MiRNA-155 Regulates the Th17/Treg Ratio by Targeting SOCS1 in Severe Acute Pancreatitis

Dongyan Wang, Maochun Tang, Pengfei Zong, Hua Liu, Ting Zhang, Yu Liu, Yan Zhao, Dongyan Wang, Maochun Tang, Pengfei Zong, Hua Liu, Ting Zhang, Yu Liu, Yan Zhao

Abstract

Acute pancreatitis (AP) is a serious condition associated with intestinal barrier disruption or inflammation of the pancreatic tissue. Specific microRNAs are involved in the pathogenesis of AP, during which IL-17-producing CD4+ T helper (Th17) cells accumulate in the pancreas. In this study, significantly increased levels of miR-155 were detected in clinical samples from patients with AP, and overexpression of miR-155 correlated with severe AP (SAP). To identify the effect of miR-155 on T cell differentiation, we isolated CD4+ T lymphocytes and in vitro experiments showed that inhibition of miR-155 significantly reversed the stress-induced increase in the Th17/Treg ratio. The results also showed that miR-155 increased the Th17-mediated inflammatory response by targeting SOCS1. The interaction between miR-155 and the 3'-UTR of SOCS1 was confirmed by a dual luciferase reporter assay and RT-PCR. Experimental AP of varying severity was induced in BALB/c mice by caerulein hyperstimulation and miR-155 expression was found to increase with disease progression. Inhibition of miR-155 expression significantly improved the pathology of the pancreas. We also observed downregulation of expression of inflammatory factors, IL-17, SOCS1 and phosphorylated STAT1 after miR-155 inhibition. In summary, miR-155 regulates the Th17/Treg ratio by targeting SOCS1, most probably via direct binding to its 3'-UTR region, indicating that this microRNA may be a potential biomarker and/or therapeutic target for AP.

Keywords: CD4+ T cells; SOCS1; Th17; acute pancreatitis; miRNA-155.

