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Interleukin 1β Mediates Intestinal Inflammation in Mice and Patients With Interleukin 10 Receptor Deficiency

2016; Elsevier BV; Volume: 151; Issue: 6 Linguagem: Inglês

10.1053/j.gastro.2016.08.055

1528-0012

Dror S. Shouval, Amlan Biswas, Yu Hui Kang, Alexandra Griffith, Liza Konnikova, Iván Mascanfroni, Naresh Singh Redhu, Sandra M. Frei, Michael Field, Andria Doty, Jeffrey D. Goldsmith, Atul K. Bhan, Anthony M. Loizides, Batia Weiss, Baruch Yerushalmi, Tadahiro Yanagi, Xiuli Lui, Francisco J. Quintana, Aleixo M. Muise, Christoph Klein, Bruce Horwitz, Sarah C. Glover, Athos Bousvaros, Scott B. Snapper,

Immune Cell Function and Interaction

Interleukin 10 receptor (IL10R)−deficient mice develop spontaneous colitis and, similarly, patients with loss-of-function mutations in IL10R develop severe infant-onset inflammatory bowel disease. Loss of IL10R signaling in mouse and human macrophages is associated with increased production of interleukin 1β. We demonstrated that innate immune production of IL1β mediates colitis in IL10R-deficient mice. Transfer of Il1r1−/− CD4+ T cells into Rag1−/−/Il10rb−/− mice reduced the severity of their colitis (compared to mice that received CD4+ T cells that express IL1R), accompanied by decreased production of interferon gamma, tumor necrosis factor−α, and IL17A. In macrophages from mice without disruption of IL10R signaling or from healthy humans (controls), incubation with IL10 reduced canonical activation of the inflammasome and production of IL1β through transcriptional and post-translational regulation of NLRP3. Lipopolysaccharide and adenosine triphosphate stimulation of macrophages from Il10rb−/− mice or IL10R-deficient patients resulted in increased production of IL1β. Moreover, in human IL10R-deficient macrophages, lipopolysaccharide stimulation alone triggered IL1β secretion via non-canonical, caspase 8−dependent activation of the inflammasome. We treated 2 IL10R-deficient patients with severe and treatment-refractory infant-onset inflammatory bowel disease with the IL1−receptor antagonist anakinra. Both patients had marked clinical, endoscopic, and histologic responses after 4–7 weeks. This treatment served as successful bridge to allogeneic hematopoietic stem cell transplantation in 1 patient. Our findings indicate that loss of IL10 signaling leads to intestinal inflammation, at least in part, through increased production of IL1 by innate immune cells, leading to activation of CD4+ T cells. Agents that block IL1 signaling might be used to treat patients with inflammatory bowel disease resulting from IL10R deficiency. Interleukin 10 receptor (IL10R)−deficient mice develop spontaneous colitis and, similarly, patients with loss-of-function mutations in IL10R develop severe infant-onset inflammatory bowel disease. Loss of IL10R signaling in mouse and human macrophages is associated with increased production of interleukin 1β. We demonstrated that innate immune production of IL1β mediates colitis in IL10R-deficient mice. Transfer of Il1r1−/− CD4+ T cells into Rag1−/−/Il10rb−/− mice reduced the severity of their colitis (compared to mice that received CD4+ T cells that express IL1R), accompanied by decreased production of interferon gamma, tumor necrosis factor−α, and IL17A. In macrophages from mice without disruption of IL10R signaling or from healthy humans (controls), incubation with IL10 reduced canonical activation of the inflammasome and production of IL1β through transcriptional and post-translational regulation of NLRP3. Lipopolysaccharide and adenosine triphosphate stimulation of macrophages from Il10rb−/− mice or IL10R-deficient patients resulted in increased production of IL1β. Moreover, in human IL10R-deficient macrophages, lipopolysaccharide stimulation alone triggered IL1β secretion via non-canonical, caspase 8−dependent activation of the inflammasome. We treated 2 IL10R-deficient patients with severe and treatment-refractory