Jianping He

Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon

Blood monocytes differentiate into distinct colonic macrophage or dendritic cell subsets depending on the presence or absence of inflammation

Critical Role of STAT5 Transcription Factor Tetramerization for Cytokine Responses and Normal Immune Function

Cytokine-activated STAT proteins dimerize and bind to high-affinity motifs, and N-terminal domain-mediated oligomerization of dimers allows tetramer formation and binding to low-affinity tandem motifs, but the functions of dimers versus tetramers are unknown. We generated Stat5a-Stat5b double knockin (DKI) N-domain mutant mice in which STAT5 proteins form dimers but not tetramers, identified cytokine-regulated genes whose expression required STAT5 tetramers, and defined dimer versus tetramer consensus motifs. Whereas Stat5-deficient mice exhibited perinatal lethality, DKI mice were viable; thus, STAT5 dimers were sufficient for survival. Nevertheless, STAT5 DKI mice had fewer CD4(+)CD25(+) T cells, NK cells, and CD8(+) T cells, with impaired cytokine-induced and homeostatic proliferation of CD8(+) T cells. Moreover, DKI CD8(+) T cell proliferation after viral infection was diminished and DKI Treg cells did not efficiently control colitis. Thus, tetramerization of STAT5 is critical for cytokine responses and normal immune function, establishing a critical role for STAT5 tetramerization in vivo.

The Cytokines IL-21 and GM-CSF Have Opposing Regulatory Roles in the Apoptosis of Conventional Dendritic Cells

Interleukin-21 (IL-21) has broad actions on T and B cells, but its actions in innate immunity are poorly understood. Here we show that IL-21 induced apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3. Expression of IL-21 in vivo decreased cDC numbers, and this was prevented by GM-CSF. Moreover, repetitive α-galactosylceramide injection of mice induced IL-21 but decreased GM-CSF production by natural killer T (NKT) cells, correlating with decreased cDC numbers. Furthermore, adoptive transfer of wild-type CD4+ T cells caused more severe colitis with increased DCs and interferon-γ (IFN-γ)-producing CD4+ T cells in Il21r(-/-)Rag2(-/-) mice (which lack T cells and have IL-21-unresponsive DCs) than in Rag2(-/-) mice. Thus, IL-21 and GM-CSF exhibit cross-regulatory actions on gene regulation and apoptosis, regulating cDC numbers and thereby the magnitude of the immune response.

Type I interferons produced by hematopoietic cells protect mice against lethal infection by mammalian reovirus

We defined the function of type I interferons (IFNs) in defense against reovirus strain type 1 Lang (T1L), which is a double-stranded RNA virus that infects Peyers patches (PPs) after peroral inoculation of mice. T1L induced expression of mRNA for IFN-α, IFN-β, and Mx-1 in PPs and caused localized intestinal infection that was cleared in 10 d. In contrast, T1L produced fatal systemic infection in IFNαR1 knockout (KO) mice with extensive cell loss in lymphoid tissues and necrosis of the intestinal mucosa. Studies of bone-marrow chimeric mice indicated an essential role for hematopoietic cells in IFN-dependent viral clearance. Dendritic cells (DCs), including conventional DCs (cDCs), were the major source of type I IFNs in PPs of reovirus-infected mice, whereas all cell types expressed the antiviral protein Mx-1. Neither NK cells nor signaling via Toll-like receptor 3 or MyD88 were essential for viral clearance. These data demonstrate a requirement for type I IFNs in the control of an intestinal viral infection and indicate that cDCs are a significant source of type I IFN production in vivo. Therefore, innate immunity in PPs is an essential component of host defense that limits systemic spread of pathogens that infect the intestinal mucosa.