Figures

FIGURE 1
FIGURE 1
MiR-155 regulates AP pathogenesis. (A) Quantitative RT-PCR analyses of miR-155 expression in the sera of AP patients. Data are presented as the mean ± SD. ∗∗P < 0.01, ∗∗∗P < 0.001. (B) Western blot analysis of SOCS1 expression in CD4+ T cells isolated from the different groups of AP patients. GAPDH was detected as an endogenous control. (C) The relative SOCS1 expression was determined by densitometric analysis. Data are presented as the mean ± SD. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. AP group. ###P < 0.001 vs. MAP group. (D–F) ELISA to determine the concentrations of inflammatory factors IL-6, IL-13 and TNF-α in the sera from the different groups of AP patients. Data are presented as the mean ± SD. ∗P < 0.05, ∗∗∗P < 0.001 vs. AP group. ##P < 0.01 vs. MAP group. (G) Representative flow cytometric analyses of IL-17 and Foxp3 cells among CD4+ gated cells from different groups of AP patients. (H) The percentage of IL-17+ cells. Data are presented as the mean ± SD. ∗∗∗P < 0.001 vs. AP group. ###P < 0.01 vs. MAP group. AP, mild AP; MAP, moderate AP; SAP, severe AP.
FIGURE 2
FIGURE 2
MiR-155 promotes the generation of Th17 cells. CD4+ T cells from AP patients were first pretreated with miR-155 mimics or miR-155 inhibitor for 24 h. (A) Flow cytometric analyses of CD4+ T cells after 3 days of induction under Th17-polarizing conditions. (B) The percentage of IL-17+ cells. Data are presented as the mean ± SD. ∗∗∗P < 0.001 vs. control. ###P < 0.01 vs. miR-155 overexpression group. (C) ELISA to detect IL-17 in the culture supernatants of the pretreated CD4+ T cells (n = 5 per group). Data are presented as the mean ± SD. ∗∗∗P < 0.001 vs. control. ###P < 0.01 vs. miR-155 overexpression group. (D) Western blot analysis of SOCS1 expression in the pretreated CD4+ T cells (n = 3). GAPDH was detected as an endogenous control. (E) The relative SOCS1 expression was determined by densitometric analysis. Data are presented as the mean ± SD. ∗∗∗P < 0.001 vs. control. ###P < 0.01 vs. miR-155 overexpression group.
FIGURE 3
FIGURE 3
Inhibition of miR-155 expression decreased the conversion of CD4+ T cells to Th17 cells following caerulein induction. CD4+ T cells were first induced with caerulein (2 μM) to simulate AP for 48 h and were pretreated with or without miR-155 inhibitor. (A) Flow cytometric analyses of IL-17 cells (n = 3). (B) The percentage of IL-17+ cells. Data are presented as the mean ± SD. ∗∗∗P < 0.001 vs. control. (C) ELISA of IL-17 in the culture supernatants (n = 5 per group). Data are presented as the mean ± SD. ∗∗∗P < 0.001 vs. control. (D) Western blot analysis of the expression of SOCS1 in the pretreated CD4+ T cells (n = 3). GAPDH was detected as an endogenous control. (E) The relative SOCS1 expression was determined by densitometric analysis. Data are presented as the mean ± SD. ∗∗∗P < 0.001 vs. control.
FIGURE 4
FIGURE 4
SOCS1 is a direct target of miR-155. (A) Illustration of the sequence match between miR-155 and SOCS1 mRNA determined using TargetScan. (B) Luciferase activity of reporter vectors containing wild-type (WT) or mutated (MUT) SOCS1 3′-UTR co-transfected with miR-155 or control miR (ctrl; n = 3 per group). Data are presented as means ± SD. ∗∗∗P < 0.001 vs. control group. (C) Rt-PCR analyses for SOCS1 in CD4+ T cells 3 days post-transfection (n = 3 per group). Data are presented as means ± SD. ∗∗∗P < 0.001 vs. control group. ###P < 0.001 vs. miR-155 group.
FIGURE 5
FIGURE 5
The effect of miR-155 on histological changes in the pancreas. BALB/c mice were injected with miR-155 interference lentiviruses (anti-miR-155) or control lentiviruses (anti-ctrl) followed by 10, 50 or 250 μg/ml caerulein to model the AP, MAP and SAP stages of disease severity. (A–C) The expression of inflammatory factors IL-6 (A), TNF-α (B) and IL-13 (C) was measured by ELISA (n = 5). Data are presented as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. anti-ctrl group. (D) HE staining of the pancreatic tissue. (E) Western blot analysis of the expression levels of SOCS1, IL-17, STAT1, and pSTAT1 (n = 3). GAPDH was detected as an endogenous control.

