Colonic tolerance develops in the iliac lymph nodes and can be established independent of CD103(+) dendritic cells.

Tolerance to harmless exogenous antigens is the default immune response in the gastrointestinal tract. Although extensive studies have demonstrated the importance of the mesenteric lymph nodes (MLNs) and intestinal CD103(+) dendritic cells (DCs) in driving small intestinal tolerance to protein antigen, the structural and immunological basis of colonic tolerance remain poorly understood. We show here that the caudal and iliac lymph nodes (ILNs) are inductive sites for distal colonic immune responses and that colonic T cell-mediated tolerance induction to protein antigen is initiated in these draining lymph nodes and not in MLNs. In agreement, colonic tolerance induction was not altered by mesenteric lymphadenectomy. Despite tolerance development, CD103(+)CD11b(+) DCs, which are the major migratory DC population in the MLNs, and the tolerance-related retinoic acid-generating enzyme RALDH2 were virtually absent from the ILNs. Administration of ovalbumin (OVA) to the distal colon did increase the number of CD11c(+)MHCII(hi) migratory CD103(-)CD11b(+) and CD103(+)CD11b(-) DCs in the ILNs. Strikingly, colonic tolerance was intact in Batf3-deficient mice specifically lacking CD103(+)CD11b(-) DCs, suggesting that CD103(-) DCs in the ILNs are sufficient to drive tolerance induction after protein antigen encounter in the distal colon. Altogether, we identify different inductive sites for small intestinal and colonic T-cell responses and reveal that distinct cellular mechanisms are operative to maintain tolerance at these sites.

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-1ß (proIL-1ß) 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-1ß proteins by the ubiquitin/proteasome system. Finally, blocking IL-10R-signaling in vivo enhanced NLRP3 and proIL-1ß 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-1ß 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(+)CD45RB(high) 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.

The presence of capsule in Cryptococcus neoformans influences the gene expression profile in dendritic cells during interaction with the fungus.

The aim of this investigation was to study the effect of polysaccharide capsule on the gene expression in dendritic cells (DC) during their interaction with Cryptococcus neoformans. To this end, we used an encapsulated virulent strain of C. neoformans and a cap59 gene-disrupted acapsular avirulent strain derived from the same genetic background. DC were exposed to encapsulated and acapsular C. neoformans strains for 4 h and 18 h, and their transcriptional profiles were analyzed using the Affymetrix mouse gene chip U74Av2. A large number of DC genes were up-regulated after treatment with the acapsular strain. In particular, we observed the up-regulation of the genes involved in DC maturation, such as cell surface receptors, cytokines, and chemokines (interleukin-12 [IL-12], IL-2, IL-1alpha, IL-1beta, IL-6, IL-10, tumor necrosis factor alpha, CCR7, CCL17, CCL22, CCL3, CCL4, CCL7, and CXCL10), membrane proteins, and the genes involved in antigen processing and presentation as well as cell cycle or apoptosis. The chemokine gene expression data were confirmed by real-time reverse transcription-PCR, while the expression of cytokine genes was correlated with their secretion. A completely different pattern of gene expression was observed for DC treated with an encapsulated strain of C. neoformans. In particular, no significant induction was observed in the expression of the genes mentioned above. Moreover, a number of genes, such as those coding for chemokines, were down-regulated. These results suggest that the polysaccharide capsule shrouding the cell wall of C. neoformans plays a fundamental role in inducing DC response, highlighting the molecular basis of the true nature of immune silencing exerted by capsular material.

Innate and adaptive mechanisms to control [corrected] pathological intestinal inflammation.

The intestine is the home of a tremendous number of commensal organisms that have a primary role in host metabolism. As a consequence, the gut mucosa has evolved multiple layers of protection. This review highlights both innate and adaptive mechanisms that prevent bacterial invasion and abnormal intestinal inflamamation, how a failure of these mechanisms may be involved in the pathogenesis of inflammatory bowel diseases, and discusses new findings implicating dendritic cells as central to the induction of active mucosal tolerance to commensal bacteria.

Activation of the formyl peptide receptor by the HIV-derived peptide T-20 suppresses interleukin-12 p70 production by human monocytes.

