ARTICLES KLF6 contributes to myeloid cell plasticity in the pathogenesis of intestinal inflammation WA Goodman1, S Omenetti1, D Date2, L Di Martino2, C De Salvo1, G-D Kim2, S Chowdhry2,3, G Bamias4, F Cominelli2,3, TT Pizarro1,2,5 and GH Mahabeleshwar1,2,5 Inflammatory bowel disease (IBD) is associated with dysregulated macrophage responses, such that quiescent macrophages acquire a pro-inflammatory activation state and contribute to chronic intestinal inflammation. The transcriptional events governing macrophage activation and gene expression in the context of chronic inflammation such as IBD remain incompletely understood. Here, we identify Kruppel-like transcription factor-6 (KLF6) as a critical regulator of pathogenic myeloid cell activation in human and experimental IBD. We found that KLF6 was significantly upregulated in myeloid cells and intestinal tissue from IBD patients and experimental models of IBD, particularly in actively inflamed regions of the colon. Using complementary gain- and loss-of-function studies, we observed that KLF6 promotes pro-inflammatory gene expression through enhancement of nuclear factor jB (NFjB) signaling, while simultaneously suppressing anti-inflammatory gene expression through repression of signal transducer and activator of transcription 3 (STAT3) signaling. To study the in vivo role of myeloid KLF6, we treated myeloid-specific KLF6-knockout mice (Mac-KLF6-KO) with dextran sulfate sodium (DSS) and found that Mac-KLF6-KO mice were protected against chemically-induced colitis; this highlights the central role of myeloid KLF6 in promoting intestinal inflammation. Collectively, our results point to a novel gene regulatory program underlying pathogenic, pro-inflammatory macrophage activation in the setting of chronic intestinal inflammation. INTRODUCTION function,3 intestinal mucosal macrophages are required for The human gastrointestinal tract is home to more than one shaping immune responses to the continuous barrage of hundred trillion commensal microbes,1 highlighting the need antigenic challenges that breach the intestinal epithelium. to discriminate between beneficial organisms and pathogenic, The requirement of macrophages for maintenance of invading microbes. The innate immune system of the intestinal intestinal homeostasis was established by seminal work in mucosa has therefore evolved to provide a rapid, first-line which deletion of macrophages4,5 or toll-like receptors and defense against harmful, infectious organisms through the their common signaling adapter, MyD88,6 resulted in severe recognition of conserved molecular patterns unique to invading morbidity and mortality in response to dextran sulfate sodium pathogens, such as endotoxins and nucleic acids. Pattern (DSS)-induced colitis. These studies suggest that under non- recognition receptor-bearing macrophages situated in the sub- inflamed conditions, intestinal macrophages recognize com- epithelial lamina propria of the small and large intestine are mensal microbes through pattern recognition receptor inter- central to this process, and in fact represent the largest pool of actions and regulate mucosal immunity in response to these tissue-resident macrophages in the body.2 In addition to their signals. Intestinal macrophages are also required for the essential roles in maintaining host defense, scavenging dead development of mucosal tolerance, as macrophage-deficient cells and debris, and helping to maintain epithelial barrier mice (F4/80 knockout (KO)) fail to develop tolerance or 1Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. 2Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. 3Department of Medicine, Division of Gastroenterology, University Hospitals of Cleveland, Cleveland, Ohio, USA and 4Academic Department of Gastroenterology, Ethnikon and Kapodistriakon University of Athens and Laikon Hospital, Athens, Greece. Correspondence: GH Mahabeleshwar or TT Pizarro ([email protected] or [email protected]) 5The last two authors contributed equally to this work. Received 31 July 2015; accepted 29 November 2015; published online 3 February 2016. doi:10.1038/mi.2016.1 1250 VOLUME 9 NUMBER 5 | SEPTEMBER 2016 | www.nature.com/mi ARTICLES antigen-specific regulatory T cells (Treg) following oral Mature macrophages remain transcriptionally dynamic and administration of soluble antigen.7 can alter their activation state in response to environmental Innate immunity sensing mechanisms are critical for cues such as cytokines and other soluble mediators. Changes to initiating and shaping the adaptive immune response in the the IBD cytokine environment, such as an upregulation of lamina propria8,9 and other tissues, as demonstrated by the IFNg, TNF, IL-1, and IL-6, may