References

    1. Ammori B. J. (2003). Role of the gut in the course of severe acute pancreatitis. Pancreas 26 122–129. 10.1097/00006676-200303000-00006
    1. Ammori B. J., Leeder P. C., King R. F., Barclay G. R., Martin I. G., Larvin M., et al. (1999). Early increase in intestinal permeability in patients with severe acute pancreatitis: correlation with endotoxemia, organ failure, and mortality. J. Gastrointest. Surg. 3 252–262. 10.1016/S1091-255X(99)80067-5
    1. Bhatia M. (2004). Apoptosis versus necrosis in acute pancreatitis. Am. J. Physiol. Gastrointest. Liver Physiol. 286 G189–G196. 10.1152/ajpgi.00304.2003
    1. Capurso G., Zerboni G., Signoretti M., Valente R., Stigliano S., Piciucchi M., et al. (2012). Role of the gut barrier in acute pancreatitis. J. Clin. Gastroenterol. 46(Suppl.), S46–S51. 10.1097/MCG.0b013e3182652096
    1. Chen Y., Li L., Lu Y., Su Q., Sun Y., Liu Y., et al. (2015). Upregulation of miR-155 in CD4(+) T cells promoted Th1 bias in patients with unstable angina. J. Cell. Physiol. 230 2498–2509. 10.1002/jcp.24987
    1. Clark J. A., Coopersmith C. M. (2007). Intestinal crosstalk: a new paradigm for understanding the gut as the “motor” of critical illness. Shock 28 384–393. 10.1097/shk.0b013e31805569df
    1. Cobb B. S., Hertweck A., Smith J., O’Connor E., Graf D., Cook T., et al. (2006). A role for Dicer in immune regulation. J. Exp. Med. 203 2519–2527. 10.1084/jem.20061692
    1. Du C., Liu C., Kang J., Zhao G., Ye Z., Huang S., et al. (2009). MicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of multiple sclerosis. Nat. Immunol. 10 1252–1259. 10.1038/ni.1798
    1. Dudda J. C., Salaun B., Ji Y., Palmer D. C., Monnot G. C., Merck E., et al. (2013). MicroRNA-155 is required for effector CD8+ T cell responses to virus infection and cancer. Immunity 38 742–753. 10.1016/j.immuni.2012.12.006
    1. Frobose H., Rønn S. G., Heding P. E., Mendoza H., Cohen P., Mandrup-Poulsen T., et al. (2006). Suppressor of cytokine signaling-3 inhibits interleukin-1 signaling by targeting the TRAF-6/ TAK1 complex. Mol. Endocrinol. 20 1587–1596. 10.1210/me.2005-0301
    1. Fujimoto M., Naka T. (2010). SOCS1, a negative regulator of cytokine signals and TLR responses, in human liver diseases. Gastroenterol. Res. Pract. 2010:470468. 10.1155/2010/470468
    1. Hovsepian E., Penas F., Siffo S., Mirkin G. A., Goren N. B. (2013). IL-10 inhibits the NF-kappaB and ERK/MAPK-mediated production of pro-inflammatory mediators by up-regulation of SOCS-3 in Trypanosoma cruzi-infected cardiomyocytes. PLoS One 8:e79445. 10.1371/journal.pone.0079445
    1. Jamshidian A., Kazemi M., Shaygannejad V., Salehi M. (2015). The effect of plasma exchange on the expression of FOXP3 and RORC2 in relapsed multiple sclerosis patients. Iran. J. Immunol. 12 311–318.
    1. Jamshidian A., Shaygannejad V., Pourazar A., Zarkesh-Esfahani S. H., Gharagozloo M. (2013). Biased Treg/Th17 balance away from regulatory toward inflammatory phenotype in relapsed multiple sclerosis and its correlation with severity of symptoms. J. Neuroimmunol. 262 106–112. 10.1016/j.jneuroim.2013.06.007
    1. Jia R., Tang M., Qiu L., Sun R., Cheng L., Ma X., et al. (2015). Increased interleukin-23/17 axis and C-reactive protein are associated with severity of acute pancreatitis in patients. Pancreas 44 321–325. 10.1097/MPA.0000000000000284