infant-onset inflammatory bowel disease with the IL1−receptor antagonist anakinra. Both patients had marked clinical, endoscopic, and histologic responses after 4–7 weeks. This treatment served as successful bridge to allogeneic hematopoietic stem cell transplantation in 1 patient. Our findings indicate that loss of IL10 signaling leads to intestinal inflammation, at least in part, through increased production of IL1 by innate immune cells, leading to activation of CD4+ T cells. Agents that block IL1 signaling might be used to treat patients with inflammatory bowel disease resulting from IL10R deficiency. See editorial on page 1061. See editorial on page 1061. Interleukin-10 (IL10) is a key immunoregulatory cytokine.1Shouval D.S. et al.Adv Immunol. 2014; 122: 177-210Crossref PubMed Scopus (225) Google Scholar Patients with loss-of-function mutations in IL10 receptor (IL10R) genes develop severe inflammatory bowel disease in the first months of life2Kotlarz D. Beier R. Murugan D. et al.Gastroenterology. 2012; 143: 347-355Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 3Glocker E.O. Kotlarz D. Boztug K. et al.N Engl J Med. 2009; 361: 2033-2045Crossref PubMed Scopus (1139) Google Scholar and, similarly, Il10rb−/− mice develop spontaneous colitis.4Shouval D.S. et al.Immunity. 2014; 40: 706-719Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar We and others have recently reported that IL10R deficiency in innate immune cells results in severe colitis and an intrinsic defect in generation and function of anti-inflammatory macrophages.4Shouval D.S. et al.Immunity. 2014; 40: 706-719Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 5Zigmond E. et al.Immunity. 2014; 40: 720-733Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 6Li B. et al.Mucosal Immunol. 2014; 7: 869-878PubMed Scopus (134) Google Scholar, 7Li B. et al.Nat Commun. 2015; 6: 6131Crossref Scopus (50) Google Scholar The mechanisms driving this hyperinflammatory state are unclear. IL1β is a potent inflammatory cytokine that is produced in a 2-step process8Garlanda C. et al.Immunity. 2013; 39: 1003-1018Abstract Full Text Full Text PDF PubMed Scopus (1419) Google Scholar: induction of pro-IL1β and activation of the inflammasome, which is necessary for conversion of pro-caspase-1 to caspase-1 that cleaves pro-IL1β into its mature form.9Lamkanfi M. et al.Cell. 2014; 157: 1013-1022Abstract Full Text Full Text PDF PubMed Scopus (1835) Google Scholar Recent work implicates IL1 in the development of colitis and TH17-associated responses in the gut.10Coccia M. et al.J Exp Med. 2012; 209: 1595-1609Crossref PubMed Scopus (452) Google Scholar We hypothesized that IL10R deficiency results in dysregulated inflammasome activation, leading to excessive IL1 secretion and subsequent mucosal inflammation. Rag1−/−Il10rb−/− mice develop severe colitis after transfer of unfractionated wild-type (WT) CD4+ T cells associated with increased IL1β in the colon (Supplementary Figure 1). To assess the role of innate immune IL1β production in mediating colitis in IL10R deficiency through modulation of effector CD4+ T cells, we compared transfer of WT and Il1r−/− total CD4+ T cells into Rag1−/−Il10rb−/− mice. Mice adoptively transferred with Il1r−/− cells displayed an attenuated course of colitis, accompanied by a significant reduction in the number of interferon gamma, tumor necrosis factor−α, and IL17A-producing CD4+ T cells, and an increase in regulatory T cell frequency in the lamina propria (Figure 1A−D, Supplementary Figure 2). Overall, our data indicate that production of innate IL1 facilitates the ability of CD4+ T cells to induce colitis in Rag1−/−Il10rb−/− mice. We next sought to determine whether neutralizing IL1 may be beneficial for controlling inflammation in patients with IL10R deficiency. Two patients aged 2 and 28 years with history of severe infant-onset medical-refractory colitis and fistulizing disease due to loss-of-function mutations in IL10RA were treated with anakinra, an