Disease Risk−Associated Genetic Variants in STAT1 and STAT4 Function in a Complementary Manner to Increase Pattern-Recognition Receptor−Induced This information is current as Outcomes in Human Macrophages of September 26, 2021. Matija Hedl, Rui Sun and Clara Abraham J Immunol published online 13 July 2020 http://www.jimmunol.org/content/early/2020/07/31/jimmun ol.1901112 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2020/07/10/jimmunol.190111 Material 2.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published July 31, 2020, doi:10.4049/jimmunol.1901112 The Journal of Immunology Disease Risk–Associated Genetic Variants in STAT1 and STAT4 Function in a Complementary Manner to Increase Pattern-Recognition Receptor–Induced Outcomes in Human Macrophages Matija Hedl,1 Rui Sun,1 and Clara Abraham STAT proteins can regulate both pro- and anti-inflammatory cytokine signaling. Therefore, identifying consequences of modu- lating expression of a given STAT is ultimately critical for determining its potential as a therapeutic target and for defining the mechanisms through which immune-mediated disease variants in STAT genes contribute to disease pathogenesis. Genetic variants in the STAT1/STAT4 region are associated with multiple immune-mediated diseases, including inflammatory bowel disease (IBD). These diseases are characterized by dysregulated cytokine secretion in response to pattern-recognition receptor (PRR) stimula- Downloaded from tion. We found that the common IBD-associated rs1517352 C risk allele increased both STAT1 and STAT4 expression in human monocyte-derived macrophages (MDMs). We therefore hypothesized that the STAT1/STAT4 variant might regulate PRR-initiated responses in a complementary and cooperative manner because of the important role of autocrine/paracrine cytokines in mod- ulating PRR-initiated signaling. STAT1 and STAT4 were required for PRR- and live bacterial-induced secretion of multiple cytokines. These outcomes were particularly dependent on PRR-initiated autocrine/paracrine IL-12–induced STAT4 activation http://www.jimmunol.org/ to generate IFN-g, with autocrine IFN-g then signaling through STAT1. STAT1 and STAT4 also promoted bacterial-induced cytokines in intestinal myeloid cells and PRR-enhanced antimicrobial pathways in MDMs. Importantly, MDMs from rs1517352 C IBD risk allele carriers demonstrated increased TLR4-, IFN-g– and IL-12–induced STAT1 and STAT4 phosphorylation and cytokine secretion and increased TLR4-enhanced antimicrobial pathways. Taken together, STAT1 and STAT4 expression is coregulated by a shared genetic region, and STAT1/STAT4-immune disease–associated variants modulate IFN-g– and IL-12– associated outcomes, and in turn, PRR-induced outcomes, highlighting that these genes cooperate to regulate pathways relevant to disease pathogenesis. The Journal of Immunology, 2020, 205: 000–000. roper responses to microbial products detected by pattern- pathways is that these proteins can mediate responses to both by guest on September 26, 2021 recognition receptors (PRRs) on myeloid cells are critical proinflammatory and anti-inflammatory cytokines. As such, it is P for immune homeostasis at mucosal surfaces such as the critical to clearly define the distinct contributions of each of these intestine. A key PRR-induced response is cytokine secretion, and family members to this cytokine balance. Furthermore, mouse and this cytokine secretion can, in turn, dramatically amplify the initial human cells can show significant differences in inflammatory path- response to microbial products (1). Consistently, one of the hall- ways (2), such it is important to understand how these STAT proteins marks of immune-mediated diseases, including inflammatory regulate outcomes in human myeloid-derived cells. Genetic variants bowel disease (IBD), is dysregulated responses to and/or pro- in these genes can help elucidate consequences of this regulation. duction of cytokines (1). STAT proteins are critical for mediating Common genetic variants in the STAT1/STAT4 region confer responses to cytokines, such that the JAK-STAT pathway is being altered susceptibility to IBD (3) and systemic lupus erythematosus actively investigated as a therapeutic target in immune-mediated (SLE) (4). STAT1 and STAT4 can complement each other as diseases. However, an important