It has been proposed that in the early stages of human immunodeficiency (HIV) infection, before the loss of CD4(+) T cells, inhibition of IL-12 production from host antigen-presenting cells plays a critical role in the suppression of T-helper cell type 1 responses. Activation of the G(i)-protein-coupled high-affinity N-formyl peptide receptor by f-met-leu-phe and HIV-derived peptide T-20-suppressed IL-12 p70 production from human monocytes in response to both T-cell-dependent and T-cell-independent stimulation are reported. Activation of the low-affinity N-formyl peptide receptor by the HIV-derived F-peptide suppressed IL-12 production more modestly. This suppression was pertussis toxin sensitive and was selective for IL-12; the production of IL-10, transforming growth factor-beta, and tumor necrosis factor-alpha was unaltered. The production of IL-12 p70 by dendritic cells was unaffected by these peptides despite functional expression of the high-affinity fMLP receptor. These findings provide a potential direct mechanism for HIV-mediated suppression of IL-12 production and suggest a broader role for G-protein-coupled receptors in the regulation of innate immune responses. (Blood. 2001;97:3531-3536)

Unique functions of CD11b+, CD8 alpha+, and double-negative Peyer's patch dendritic cells.

We have recently demonstrated the presence of three populations of dendritic cells (DC) in the murine Peyer's patch. CD11b(+)/CD8alpha(-) (myeloid) DCs are localized in the subepithelial dome, CD11b(-)/CD8alpha(+) (lymphoid) DCs in the interfollicular regions, and CD11b(-)/CD8alpha(-) (double-negative; DN) DCs at both sites. We now describe the presence of a novel population of intraepithelial DN DCs within the follicle-associated epithelium and demonstrate a predominance of DN DCs only in mucosal lymphoid tissues. Furthermore, we demonstrate that all DC subpopulations maintain their surface phenotype upon maturation in vitro, and secrete a distinct pattern of cytokines upon exposure to T cell and microbial stimuli. Only myeloid DCs from the PP produce high levels of IL-10 upon stimulation with soluble CD40 ligand(-) trimer, or Staphylococcus aureus and IFN-gamma. In contrast, lymphoid and DN, but not myeloid DCs, produce IL-12p70 following microbial stimulation, whereas no DC subset produces IL-12p70 in response to CD40 ligand trimer. Finally, we show that myeloid DCs from the PP are particularly capable of priming naive T cells to secrete high levels of IL-4 and IL-10, when compared with those from nonmucosal sites, while lymphoid and DN DCs from all tissues prime for IFN-gamma production. These findings thus suggest that DC subsets within mucosal tissues have unique immune inductive capacities.

Regulation of interleukin-12 production by G-protein-coupled receptors.

G-protein-coupled receptors have long been known to play a critical role in the recruitment and migration of leukocytes to areas of inflammation. This review will focus, however, on emerging data that G-protein-coupled receptors can modulate cytokine production by antigen-presenting cells including interleukin-12 and tumor necrosis factor-alpha and may therefore play a significant role in the regulation of innate and acquired immunity.

Antigen-induced mucosal T cell activation is followed by Th1 T cell suppression in continuously fed ovalbumin TCR-transgenic mice.

We investigated kinetics and dose-dependent features of mucosal and peripheral immune responses following oral antigen application in a TCR-transgenic mouse model. Ovalbumin (OVA) TCR-transgenic mice were fed OVA at different doses (5-250 mg) and various frequencies (one to seven times, or continuous feeding). Low- and medium-dose (10, 100 mg) OVA feeding resulted in priming of immune responses, i.e. increased antigen-specific proliferation as well as IL-2, IL-4 and IFN-gamma secretion upon in vitro restimulation in Peyer's patches and spleen. Immune responses were suppressed with doses of one or three times 250 mg OVA feeding in the spleen. However, only the highest OVA feeding doses (7x250 mg OVA) or continuous feeding (5 mg daily in the drinking water over a 12-week period) actively suppressed immune responses and were associated with production of TGF-beta and IL-10 in the spleen and Peyer's patches. Thus, the cell population generated by continuous antigen feeding was characterized by production of suppressive cytokines and seems to be based on a counter-regulation with Th1 cytokines. These data further define the regulation of suppressive immune functions following antigen feeding in the periphery and the mucosal immune system.