IL1 receptor antagonist. Both patients displayed marked clinical, endoscopic, and histologic improvement in response to therapy after 4–7 weeks (Figure 1E; Supplementary Material). Analysis of intestinal biopsies in both patients showed a substantial reduction in inflammatory transcripts (Figure 1F). The clinical improvement in the first patient permitted a reduction in her steroid requirement and a successful bridge to allogeneic hematopoietic stem cell transplantation. The second patient is currently being evaluated for transplantation. This treatment strategy, to our knowledge, is the first therapeutic approach leading to reduced intestinal inflammation in the setting of IL10R deficiency. To assess the role of IL10 in regulating IL1β production, bone-marrow−derived macrophages from WT and Il10rb−/− mice and monocyte-derived macrophages from 10 control subjects and 5 IL10R-deficient patients (patients 1–5 in Supplementary Table 1) were stimulated with lipopolysaccharide (LPS). Adenosine triphosphate (ATP) was used as a secondary signal for inflammasome activation. In both murine and human IL10R-deficient macrophages, LPS+ATP triggered increased IL1β production that was not suppressed by IL10 pretreatment compared to WT and control macrophages, respectively (Figure 2A−D). Similarly, stimulation of human control macrophages with LPS+ATP in the presence of a blocking IL10R1 antibody also resulted in increased production of IL1β (Figure 2C and D). Western blot analyses demonstrated that IL10 suppresses production of pro-IL1β and conversion of pro-caspase-1 to its mature form in both mouse and human macrophages (Figure 2B and D). In contrast to murine macrophages, in human IL10R-deficient macrophages, LPS stimulation alone, in the absence of ATP, resulted in IL1β production (Figure 2C and D), suggesting that addition of a secondary inflammasome activation signal is not required in the setting of human IL10R deficiency. Overall, these data indicate that IL10R signaling regulates inflammasome-dependent IL1β production in murine and human macrophages. To understand how IL10 regulates IL1β production, we examined the effect of IL10 on the expression of NLRP3 and ASC, 2 important components of the inflammasome complex. While LPS stimulation up-regulated expression of murine NLRP3 in WT macrophages, NLPR3 messenger RNA and protein levels were inhibited by IL10 (Supplementary Figure 3). This inhibition of NLRP3 expression was IL10-dependent, as it was abrogated in murine IL10R-deficient macrophages and in human macrophages in the presence of blocking IL10R1 antibodies (Figure 2E and Supplementary Figure 3). ASC expression was unchanged after LPS or IL10 stimulation (Supplementary Figure 3). Interestingly, treatment with MG132, an inhibitor of proteasome- and autophagy-mediated proteolysis, partially blocked IL10-dependent down-regulation of NLRP3 in murine and human WT/control macrophages (Figure 2E), indicating that IL10 modulates NLRP3 fate by enhancing proteasomal degradation. We demonstrated that IL10 promotes K48-linked polyubiquitination of the NLRP3 complex, further supporting a role for IL10 in regulating inflammasome protein degradation (Supplementary Figure 3). Together these data identify IL10 as a critical transcriptional and post-translational regulator of NLRP3-mediated inflammasome activation. One of the main differences between human and murine IL10R-deficient macrophages was the ability of the former to produce IL1β after LPS activation, without a secondary inflammasome activation trigger (such as ATP). Recently, it was shown that human monocytes can produce IL1β in response to LPS alone through a TLR4-TRIF-RIPK1-FADD-CASP8−mediated non-canonical (alternative) activation of the inflammasome.11Gaidt M.M. et al.Immunity. 