Angela Schippers

Intestinal Tolerance Requires Gut Homing and Expansion of FoxP3+ Regulatory T Cells in the Lamina Propria

Tolerance to food antigen manifests in the absence and/or suppression of antigen-specific immune responses locally in the gut but also systemically, a phenomenon known as oral tolerance. Oral tolerance is thought to originate in the gut-draining lymph nodes, which support the generation of FoxP3(+) regulatory T (Treg) cells. Here we use several mouse models to show that Treg cells, after their generation in lymph nodes, need to home to the gut to undergo local expansion to install oral tolerance. Proliferation of Treg cells in the intestine and production of interleukin-10 by gut-resident macrophages was blunted in mice deficient in the chemokine (C-X3-C motif) receptor 1 (CX3CR1). We propose a model of stepwise oral tolerance induction comprising the generation of Treg cells in the gut-draining lymph nodes, followed by migration into the gut and subsequent expansion of Treg cells driven by intestinal macrophages.

Monocytes/macrophages and/or neutrophils are the target of IL-10 in the LPS endotoxemia model

IL‐10 is a potent regulator of the innate and adaptive immune responses. Several cell types produce IL‐10 and its receptor chains and these may regulate different immune responses. Here we report that inactivation of the IL‐10 receptor (IL‐10R1) gene in mice leads to an increased susceptibility to chemically induced colitis as in the classical IL‐10‐deficient mutant. To identify the cells regulated by IL‐10 in immune responses, we generated several cell type specific IL‐10R1‐deficient mutants. We show that, in an IL‐10‐dependent LPS model of endotoxemia, dampening of the immune response requires expression of IL‐10R1 in monocytes/macrophages and/or neutrophils but not in T cells nor B cells. As the macrophage and/or neutrophil‐specific IL‐10‐deficient mutants also display the same phenotype, our results suggest that an autocrine loop in monocytes/macrophages is the most probable mechanism for the regulation of an LPS‐induced septic shock. In contrast, in an IL‐10‐regulated T‐cell response to Trichuris muris infection, IL‐10 acting on T cells or monocytes/macrophages/neutrophils is not critical for the control of the infection.

Sphingosine-1 Phosphate Signaling Regulates Positioning of Dendritic Cells within the Spleen

A successful execution and balance of adaptive immune responses requires a controlled positioning and navigation of dendritic cells (DC) into and inside secondary lymphoid organs. Whereas mechanisms were identified governing the migration of DC from peripheral nonlymphoid organs into their draining lymph nodes, little is known about the molecular cues controlling the proper positioning of spleen or lymph node resident DC. In this study, we show that the sphingosine-1 phosphate (S1P) receptor 1 influences the positioning of immature DC inside the murine spleen. Following treatment with FTY720 or SEW2871, drugs known to interfere with S1P1-mediated signaling, the 33D1+ DC subpopulation homogeneously redistributes from the bridging channels to the marginal zone. In contrast, the CD205+ DC subset remains associated with the T cell zone. Upon in vivo LPS treatment, the maturing DC assemble in the T cell zone. The LPS-driven redistribution occurs in the absence of CCR7 and cannot be prevented by FTY720, indicating that guiding mechanisms differ between immature and mature DC. Along with the observed DC subtype-specific S1P receptor expression pattern as well as the profound up-regulation of S1P1 and S1P3 accompanying DC maturation, these results suggest a decisive contribution of S1P signaling to intrasplenic DC motility and migration.

Differential Molecular and Anatomical Basis for B Cell Migration into the Peritoneal Cavity and Omental Milky Spots

The constitutive migration of B cells from the circulation into the peritoneal cavity and back is essential for peritoneal B cell homeostasis and function. However, the molecular machinery and the anatomical basis for these migratory processes have hardly been investigated. In this study, we analyze the role of integrins as well as the role of the omentum for B2 cell migration into and out of the peritoneal cavity of mice. We demonstrate that α4β7 integrin-mucosal addressin cell adhesion molecule 1 interaction enables B2 cell migration from the circulation into omental milky spots but not into the peritoneum. In contrast, α4β1 integrin mediates direct entry of B2 cells into the peritoneal cavity as well as their retention at that site, limiting B2 cell egress via the draining parathymic lymph nodes. Surgical removal of the omentum results in a 40% reduced immigration of B2 cells from the circulation into the peritoneum but does not impair B cell exit from this compartment. In conclusion, these data reveal the existence of alternative routes for B2 cell entry into the peritoneal cavity and identify integrins as key factors for peritoneal B2 cell homeostasis, mediating B2 cell migration into and out of the peritoneal cavity as well as their retention at this site.