therefore divert macrophages ability of ‘‘classically’’ (M1) and ‘‘alternatively’’ (M2) activated away from inflammatory anergy and toward a pathogenic, M1 macrophages to promote Th1 and Th2 responses, respectively. phenotype. Given the plasticity of macrophage phenotypes and Although M1/M2 macrophage classification remains widely functions, it is critical to identify molecular events governing used, these broad categories encompass many different the loss of inflammatory anergy and the ‘‘switch’’ to pathogenic, phenotypic and functional subsets.10 Macrophages are differ- M1 phenotypes that occurs in the setting of chronical intestinal entiated and activated by many different stimuli, including inflammation such as IBD. cytokines, toll-like receptor agonists, and other exogenous Kruppel-like transcription factors (KLFs) are a subclass stimuli. Responses to stimulation by these agents, alone or in of zinc-finger family transcription factors that regulate combination, can lead to a spectrum of activation states not critical cellular processes, including development, differentia- restricted to the classic M1/M2 classificiations. Recent efforts tion, proliferation, and programmed cell death.23–25 Previous have therefore led to the development of a new consensus studies have indicated that KLF2 (ref. 26) and KLF4 (ref. 27) framework for the classification of activated macrophages, have important roles in macrophage inflammatory gene aimed at standardizing macrophage nomenclature.10 expression and function; however, it has been recently Under homeostatic conditions, intestinal macrophages shown that KLF6 is the most highly expressed KLF family remain ‘‘inflammation anergic’’11 in response to strong signals member in primary macrophages, where it simultaneously from anti-inflammatory interleukin (IL)-10 and TGFb (trans- promotes M1 activation and inhibits M2 activation.28 KLF6 forming growth factor beta), displaying many phenotypic and has also been recently linked to the pathogenesis of human functional features of M2 macrophages. Although these cells inflammatory diseases, including psoriasis29 and hepatic express pattern recognition receptors and display robust steatosis.30 Nonetheless, the role of myeloid-derived KLFs, phagocytic activity, they fail to mount inflammatory responses including KLF6, in the pathogenesis of IBD has never been to exogenous antigens;12 this unique feature allows intestinal investigated. macrophages to effectively eliminate foreign invaders Our current studies seek to investigate the contribution of while simultaneously maintaining immune quiescence. macrophage KLF6 to the loss of macrophage inflammatory Myeloid-specific deletion of M2-associated molecules such anergy in IBD. We demonstrate a significant upregulation of as IL-10Ra,13 signal transducer and activator of transcription 3 KLF6 in intestinal myeloid cells from human and experimental (STAT3),14 and peroxisome proliferator-activated receptor IBD subjects, as well as a central role for KLF6 in controlling gamma (PPARg)15 leads to a significant exacerbation of macrophage responses to pro- and anti-inflammatory stimuli experimental colitis, highlighting the immunoprotective role associated with the development and resolution of intestinal conferred by these molecules under intestinal homeostasis. inflammation. Thus, we propose that KLF6 may function as a In contrast, intestinal macrophages of inflammatory bowel molecular switch, diverting intestinal macrophages away disease (IBD) patients display an impairment in inflammatory from protective, inflammatory anergic phenotypes and toward anergy and an increase in pro-inflammatory, M1 activation.16 pathogenic, M1 phenotypes in response to the local IBD- This process is thought to occur due to high levels of cytokines, associated cytokine milieu. such as interferon gamma (IFNg), tumor necrosis factor (TNF), IL-1, and IL-6, in the gut mucosa that can divert infiltrating RESULTS 3 monocytes towards a pathogenic, M1 macrophage phenotype. Myeloid KLF6 is upregulated in human and experimental These cells exhibit increased activation of nuclear factor kB IBD (NFkB), enhanced sensitivity to toll-like receptor ligands, Our previous work demonstrated that KLF6 is the most highly and elevated expression of pro-inflammatory cytokines, expressed Kruppel-like transcription factor in macrophages chemokines, matrix metalloproteinases, and reactive oxygen and contributes to pro-inflammatory macrophage activation species /nitric oxide synthases,17–19 leading to inappropriate and function.28 Given the role of activated macrophages in inflammatory responses directed against non-harmful human and experimental IBD,12 we sought to investigate commensal microbes and