Rosa Varona

Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis.

Intestinal homeostasis is critical for efficient energy extraction from food and protection from pathogens. Its disruption can lead to an array of severe illnesses with major impacts on public health, such as inflammatory bowel disease characterized by self-destructive intestinal immunity. However, the mechanisms regulating the equilibrium between the large bacterial flora and the immune system remain unclear. Intestinal lymphoid tissues generate flora-reactive IgA-producing B cells, and include Peyers patches and mesenteric lymph nodes, as well as numerous isolated lymphoid follicles (ILFs). Here we show that peptidoglycan from Gram-negative bacteria is necessary and sufficient to induce the genesis of ILFs in mice through recognition by the NOD1 (nucleotide-binding oligomerization domain containing 1) innate receptor in epithelial cells, and β-defensin 3- and CCL20-mediated signalling through the chemokine receptor CCR6. Maturation of ILFs into large B-cell clusters requires subsequent detection of bacteria by toll-like receptors. In the absence of ILFs, the composition of the intestinal bacterial community is profoundly altered. Our results demonstrate that intestinal bacterial commensals and the immune system communicate through an innate detection system to generate adaptive lymphoid tissues and maintain intestinal homeostasis.

In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells

The nuclear hormone receptor retinoic acid receptor–related orphan receptor γt (RORγt) is required for the generation of T helper 17 cells expressing the proinflammatory cytokine interleukin (IL)-17. In vivo, however, less than half of RORγt+ T cells express IL-17. We report here that RORγt+ Tαβ cells include Foxp3+ cells that coexist with IL-17–producing RORγt+ Tαβ cells in all tissues examined. The Foxp3+ RORγt+ Tαβ express IL-10 and CCL20, and function as regulatory T cells. Furthermore, the ratio of Foxp3+ to IL-17–producing RORγt+ Tαβ cells remains remarkably constant in mice enduring infection and inflammation. This equilibrium is tuned in favor of IL-10 production by Foxp3 and CCL20, and in favor of IL-17 production by IL-6 and IL-23. In the lung and skin, the largest population of RORγt+ T cells express the γδ T cell receptor and produce the highest levels of IL-17 independently of IL-6. Thus, potentially antagonistic proinflammatory IL-17–producing and regulatory Foxp3+ RORγt+ T cells coexist and are tightly controlled, suggesting that a perturbed equilibrium in RORγt+ T cells might lead to decreased immunoreactivity or, in contrast, to pathological inflammation.

CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses

CCR6 expression in dendritic, T, and B cells suggests that this beta-chemokine receptor may regulate the migration and recruitment of antigen-presenting and immunocompetent cells during inflammatory and immunological responses. Here we demonstrate that CCR6-/- mice have underdeveloped Peyers patches, in which the myeloid CD11b+ CD11c+ dendritic-cell subset is not present in the subepithelial dome. CCR6-/- mice also have increased numbers in T-cell subpopulations within the intestinal mucosa. In 2,4-dinitrofluorobenzene-induced contact hypersensitivity (CHS) studies, CCR6-/- mice developed more severe and more persistent inflammation than wild-type (WT) animals. Conversely, in a delayed-type hypersensitivity (DTH) model induced with allogeneic splenocytes, CCR6-/- mice developed no inflammatory response. The altered responses seen in the CHS and DTH assays suggest the existence of a defect in the activation and/or migration of the CD4(+) T-cell subsets that downregulate or elicit the inflammation response, respectively. These findings underscore the role of CCR6 in cutaneous and intestinal immunity and the utility of CCR6-/- mice as a model to study pathologies in these tissues. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

CCR6 regulates EAE pathogenesis by controlling regulatory CD4+ T-cell recruitment to target tissues.