Primary role for Gi protein signaling in the regulation of interleukin 12 production and the induction of T helper cell type 1 responses.

We explored the role of Gi protein signaling in the regulation of interleukin (IL)-12 production and T helper cell type 1 (Th1) T cell differentiation. In initial studies, we showed that treatment of normal mice with pertussis toxin (PT), which inhibits Gi protein signaling, enhanced the capacity of splenocytes to produce IL-12 in response to both microbial and nonmicrobial stimuli. In addition, PT treatment increased the production of tumor necrosis factor (TNF)-alpha and IL-10 by stimulated cells. These findings were corroborated by the fact that untreated Gi2alpha(2/-) mice exhibited enhanced production of IL-12 and TNF-alpha by splenocytes, and of IL-12 p40 by purified spleen CD8alpha(+) lymphoid dendritic cells. Finally, we showed that while normal BALB/c mice infected with Leishmania major exhibited a nonhealing phenotype, those treated with PT when infection was initiated exhibited a healing phenotype along with an enhancement of leishmania-specific Th1 responses in draining lymph nodes. Further, healing was prevented by coadministration of anti-IL-12 and PT. These data demonstrate that endogenous Gi protein signaling has a primary role in the regulation of IL-12 production and the induction of Th1 responses in vivo.

Localization of distinct Peyer's patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3alpha, MIP-3beta, and secondary lymphoid organ chemokine.

We describe the anatomical localization of three distinct dendritic cell (DC) subsets in the murine Peyer's patch (PP) and explore the role of chemokines in their recruitment. By two-color in situ immunofluorescence, CD11b(+) myeloid DCs were determined to be present in the subepithelial dome (SED) region, whereas CD8alpha(+) lymphoid DCs are present in the T cell-rich interfollicular region (IFR). DCs that lack expression of CD8alpha or CD11b (double negative) are present in both the SED and IFR. By in situ hybridization, macrophage inflammatory protein (MIP)-3alpha mRNA was dramatically expressed only by the follicle-associated epithelium overlying the SED, while its receptor, CCR6, was concentrated in the SED. In contrast, CCR7 was expressed predominantly in the IFR. Consistent with these findings, reverse transcriptase polymerase chain reaction analysis and in vitro chemotaxis assays using freshly isolated DCs revealed that CCR6 was functionally expressed only by DC subsets present in the SED, while all subsets expressed functional CCR7. Moreover, none of the splenic DC subsets migrated toward MIP-3alpha. These data support a distinct role for MIP-3alpha/CCR6 in recruitment of CD11b(+) DCs toward the mucosal surfaces and for MIP-3beta/CCR7 in attraction of CD8alpha(+) DCs to the T cell regions. Finally, we demonstrated that all DC subsets expressed an immature phenotype when freshly isolated and maintained expression of subset markers upon maturation in vitro. In contrast, CCR7 expression by myeloid PP DCs was enhanced with maturation in vitro. In addition, this subset disappeared from the SED and appeared in the IFR after microbial stimulation in vivo, suggesting that immature myeloid SED DCs capture antigens and migrate to IFR to initiate T cell responses after mucosal microbial infections.

Selective suppression of IL-12 production by chemoattractants.

We investigated the ability of chemoattractants to affect IL-12 production by human monocytes and dendritic cells. We found that pretreatment of monocytes with macrophage chemoattractant proteins (MCP-1 to -4), or C5a, but not stromal-derived factor-1, macrophage inflammatory protein-1alpha, RANTES, or eotaxin, inhibited IL-12 p70 production in response to stimulation with Staphylococcus aureus, Cowan strain 1 (SAC), and IFN-gamma. The production of TNF-alpha and IL-10, however, was minimally affected by any of the chemoattractants. The degree of inhibition of IL-12 p70 production by MCP-1 to -4 was donor dependent and was affected by the autocrine inhibitory effects of IL-10. In contrast, C5a profoundly suppressed IL-12 production in an IL-10-independent fashion. Neither TGF-beta1 nor PGE2 was important for the suppression of IL-12 by any of the chemoattractants tested. The accumulation of mRNA for both IL-12 p35 and p40 genes was inhibited by chemokine pretreatment. Interestingly, MCP-1 to -4 and C5a did not suppress IL-12 production by monocyte-derived dendritic cells (DC) stimulated with CD40 ligand and IFN-gamma or by SAC and IFN-gamma, suggesting that these factors may act at the site of inflammation to suppress IL-12 and IFN-gamma production rather than in the lymph node to affect T cell priming. Despite the inability of C5a to inhibit IL-12 production by DCs, the receptor for C5a (CD88) was expressed by these cells, and recombinant C5a induced a Ca2+ flux. Taken together, these results define a range of chemoattractant molecules with the ability to suppress IL-12 production by human monocytes and have broad implications for the regulation of immune responses in vivo.