    1. Juvonen P. O., Alhava E. M., Takala J. A. (2000). Gut permeability in patients with acute pancreatitis. Scand. J. Gastroenterol. 35 1314–1318. 10.1080/003655200453683
    1. Kurowska-Stolarska M., Alivernini S., Ballantine L. E., Asquith D. L., Millar N. L., Gilchrist D. S., et al. (2011). MicroRNA-155 as a proinflammatory regulator in clinical and experimental arthritis. Proc. Natl. Acad. Sci. U.S.A. 108 11193–11198. 10.1073/pnas.1019536108
    1. Lochner M., Wang Z., Sparwasser T. (2015). The special relationship in the development and function of T helper 17 and regulatory T cells. Prog. Mol. Biol. Transl. Sci. 136 99–129. 10.1016/bs.pmbts.2015.07.013
    1. Lu L. F., Thai T. H., Calado D. P., Chaudhry A., Kubo M., Tanaka K., et al. (2009). Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein. Immunity 30 80–91. 10.1016/j.immuni.2008.11.010
    1. McBerry C., Gonzalez R. M., Shryock N., Dias A., Aliberti J. (2012). SOCS2-induced proteasomedependent TRAF6 degradation: a common anti-inflammatory pathway for control of innate immune responses. PLoS One 7:e38384. 10.1371/journal.pone.0038384
    1. Murugaiyan G., da Cunha A. P., Ajay A. K., Joller N., Garo L. P., Kumaradevan S., et al. (2015). MicroRNA-21 promotes Th17 differentiation and mediates experimental autoimmune encephalomyelitis. J. Clin. Invest. 125 1069–1080. 10.1172/JCI74347
    1. Mycko M. P., Cichalewska M., Machlanska A., Cwiklinska H., Mariasiewicz M., Selmaj K. W. (2012). MicroRNA-301a regulation of a T-helper17 immune response controls autoimmune demyelination. Proc. Natl. Acad. Sci. U.S.A. 109 E1248–E1257. 10.1073/pnas.1114325109
    1. Nahid M. A., Satoh M., Chan E. K. (2011). MicroRNA in TLR signaling and endotoxin tolerance. Cell. Mol. Immunol. 8 388–403. 10.1038/cmi.2011.26
    1. Niederau C., Ferrell L. D., Grendell J. H. (1985). Caerulein-induced acute necrotizing pancreatitis in mice: protective effects of proglumide, benzotript, and secretin. Gastroenterology 88 1192–1204. 10.1016/S0016-5085(85)80079-2
    1. Norman M. (1998). The role of cytokines in the pathogenesis of acute pancreatitis. Am. J. Surg. 175 76–83. 10.1016/S0002-9610(97)00240-7
    1. O’Connell R. M., Taganov K. D., Boldin M. P., Cheng G., Baltimore D. (2007). MicroRNA-155 is induced during the macrophage inflammatory response. Proc. Natl. Acad. Sci. U.S.A. 104 1604–1609. 10.1073/pnas.0610731104
    1. Pedersen I., David M. (2008). MicroRNAs in the immune response. Cytokine 43 391–394. 10.1016/j.cyto.2008.07.016
    1. Piccinini A. M., Midwood K. S. (2012). Endogenous control of immunity against infection: tenascin-C regulates TLR4-mediated inflammation via microRNA-155. Cell. Rep. 2 914–926. 10.1016/j.celrep.2012.09.005
    1. Qu X., Han J., Zhang Y., Wang Y., Zhou J., Fan H., et al. (2017). MiR-384 regulates the Th17/Treg ratio during experimental autoimmune encephalomyelitis pathogenesis. Front. Cell. Neurosci. 11:88. 10.3389/fncel.2017.00088
    1. Rahman S. H., Ammori B. J., Holmfield J., Larvin M., McMahon M. J. (2003). Intestinal hypoperfusion contributes to gut barrier failure in severe acute pancreatitis. J. Gastrointest. Surg. 7 26–35. 10.1016/S1091-255X(02)00090-2
    1. Sheedy F. J., O’Neill L. A. (2008). Adding fuel to fire: microRNAs as a new class of mediators of inflammation. Ann. Rheum. Dis. 67(Suppl. 3), iii50–iii55. 10.1136/ard.2008.100289