2016; 44: 833-846Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar To examine whether IL10 regulates alternative inflammasome signaling, we used Z-IETD-FMK, a caspase-8 small-molecule inhibitor that selectively blocks non-canonical signaling; Z-YVAD-FMK, a caspase-1 inhibitor that blocks both canonical and non-canonical signaling; and KN-62, a P2RX7 inhibitor that blocks only canonical, ATP-induced, inflammasome signaling. Blocking non-canonical inflammasome activation with either Z-IETD-FMK or Z-YVAD-FMK completely abrogated LPS-induced IL1β production in both monocyte-derived macrophages from an IL10RA-deficient patient and anti-IL10R1−treated control macrophages (Figure 2F). In contrast, LPS-induced IL1β production in these cells was not abrogated with KN-62, which blocks only canonical inflammasome activation (Figure 2F). These findings suggest that in human macrophages, IL10 can suppress production of IL1β through non-canonical activation of the inflammasome, and that in the absence of this inhibitory signal, macrophages can produce IL1β in response to LPS alone. Our results highlight the central role of IL10 in regulating inflammasome-mediated IL1β production. We demonstrate that IL1 signaling in T cells drives colitis in the absence of IL10R. Mechanistically, we show that IL10 regulates NLRP3 both through transcriptional regulation as well as proteasomal degradation, at least in part through K48-linked polyubiquitination. The murine data are consistent with recent reports that normal IL10R signaling is required to suppress NLRP3-dependent inflammasome activation and pro-IL1β production, potentially through autocrine IL10 production.7Li B. et al.Nat Commun. 2015; 6: 6131Crossref Scopus (50) Google Scholar, 12Zhang J. et al.Mucosal Immunol. 2014; 7: 1139-1150Crossref PubMed Scopus (96) Google Scholar, 13Gurung P. et al.Sci Rep. 2015; 5: 14488Crossref PubMed Scopus (105) Google Scholar, 14Guarda G. Braun M. et al.Immunity. 2011; 34: 213-223Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar, 15Filardy A.A. et al.Mucosal Immunol. 2016; 9: 850-858Crossref Scopus (33) Google Scholar We further demonstrate that intact IL10R signaling is required to suppress IL1β production in human macrophages, but, in contrast to mice, loss of signaling is sufficient to trigger release of IL1β, even in the absence of a secondary inflammasome activation signal. This is likely mediated through non-canonical inflammasome activation of caspase-8. IL10- and IL10R-deficient patients fail to respond to immunosuppressive medications.2Kotlarz D. Beier R. Murugan D. et al.Gastroenterology. 2012; 143: 347-355Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 3Glocker E.O. Kotlarz D. Boztug K. et al.N Engl J Med. 2009; 361: 2033-2045Crossref PubMed Scopus (1139) Google Scholar Our data suggest that blocking IL1 in these patients may be beneficial as a bridge to an allogeneic hematopoietic stem cell transplantation or potentially longer if a suitable donor is unavailable. In both patients, treatment was well tolerated without side effects. Though not thoroughly studied, neutralizing IL1 therapies in inflammatory bowel disease have been shown to be effective in selected cases.16Ruiz Gomez A. et al.Pediatrics. 2012; 129: e535-e539Crossref PubMed Scopus (29) Google Scholar While we demonstrated successful outcome of anakinra therapy in 2 patients, a prior report of anakinra treatment in an IL10R-deficient patient suggested it was ineffective.17Moran C.J. et al.Inflamm Bowel Dis. 2013; 19: 115-123Crossref PubMed Scopus (201) Google Scholar Given the central role of IL10 in suppressing inflammasome-mediated IL1β production, additional clinical studies are needed to assess whether IL1 blockade can be used effectively in IL10/IL10R deficiency or more broadly in inflammatory bowel disease patients. Very Early Onset Inflammatory Bowel Disease International Consortium: Dror S. Shouval, Amlan Biswas, Alexandra E. Griffith, Liza Konnikova, Michael Field, Anthony Loizides, Batia Weiss, Baruch Yerushalmi, Tadahiro Yanagi, Aleixo M. Muise, Christoph Klein, Bruce H. Horwitz, Sarah C. Glover, and Athos Bousvaros. Download .pdf (2.73 MB) Help with pdf files Supplementary Data