Commensal gut flora reduces susceptibility to experimentally induced colitis via T-cell-derived interleukin-10.

Background: Regulatory cytokines are well known to modify experimental colitis in mice. The aim of this study was to elucidate the effect of interleukin (IL)‐10 derived from different cellular sources and the effect of commensal gut flora in dextran sulfate sodium (DSS)‐induced colitis in mice. Methods: Wildtype (WT) and IL‐10 deficient (IL‐10−/−) mice either harboring a characterized specific pathogen‐free (SPF) gut flora or germfree were exposed to 2% DSS. Moreover, cell type‐specific IL‐10, IL‐4, and IL‐12 knockout mice and animals combining the T‐cell‐specific IL‐10 knockout with a deficiency in IL‐12 or IL‐4 were exposed to DSS. Results: SPF IL‐10−/− mice showed an increased susceptibility to DSS‐induced colitis compared to WT mice determined by histopathology and proinflammatory cytokine and chemokine responses. Under germfree conditions, both WT and IL‐10−/− mice were highly susceptible to DSS. IL‐10 mRNA was increased upon DSS exposure in WT SPF but not in germfree mice. Mice carrying a specific deletion of IL‐10 in T‐cells exhibited a tendency towards an enhanced susceptibility to DSS. The lack of T‐cell‐derived IL‐10 in combination with the lack of IL‐4 increased the susceptibility to DSS colitis, as did the lack of IL‐12 alone. Conclusions: IL‐10 is a crucial factor inhibiting the innate proinflammatory immune response induced by DSS. Intestinal bacteria are necessary for the induction of protective IL‐10, which is mainly T‐cell‐derived. T‐cell‐derived IL‐10 can only mediate its protective effect in a Th1‐dominated milieu. If the balance is shifted towards a Th2 response, IL‐10 is not protective. (Inflamm Bowel Dis 2011;)

Serum Response Factor Contributes Selectively to Lymphocyte Development

Serum response factor (SRF), is a crucial transcription factor for murine embryonic development and for the function of muscle cells and neurons. Gene expression data show that SRF and its transcriptional cofactors are also expressed in lymphocyte precursors and mature lymphocytes. However, the role of SRF in lymphocyte development has not been addressed in vivo so far, attributed in part to early embryonic lethality of conventional Srf-null mice. To determine the in vivo role of SRF in developing lymphocytes, we specifically inactivated the murine Srf gene during T or B cell development using lymphocyte-specific Cre transgenic mouse lines. T cell-specific Srf deletion led to a severe block in thymocyte development at the transition from CD4/CD8 double to single positive stage. The few residual T cells detectable in the periphery retained at least one functional Srf allele, thereby demonstrating the importance of SRF in T cell development. In contrast, deletion of Srf in developing B cells did not interfere with the growth and survival of B cells in general, yet led to a complete loss of marginal zone B cells and a marked reduction of the CD5+ B cell subset. Our study also revealed a contribution of SRF to the expression of the surface molecules IgM, CD19, and the chemokine receptor 4 in B lymphocytes. We conclude that SRF fulfills essential and distinct functions in the differentiation of different types of lymphocytes.

Mucosal Addressin Cell-Adhesion Molecule-1 Controls Plasma-Cell Migration and Function in the Small Intestine of Mice

BACKGROUND & AIMS Immunoglobulin (Ig) A secretion into the intestinal lumen is an important immune mechanism of the gastrointestinal (GI) tract. B cells proliferate and differentiate into IgA-secreting plasma cells (PC) within lymphoid organs then migrate directly into the intestinal lamina propria. We aimed to elucidate the in vivo role of the mucosal addressin cell-adhesion molecule-1 (MAdCAM-1), which is constitutively expressed in the GI-associated lymphoid tissue, in B-cell migration. METHODS We generated MAdCAM-1-deficient mice (MAdCAM(Delta)) and evaluated the B-cell compartment of the GI-associated lymphoid tissue. We also assessed PC migration to the small intestine and the intestinal immune response after oral immunization. RESULTS In MAdCAM(Delta) mice, the size of Peyers patches was drastically reduced, compared with that of wild-type mice; this difference was detectable by 3 days after birth, indicating that MAdCAM-1 is dispensable for embryonic Peyers patch development but mediates recruitment of lymphocytes into this lymphoid organ at later stages. Moreover, antigen-specific, IgA-positive PC were severely compromised in their migration to the small intestine; accordingly, there was a reduced number of IgA-secreting PC in the lamina propria of the small intestine. The MAdCAM(Delta) mice were unable to mount a normal intestinal IgA response after oral immunization with cholera toxin. CONCLUSION These data provide in vivo evidence that MAdCAM-1 is required for the localization and function of IgA-secreting PC in the intestine.