    1. Singh U. P., Murphy A. E., Enos R. T., Shamran H. A., Singh N. P., Guan H., et al. (2014). miR-155 deficiency protects mice from experimental colitis by reducing T helper type 1/type 17 responses. Immunology 143 478–489. 10.1111/imm.12328
    1. Suzuki A., Hanada T., Mitsuyama K., Yoshida T., Kamizono S., Hoshino T., et al. (2001). CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation. J. Exp. Med. 193 471–481. 10.1084/jem.193.4.471
    1. Tarassishin L., Loudig O., Bauman A., Shafit-Zagardo B., Suh H. S., Lee S. C. (2011). Interferon regulatory factor 3 inhibits astrocyte inflammatory gene expression through suppression of the proinflammatory miR-155 and miR-155∗. Glia 59 1911–1922. 10.1002/glia.21233
    1. Tian R., Wang R. L., Xie H., Jin W., Yu K. L. (2013). Overexpressed miRNA-155 dysregulates intestinal epithelial apical junctional complex in severe acute pancreatitis. World J. Gastroenterol. 19 8282–8291. 10.3748/wjg.v19.i45.8282
    1. Tili E., Michaille J. J., Cimino A., Costinean S., Dumitru C. D., Adair B., et al. (2007). Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock. J. Immunol. 179 5082–5089. 10.4049/jimmunol.179.8.5082
    1. Tzartos J. S., Friese M. A., Craner M. J., Palace J., Newcombe J., Esiri M. M., et al. (2008). Interleukin-17 production in central nervous system-infiltrating T cells and glial cells is associated with active disease in multiple sclerosis. Am. J. Pathol. 172 146–155. 10.2353/ajpath.2008.070690
    1. Venkatesha S. H., Dudics S., Weingartner E., So E. C., Pedra J., Moudgil K. D. (2015). Altered Th17/Treg balance and dysregulated IL-1β response influence susceptibility/resistance to experimental autoimmune arthritis. Int. J. Immunopathol. Pharmacol. 28 318–328. 10.1177/0394632015595757
    1. Wu B. U., Banks P. A. (2013). Clinical management of patients with acute pancreatitis. Gastroenterology 144 1272–1281. 10.1053/j.gastro.2013.01.075
    1. Wu J., Ma C., Wang H., Wu S., Xue G., Shi X., et al. (2014). A MyD88-JAK1-STAT1 complex directly induces SOCS-1expression in macrophages infected with Group A Streptococcus. Cell. Mol. Immunol. 12 373–383. 10.1038/cmi.2014.107
    1. Yao R., Ma Y. L., Liang W., Li H. H., Ma Z. J., Yu X., et al. (2012). MicroRNA-155 modulates Treg and Th17 cells differentiation and Th17 cell function by targeting SOCS1. PLoS One 7:e46082. 10.1371/journal.pone.0046082
    1. Yu H., Liu Y., McFarland B. C., Deshane J. S., Hurst D. R., Ponnazhagan S., et al. (2015). SOCS3 deficiency in myeloid cells promotes tumor development: involvement of STAT3 activation and myeloid-derived suppressor cells. Cancer Immunol. Res. 3 727–740. 10.1158/2326-6066.CIR-15-0004
    1. Yu J. H., Kim K. H., Kim H. (2008). SOCS3 and PPAR-gamma ligands inhibit the expression of IL-6 and TGF-beta1 by regulating JAK2/STAT3 signaling in pancreas. Int. J. Biochem. Cell. Biol. 40 677–688. 10.1016/j.biocel.2007.10.007
    1. Zhu E., Wang X., Zheng B., Wang Q., Hao J., Chen S., et al. (2014). miR-20b suppresses Th17 differentiation and the pathogenesis of experimental autoimmune encephalomyelitis by targeting RORγt and STAT3. J. Immunol. 192 5599–5609. 10.4049/jimmunol.1303488
    1. Zhu S., Pan W., Song X., Liu Y., Shao X., Tang Y., et al. (2012). The microRNA miR-23b suppresses IL-17-associated autoimmune inflammation by targeting TAB2, TAB3 and IKK-α. Nat. Med. 18 1077–1086. 10.1038/nm.2815

Source: PubMed

Sponsors and Collaborators

Medical Conditions

Drug Interventions

3
Subscribe