The T‐cell subsets, characterized by their cytokine production profiles and immune regulatory functions, depend on correct in vivo location to interact with accessory or target cells for effective immune responses. Differentiation of naive CD4+ T cells into effectors is accompanied by sequentially regulated expression of the chemokine receptors responsible for cell recruitment to specific tissues. We studied CCR6 function in EAE, a CD4+ T‐cell‐mediated CNS disease characterized by mononuclear infiltration and demyelination. CCR6−/− mice showed an altered time course of EAE development, with delayed onset, a higher clinical score, and more persistent symptoms than in controls. An imbalanced cytokine profile and reduced Foxp3+ cell frequency characterized CNS tissues from CCR6−/− compared with CCR6+/+ mice during the disease effector phase. Transfer of CCR6+/+ Treg to CCR6−/− mice the day before EAE induction reduced the clinical score associated with an increased in infiltrating Foxp3+ cells and recovery of the cytokine balance in CCR6−/− mouse CNS. Competitive assays between CCR6+/+ and CCR6−/− Treg adoptively transferred to CCR6−/− mice showed impaired ability of CCR6−/− Treg to infiltrate CNS tissues in EAE‐affected mice. Our data indicate a CCR6 requirement by CD4+ Treg to downregulate the CNS inflammatory process and neurological signs associated with EAE.

Absence of CCR8 Does Not Impair the Response to Ovalbumin-Induced Allergic Airway Disease

Interaction of chemokines with their specific receptors results in tight control of leukocyte migration and positioning. CCR8 is a chemokine receptor expressed mainly in CD4+ single-positive thymocytes and Th2 cells. We generated CCR8-deficient mice (CCR8−/−) to study the in vivo role of this receptor, and describe in this study the CCR8−/− mouse response in OVA-induced allergic airway disease using several models, including an adoptive transfer model and receptor-blocking experiments. All CCR8−/− mice developed a pathological response similar to that of wild-type animals with respect to bronchoalveolar lavage cell composition, peripheral blood and bone marrow eosinophilia, lung infiltrates, and Th2 cytokine levels in lung and serum. The results contrast with a recent report using one of the OVA-induced asthma models studied here. Similar immune responses were also observed in CCR8−/− and wild-type animals in a different model of ragweed allergen-induced peritoneal eosinophilic inflammation, with an equivalent number of eosinophils and analogous increased levels of Th2 cytokines in peritoneum and peripheral blood. Our results show that allergic diseases course without critical CCR8 participation, and suggest that further work is needed to unravel the in vivo role of CCR8 in Th2-mediated pathologies.

Molecular cloning, functional characterization and mRNA expression analysis of the murine chemokine receptor CCR6 and its specific ligand MIP‐3α1

We have cloned the murine CCR6 receptor and its ligand, the β‐chemokine mMIP‐3α. Calcium mobilization assays performed with mCCR6 transfectants showed significant responses upon addition of mMIP‐3α. Murine MIP‐3α RNA is expressed in thymus, small intestine and colon, whereas mCCR6 RNA is expressed in spleen and lymph nodes. RT‐PCR analysis of FACS‐sorted lymphoid and antigen presenting cell subsets showed mCCR6 expression mainly in B cells, CD8− splenic dendritic cells and CD4+ T cells. The cloning and functional characterization of the mCCR6 and mMIP‐3α will allow the study of the role of these proteins in mouse models of inflammation and immunity.

CCR6 has a non‐redundant role in the development of inflammatory bowel disease

Antigen‐loaded tissues such as the intestinal mucosa must simultaneously elicit appropriate immune response to innocuous bacteria and food proteins, and to potentially harmful antigens. Impairment of the mechanisms controlling this response may mediate the excessive immune reaction that can lead to tissue destruction and inflammatory intestinal diseases, including inflammatory bowel disease. The intestinal epithelium influences local immune responses through the expression of adhesion molecules, costimulatory factors, cytokines and chemokines. CCL20, a β‐chemokine expressed in epithelia from colon and other intestinal tissue, plays a role in immune responses of intestinal mucosa, as deduced from the defects in intestinal leukocyte homeostasis shown by mice lacking CCR6, the CCL20 receptor. We studied the response of CCR6‐deficient mice in two models of inflammatory bowel disease. The data show that absence of CCR6 resulted in less severe intestinal pathology in animals treated with dextran sodium sulfate. Conversely, CCR6 deficiency alters leukocyte homeostasis and the cytokine environment in the intestinal mucosa; these changes are sufficient to confer susceptibility to trinitrobenzene sulfonic acid‐induced intestinal inflammation in the otherwise resistant C57BL/6J mouse strain. These results suggest that the CCR6/CCL20 axis has a critical, non‐redundant role in the in vivo control of immune responses in the intestine.

