this study indicate that miR155 contributes to susceptibility to CL caused by L. major by promoting disease-exacerbating Th2 response and attenuating DC activation and function. Furthermore, they also suggest that miR155 is not required for the induction of a Th1 response and IFN-γ production by NK cells and CD8+ T cells during L. major infection. Together, these findings suggest that miR155 plays different roles in regulating immune response in visceral leishmaniasis and CL. We thank the Department of Pathology core facility at The Ohio State University for allowing us to use the flow-cytometry machines. A.R.S. conceived the study. S.V., C.V., and G.N. designed the study, performed the experiments, and analyzed the data. S.V., A.R.S., and S.O. wrote and edited the manuscript. A.R.S. provided administrative and funding support.

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