Restoration of lymphoid populations in a murine model of X-linked severe combined immunodeficiency by a gene-therapy approach.

X-linked severe combined immunodeficiency (XSCID) is a life-threatening syndrome in which both cellular and humoral immunity are profoundly compromised. This disease results from mutations in the IL2RG gene, which encodes the common cytokine receptor gamma chain, gamma(c). Previously, we generated gamma(c)-deficient mice as a murine model of XSCID. We have now used lethally irradiated gamma(c)-deficient mice to evaluate a gene therapeutic approach for treatment of this disease. Transfer of the human gamma(c) gene to repopulating hematopoietic stem cells using an ecotropic retrovirus resulted in an increase in T cells, B cells, natural killer (NK) cells, and intestinal intraepithelial lymphocytes, as well as normalization of the CD4:CD8 T-cell ratio and of serum Ig levels. In addition, the restored cells could proliferate in response to interleukin-2 (IL-2). Thus, our results provide added support that gene therapy is a feasible therapeutic strategy for XSCID. Moreover, because we used a vector directing expression of human gamma(c) to correct a defect in gamma(c)-deficient mice, these data also indicate that human gamma(c) can cooperate with the distinctive cytokine receptor chains such as IL-2Rbeta and IL-7Ralpha to mediate responses to murine cytokines in vivo.

Freshly isolated Peyer's patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells.

Orally administered antigens often generate immune responses that are distinct from those injected systemically. The role of antigen-presenting cells in determining the type of T helper cell response induced at mucosal versus systemic sites is unclear. Here we examine the phenotypic and functional differences between dendritic cells (DCs) freshly isolated from Peyer's patches (PP) and spleen (SP). Surface phenotypic analysis of CD11c(+) DC populations revealed that PP DCs expressed higher levels of major histocompatibility complex class II molecules, but similar levels of costimulatory molecules and adhesion molecules compared with SP DCs. Freshly isolated, flow cytometrically sorted 98-100% pure CD11c(+) DC populations from PP and SP were compared for their ability to stimulate naive T cells. First, PP DCs were found to be much more potent in stimulating allogeneic T cell proliferation compared with SP DCs. Second, by using naive T cells from ovalbumin peptide-specific T cell receptor transgenic mice, these ex vivo DCs derived from PP, but not from SP, were found to prime for the production of interleukin (IL)-4 and IL-10 (Th2 cytokines). In addition, PP DCs were found to prime T cells for the production of much lower levels of interferon (IFN)-gamma (Th1) compared with SP DCs. The presence of neutralizing antibody against IL-10 in the priming culture dramatically enhanced IFN-gamma production by T cells stimulated with PP DCs. Furthermore, stimulation of freshly isolated PP DCs via the CD40 molecule resulted in secretion of high levels of IL-10, whereas the same stimulus induced no IL-10 secretion from SP DCs. These results suggest that DCs residing in different tissues are capable of inducing distinct immune responses and that this may be related to the distinct cytokines produced by the DCs from these tissues.

Extinction of IL-12 signaling promotes Fas-mediated apoptosis of antigen-specific T cells.