Grass tablet sublingual immunotherapy downregulates the TH2 cytokine response followed by regulatory T-cell generation

BACKGROUND Sublingual administration of Phleum pratense allergen immunotherapy (SLIT) tablets is a clinically efficient treatment for grass pollen-induced rhinoconjunctivitis. This immunotherapy downregulates TH2 immune responses, induces tolerogenic pathways, and increases regulatory T cells. However, associated immune response markers of allergen desensitization remain undefined. OBJECTIVE We sought to characterize the kinetics of individual changes in the immunologic response to grass tablet SLIT. METHODS We evaluated the systemic effects of SLIT in a longitudinal analysis of humoral and cellular immune parameters in peripheral blood samples. RESULTS Grass tablet SLIT administration induced a 2-phase systemic humoral and cellular response. The TH2 response was initially exacerbated and detected as increased allergen-specific IgE (sIgE) and IgG4 (sIgG4) levels and an increase in IL-4-producing cells, followed by downregulation of the TH2 response with a shift toward a TH1 cytokine profile. T cells with a regulatory phenotype were also elicited. Statistical correlations between immunologic measurements for each patient throughout therapy indicated that TH2 response downregulation and reduction of the immediate SLIT-induced IgE response were associated with increased allergen-specific IgG4 synthesis early in therapy. TH2 response downregulation by month 4 correlated with increased frequency of CD4(+) T cells with a regulatory phenotype by 12 months. CONCLUSION Changes in sIgE levels after therapy were linked to a specific IgG4 response, and production of blocking antibodies correlated with TH2 response downregulation. Reduced IL-4(+) cell frequency was linked to an increase in the frequency of CD4(+) T cells with a regulatory phenotype. Changes in sIgE levels and reduced IL-4 and blocking antibody levels could thus be used as indicators of a patients immune response to therapy.

Down-regulation of the beta-chemokine receptor CCR6 in dendritic cells mediated by TNF-alpha and IL-4.

Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response. We have generated DC from human umbilical cord blood CD34+ progenitors cultured with granulocyte‐macrophage colony‐stimulating factor, tumor necrosis factor α (TNF‐α), and stem cell factor. Using an anti‐CCR6 monoclonal antibody, we observed that these cells showed maximum expression of this β‐chemokine receptor when they were immature, as determined by their relatively low expression of several DC maturation markers such as CD1a, CD11c, CD14, CD40, CD80, and CD83. Immature DC responded strongly to macrophage inflammatory protein‐3α (MIP‐3α), the CCR6 ligand, in migration and calcium mobilization assays. CCR6 expression decreased in parallel with the DC maturation induced by prolonged TNF‐α treatments. Interleukin‐4 was also able to decrease CCR6 protein levels. Our findings suggest that the MIP‐3α/CCR6 interaction plays an important role in the trafficking of immature DC to chemokine production sites such as injured or inflamed peripheral tissues, where DC undergo maturation on contact with antigens. J. Leukoc. Biol. 66: 837–844; 1999.

Functional and phenotypic analysis of thymic B cells: role in the induction of T cell negative selection

The phenotype of mouse thymic B cells and their capacity to induce T cell negative selection in vitro were analyzed. Thymic B cells expressed B cell markers such as IgM, Fcγ receptor, CD44, heat‐stable antigen, LFA‐1 and CD40. In addition, they were positive for the activation molecule CD69 and displayed high levels of B7‐2. Although thymic B cells expressed CD5 on their surface, no CD5‐specific mRNA was detected. Moreover, thymic B cells induced a stronger deletion of TCR‐transgenic (TG) thymocytes than splenic B cells, which had low CD69 and B7‐2 levels. Interestingly, CD40‐activated splenic B cells up‐regulated CD69 and B7‐2 and acquired a capacity to induce T cell deletion comparable to that of thymic B cells. Moreover, thymic B cells from CD40‐deficient mice displayed lower CD69 and B7‐2 levels than control thymic B cells, and lower capacity to induce the deletion of TCR TG thymocytes. These results support the hypothesis that CD40‐mediated activation of thymic B cells determines a high efficiency of antigen presentation, suggesting that within the thymus B cells may play an important role in the elimination of autoreactive thymocytes.