Cholera toxin inhibits IL-12 production and CD8α+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function

Prior studies have demonstrated that cholera toxin (CT) and other cAMP-inducing factors inhibit interleukin (IL)-12 production from monocytes and dendritic cells (DCs). We show that CT inhibits Th1 responses in vivo in mice infected with Toxoplasma gondii. This correlated with low serum IL-12 levels and a selective reduction in the numbers of CD8α+ conventional DCs (cDCs) in lymphoid organs. CT inhibited the function of interferon (IFN) regulatory factor (IRF) 8, a transcription factor known to positively regulate IL-12p35 and p40 gene expression, and the differentiation of CD8α+ and plasmacytoid DCs (pDCs). Fluorescence recovery after photobleaching analysis showed that exposure to CT, forskolin, or dibutyryl (db) cAMP blocked LPS and IFN-γ–induced IRF8 binding to chromatin. Moreover, CT and dbcAMP inhibited the binding of IRF8 to the IFN-stimulated response element (ISRE)–like element in the mouse IL-12p40 promoter, likely by blocking the formation of ISRE-binding IRF1–IRF8 heterocomplexes. Furthermore, CT inhibited the differentiation of pDCs from fms-like tyrosine kinase 3 ligand–treated bone marrow cells in vitro. Therefore, because IRF8 is essential for IL-12 production and the differentiation of CD8α+ cDCs and pDCs, these data suggest that CT and other Gs-protein agonists can affect IL-12 production and DC differentiation via a common mechanism involving IRF8.

Type I IFNs Regulate Effector and Regulatory T Cell Accumulation and Anti-Inflammatory Cytokine Production during T Cell–Mediated Colitis

We explored the function of endogenous type I IFNs (IFN-1) in the colon using the T cell adoptive transfer model of colitis. Colon mononuclear phagocytes (MPs) constitutively produced IFN-1 in a Toll/IL-1R domain–containing adapter-inducing IFN-β–dependent manner. Transfer of CD4+CD45RBhi T cells from wild-type (WT) or IFN-α/β receptor subunit 1 knockout (IFNAR1−/−) mice into RAG−/− hosts resulted in similar onset and severity of colitis. In contrast, RAG−/− × IFNAR1−/− double knockout (DKO) mice developed accelerated severe colitis compared with RAG−/− hosts when transferred with WT CD4+CD45RBhi T cells. IFNAR signaling on host hematopoietic cells was required to delay colitis development. MPs isolated from the colon lamina propria of IFNAR1−/− mice produced less IL-10, IL-1R antagonist, and IL-27 compared with WT MPs. Accelerated colitis development in DKO mice was characterized by early T cell proliferation and accumulation of CD11b+CD103− dendritic cells in the mesenteric lymph nodes, both of which could be reversed by systemic administration of IL-1R antagonist (anakinra). Cotransfer of CD4+CD25+ regulatory T cells (Tregs) from WT or IFNAR1−/− mice prevented disease caused by CD4+CD45RBhi T cells. However, WT CD4+CD25+Foxp3GFP+ Tregs cotransferred with CD4+CD45RBhi T cells into DKO hosts failed to expand or maintain Foxp3 expression and gained effector functions in the colon. To our knowledge, these data are the first to demonstrate an essential role for IFN-1 in the production of anti-inflammatory cytokines by gut MPs and the indirect maintenance of intestinal T cell homeostasis by both limiting effector T cell expansion and promoting Treg stability.

Involvement of Dendritic Cell Subsets in the Induction of Oral Tolerance and Immunity