Enhanced FTY720-mediated lymphocyte homing requires G alpha i signaling and depends on beta 2 and beta 7 integrin.

The immunomodulatory drug FTY720 interferes with sphingosine-1-phosphate (S1P) receptor signaling leading to lymphocyte retention in secondary lymphoid organs and consequently to profound lymphopenia in the peripheral blood. The molecular mechanisms transduced by S1P receptors upon being triggered by its native ligand, S1P, or by FTY720, are largely unknown. In this study we analyze the role of β2 and β7 integrin and their ligands ICAM-1, VCAM-1, and MadCAM-1 on lymphocyte homing in the presence of FTY720. We demonstrate that this drug facilitates homing of lymphocytes single-deficient of either β2 or β7 integrin but not of β2-deficient lymphocytes, which in addition were blocked by anti-β7 integrin Abs. Enhanced lymphocyte homing is preceded by increased adherence of integrin-deficient as well as wild-type lymphocytes to high endothelial venules (HEV) in FTY720-treated animals. Elevated adherence to HEV requires intact lymphocyte Gαi signaling that cannot be stably imprinted on lymphocytes even after prolonged exposure to FTY720. Thus, FTY720 influences lymphocyte homeostasis not only by suppressing lymphocyte egress from lymph nodes but also by facilitating lymphocyte homing across HEV in an integrin-dependent fashion.

Interleukin-2 treatment reverses effects of cAMP-responsive element modulator α-over-expressing T cells in autoimmune-prone mice.

Systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), are often characterized by a failure of self‐tolerance and result in an uncontrolled activation of B cells and effector T cells. Interleukin (IL)‐2 critically maintains homeostasis of regulatory T cells (Treg) and effector T cells in the periphery. Previously, we identified the cAMP‐responsive element modulator α (CREMα) as a major factor responsible for decreased IL‐2 production in T cells from SLE patients. Additionally, using a transgenic mouse that specifically over‐expresses CREMα in T cells (CD2CREMαtg), we provided in‐vivo evidence that CREMα indeed suppresses IL‐2 production. To analyse the effects of CREMα in an autoimmune prone mouse model we introduced a Fas mutation in the CD2CREMαtg mice (FVB/Fas–/–CD2CREMαtg). Overexpression of CREMα strongly accelerated the lymphadenopathy and splenomegaly in the FVB/Fas–/– mice. This was accompanied by a massive expansion of double‐negative (DN) T cells, enhanced numbers of interferon (IFN)‐γ‐producing T cells and reduced percentages of Tregs. Treatment of FVB/Fas–/–CD2CREMαtg mice with IL‐2 restored the percentage of Tregs and reversed increased IFN‐γ production, but did not affect the number of DNTs. Our data indicate that CREMα contributes to the failure of tolerance in SLE by favouring effector T cells and decreasing regulatory T cells, partially mediated by repression of IL‐2 in vivo.

Localization of dendritic cells in the gut epithelium requires MAdCAM-1

Directed migration of immune cells is a prerequisite for immune responses. T and B cell migration to the gut is secured by interaction of mucosal addressin cell-adhesion molecule-1 (MAdCAM-1) and β7 integrin. Here we report a novel function for MAdCAM-1: that of mediating intestinal localization of dendritic cells (DCs). In homeostasis, both MAdCAM-1-deficient and β7 integrin-deficient mice exhibit a reduced frequency of CD11c(+) cells, including CD103(+) DCs and plasmacytoid DCs (pDCs), in the gut epithelium. Deficiency of either MAdCAM-1 or β7 integrin reduces the migration efficiency of pDCs into the intestinal intraepithelial (IE) compartment. Both mouse strains display a decreased migration efficiency of precursors for conventional DCs (cDCs), from the circulation into the epithelium. By contrast, the migration of activated DCs from the small intestine to MLN is unchanged in MAdCAM-1-deficient mice. These findings suggest that MAdCAM-1 is important for the β7 integrin-dependent intestinal localization of both cDCs and pDCs.