challenge in targeting these regulators of cytokine-induced outcomes. STAT1 mediates type I IFN– and IFN-g–initiated signaling, and STAT4 can mediate signaling by type I IFNs and IL-12 and is required for IL-12– Department of Internal Medicine, Yale University, New Haven, CT 06520 induced IFN-g (5). STAT1 has been reported to promote TLR- 1M.H. and R.S. contributed equally to this work. induced outcomes in mouse macrophages (6); the role of STAT4 ORCID: 0000-0003-4054-1365 (R.S.). in PRR-initiated responses in human macrophages has not been Received for publication September 12, 2019. Accepted for publication June 8, 2020. clearly defined. Genetic associations in each STAT1 and STAT4 This work was supported by National Institutes of Health Grants R01DK099097 and have focused predominantly on rare coding mutations. As such, R01DK106593 and an ASPIRE Inflammatory Bowel Disease Pfizer Research Award. two rare coding variants resulting in defective STAT1 function Address correspondence and reprint requests to Dr. Clara Abraham, Department of result in decreased IFN-a– and IFN-g–initiated responses; infant Internal Medicine, Section of Digestive Diseases, 333 Cedar Street (LMP 1080), New carriers of these mutations can develop severe mycobacterial and Haven, CT 06520. E-mail address: [email protected] viral infections (7). In contrast, rare coding gain-of-function STAT1 The online version of this article contains supplemental material. mutations associated with STAT1 hyperactivation in T cells lead to Abbreviations used in this article: AIEC, adherent-invasive Escherichia coli; IBD, inflammatory bowel disease; MDM, monocyte-derived macrophage; MOI, multiplic- excessive IFN-g signaling, resulting in impaired Th17 immunity ity of infection; poly(I:C), polyinosinic-polycytidylic acid; PRR, pattern-recognition and chronic mucocutaneous candidiasis (8). Consistent with its role receptor; RNS, reactive nitrogen species; ROS, reactive oxygen species; siRNA, in type I IFN and IL-12 signaling, a STAT4 gain-of-function intronic small interfering RNA; SLE, systemic lupus erythematosus. polymorphism associated with SLE results in increased IFN-a re- Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 sponses in human PBMCs (9). To our knowledge, how common www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901112 2 STAT1 AND STAT4 POLYMORPHISMS MODULATE PRR-INDUCED OUTCOMES IBD-associated genetic variants in the STAT1/STAT4 region reg- Intestinal lamina propria cell isolation ulate myeloid cell outcomes has not been defined. Intestinal lamina propria cells were isolated from colonic resection The common rs1517352 polymorphism located in an intronic specimens from uninvolved intestine in non-IBD patients undergoing region of STAT4 and upstream of STAT1 is associated with IBD (3) surgery for diverticular disease or colon cancer as in Ref. 11. Briefly, and SLE (4). The C risk allele has a frequency ranging from 0.33 intestinal resections were washed and then digested (RPMI containing to 0.40 in European ancestry individuals per the Single Nucleotide 0.2 mM EDTA, 2-ME) in a 37˚C shaker to remove epithelial cells. Epithelial cells and intraepithelial lymphocytes were discarded, and Polymorphism Database. How this common polymorphism con- intestines were washed, cut into 1-mm2 pieces, and incubated in a tributes to risk is incompletely defined. One study found that the C buffer consisting of RPMI, 75 mg/ml collagenase type VIII, DNase risk allele is part of a haplotype associated with increased STAT4 (MilliporeSigma), and HEPES for 45 min in a 37˚C shaker. The cells expression in human osteoblasts and lymphoblasts and increased were filtered through a 40-mm filter, selected on a Ficoll gradient, washed, and then used in studies. anti-dsDNA in SLE patients (10). How this polymorphism regulates STAT4 protein expression, STAT4 activation, or Transfection of small interfering RNA STAT4-dependent cytokine secretion, including to PRR stim- One hundred nanomolar scrambled or ON-TARGETplus or siGENOME ulation, is not clear. Furthermore, STAT1 is located ∼50 kb SMARTpool small interfering RNA (siRNA) against STAT1, STAT4, from this polymorphism, and it is unclear if rs1517352 mod- STAT3, JAK1, TYK2, IL12RB2, IFNGR1, IFNAR, IL10RA, ERK, p38, ulates STAT1 expression or STAT1-dependent outcomes.