Abstract: Dendritic cells (DCs) play a central role in the generation of immune responses in the intestine. DCs induce differentiation and tolerance of T cells, and may have a direct role in B cell switching to IgA. Four distinct subsets of CD11c+ DCs are present in murine Peyers patches, which represent primary sites for the induction of mucosal T and B cell responses. Studies suggest that CD11b+ DCs or plasmacytoid DCs may be specialized for the induction of regulatory T cells, and CD8α+ DCs for the induction of clonal deletion in response to soluble oral antigen, while all DC subsets (including CD8α−/CD11b− DCs) may be involved in responses to pathogens. We are currently using reovirus type‐1 Lang (TIL) to explore the role of DC populations in mucosal immunity in vivo, as oral administration of live T1L to mice induces strong mucosal and systemic antiviral immune responses, whereas oral administration of inactivated T1L results in tolerance to viral proteins. We found that primary infection with T1L occurs in epithelial cells of the PP follicle‐associated epithelium, but that CD8α−/CD11b− DCs in the subepithelial dome region (SED) are loaded with T1L antigens in the absence of active DC infection. At least a portion of this antigen is associated with cell fragments from apoptotic epithelial cells, demonstrating that SED DCs cross‐present antigens from apoptotic epithelial cells. In vitro, in contrast to exposure to several TLR‐ligands or anti‐CD40, exposure to T1L does not activate DCs to mature or to produce cytokines, despite clear loading of the DCs with viral antigens. These data suggest that T1L is taken up by a “silent” receptor on DCs, and that the induction of immunity to T1L is dependent on signals from non‐DCs following active viral infection that induce DC maturation. Thus, the decision between tolerance and immunity to inactive and live virus, respectively, likely depends on whether there is active infection of epithelial cells by T1L, which results in the elaboration of molecules, such as cytokines, that induce DC maturation.

Posttranscriptional control of NLRP3 inflammasome activation in colonic macrophages

Colonic macrophages (cMPs) are important for intestinal homeostasis as they kill microbes and yet produce regulatory cytokines. Activity of the NLRP3 (nucleotide-binding leucine-rich repeat-containing pyrin receptor 3) inflammasome, a major sensor of stress and microorganisms that results in pro-inflammatory cytokine production and cell death, must be tightly controlled in the intestine. We demonstrate that resident cMPs are hyporesponsive to NLRP3 inflammasome activation owing to a remarkable level of posttranscriptional control of NLRP3 and pro-interleukin-1β (proIL-1β) protein expression, which was also seen for tumor necrosis factor-α and IL-6, but lost during experimental colitis. Resident cMPs rapidly degraded NLRP3 and proIL-1β proteins by the ubiquitin/proteasome system. Finally, blocking IL-10R-signaling in vivo enhanced NLRP3 and proIL-1β protein but not mRNA levels in resident cMPs, implicating a role for IL-10 in environmental conditioning of cMPs. These data are the first to show dramatic posttranscriptional control of inflammatory cytokine production by a relevant tissue-derived macrophage population and proteasomal degradation of proIL-1β and NLRP3 as a mechanism to control inflammasome activation, findings which have broad implications for our understanding of intestinal and systemic inflammatory diseases.

Host-dependent control of early regulatory and effector T cell differentiation underlies the genetic susceptibility of RAG2-deficient mouse strains to transfer colitis

De novo differentiation of CD4+Foxp3+ regulatory T cells (induced (i) Tregs) occurs preferentially in the gut-associated lymphoid tissues (GALT). We addressed the contribution of background genetic factors in affecting the balance of iTreg, T helper type 1 (Th1), and Th17 cell differentiation in GALT in vivo following the transfer of naive CD4+CD45RBhigh T cells to strains of RAG2-deficient mice with differential susceptibility to inflammatory colitis. iTregs represented up to 5% of CD4+ T cells in mesenteric lymph nodes of less-susceptible C57BL/6 RAG2−/− mice compared with <1% in highly susceptible C57BL/10 RAG2−/− mice 2 weeks following T-cell transfer before the onset of colitis. Early Treg induction was correlated inversely with effector cell expansion and the severity of colitis development, was controlled primarily by host and not T-cell-dependent factors, and was strongly associated with interleukin-12 (IL-12)/23 production by host CD11c+CD103+ dendritic cells. These data highlight the importance of genetic factors regulating IL-12/23 production in controlling the balance between iTreg differentiation and effector-pathogenic CD4+ T-cell expansion in lymphopenic mice and indicate a direct role for iTregs in the regulation of colonic inflammation in vivo.