In previous studies we have shown that peripheral tolerance achieved by high dose feeding of OVA to intact OVA-TCR transgenic mice was enhanced when endogenous IL-12 was neutralized simultaneously. To generalize this phenomenon, in the present study we investigated the tolerogenic mechanisms underlying the blockade of IL-12 signaling following oral and systemic Ag delivery. We found that the numbers of Ag-specific T cells in several lymphoid organs were significantly reduced due to T cell apoptosis following oral OVA or systemic OVA administration when combined with anti-IL-12 injection, but there was no decrease in T cell numbers for OVA-fed, OVA-injected, or anti-IL-12 alone-treated mice compared with those in untreated control mice. In addition, mostly Fas+ T cells were subject to apoptotic deletion in the OVA- plus anti-IL-12-treated groups, and an enhanced cell death of T cells upon OVA restimulation in vitro could be partially reversed by blockade of the Fas/Fas ligand interaction. Finally, in a murine model of superantigen-driven T cell expansion and deletion, we observed no deletional effects of anti-IL-12 treatment on CD4+ cells in Fas-deficient (MRL/lpr) mice, but did find these effects in MRL wild-type mice. In summary, our data suggest that in the course of Ag-induced cell proliferation of Th1 cells, signaling through IL-12 is required to prevent an induction of Fas-mediated apoptosis. Thus, the use of anti-IL-12 may be potentially useful in modulating peripheral immune responses by promotion of Fas-mediated cell death.

Mucosal immunity and inflammation. I. Mucosal dendritic cells: their specialized role in initiating T cell responses.

Dendritic cells (DCs) are the most competent antigen-presenting cells known for the induction of primary T cell responses. Functional studies of tissue-resident DCs have been impaired by the rarity of these cells in any given organ. Recent development of isolation procedures allowing extraction of highly purified fresh DC populations has made it possible to study mucosal DCs in distinct mucosa-associated lymphoid tissues. Here, we discuss several recent studies by us and others that describe the tissue-specific phenotype and function of mucosal DCs and speculate on the mechanism by which the resident DCs regulate tissue-specific T cell responses.

[Mechanisms and applications of oral tolerance]. / Mechanismen und Anwendungen oraler Toleranz.

The term oral tolerance (OT) describes the antigen-specific suppression of immune responses following the feeding of the antigen. While some common features with other forms of induction of systemic tolerance have been disclosed, OT can be distinguished by certain immunologic characteristics. Thus, work in experimental animal models revealed the importance of intestinal antigen processing, especially antigen processing in the Peyer's patches, in inducing OT. It has become clear that suppressive T cell cytokines derived from mucosal sites play a major role in mediating OT. The variation of the dose of fed antigen and the modulation of the cytokine milieu both have influences on the underlying immunologic mechanisms active suppression, clonal anergy and clonal deletion following oral antigen uptake. In several animal models of autoimmunity the disease activity can be suppressed by feeding oral autoantigen. Based on these results, recent clinical studies have begun to explore OT as a means to treat autoimmune disorders such as multiple sclerosis, rheumatoid arthritis and diabetes.

Cholera toxin suppresses interleukin (IL)-12 production and IL-12 receptor beta1 and beta2 chain expression.

Cholera toxin (CT) is a potent mucosal vaccine adjuvant, which has been shown to induce T helper cell type 2 (Th2) responses in systemic and mucosal tissues. We report that CT inhibits the production of interleukin (IL)-12, a major Th2 counterregulatory cytokine. IL-12 p70 production by stimulated human monocytes was inhibited by CT in a dose-dependent manner. This suppression occurred at the level of gene transcription, was maximal at low concentrations of CT, and was dependent on the A subunit of the toxin, since purified CT B subunit had minimal effect. CT also inhibited the production of IL-12 p70 by monocyte-derived dendritic cells, as well as the production of tumor necrosis factor alpha, but not IL-10, IL-6, or transforming growth factor (TGF)-beta1, by stimulated monocytes. The effects of CT were not due to autocrine production of IL-10, TGF-beta1, or prostaglandin E2. CT inhibited the production of IFN-gamma by anti-CD3-stimulated human peripheral blood mononuclear cell, due in part to suppression of IL-12 production, but also to the inhibition of expression of the beta1 and beta2 chains of the IL-12 receptor on T cells. In vivo, mice given CT before systemic challenge with lipopolysaccharide had markedly reduced serum levels of IL-12 p40 and interferon gamma. These data demonstrate two novel mechanisms by which CT can inhibit Th1 immune responses, and help explain the ability of mucosally administered CT to enhance Th2-dependent immune responses.