Jennifer L. Gommerman

Notch2 Receptor Signaling Controls Functional Differentiation of Dendritic Cells in the Spleen and Intestine

Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b(+) DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esam(hi) DC subset required lymphotoxin beta receptor signaling, proliferated in situ, and facilitated CD4(+) T cell priming. The Notch-independent Esam(lo) DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b(+)CD103(+) DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4(+) T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b(+) DC subsets in the spleen and intestine.

Notch2-dependent classical dendritic cells orchestrate intestinal immunity to attaching-and-effacing bacterial pathogens

Defense against attaching-and-effacing bacteria requires the sequential generation of interleukin 23 (IL-23) and IL-22 to induce protective mucosal responses. Although CD4+ and NKp46+ innate lymphoid cells (ILCs) are the critical source of IL-22 during infection, the precise source of IL-23 is unclear. We used genetic techniques to deplete mice of specific subsets of classical dendritic cells (cDCs) and analyzed immunity to the attaching-and-effacing pathogen Citrobacter rodentium. We found that the signaling receptor Notch2 controlled the terminal stage of cDC differentiation. Notch2-dependent intestinal CD11b+ cDCs were an obligate source of IL-23 required for survival after infection with C. rodentium, but CD103+ cDCs dependent on the transcription factor Batf3 were not. Our results demonstrate a nonredundant function for CD11b+ cDCs in the response to pathogens in vivo.

Nod2-Dependent Th2 Polarization of Antigen-Specific Immunity

While a number of microbial-associated molecular patterns have been known for decades to act as adjuvants, the mechanisms and the signaling pathways underlying their action have remained elusive. Here, we examined the unfolding of the adaptive immune response induced by Nod2 in vivo upon activation by its specific ligand, muramyl dipeptide, a component of peptidoglycan. Our findings demonstrate that this bacterial sensor triggers a potent Ag-specific immune response with a Th2-type polarization profile, characterized by the induction of IL-4 and IL-5 by T cells and IgG1 Ab responses. Nod2 was also found to be critical for the induction of both Th1- and Th2-type responses following costimulation with TLR agonists. Importantly, the synergistic responses to Nod2 and TLR agonists seen in vivo were recapitulated by dendritic cells in vitro, suggesting that these cells likely play a central role in the integration of Nod2- and TLR-dependent signals for driving the adaptive immune response. Taken together, our results identify Nod2 as a critical mediator of microbial-induced potentiation and polarization of Ag-dependent immunity. Moreover, these findings affect our understanding of Crohn’s diseases pathogenesis, where lack of Nod2-dependent Th2 signaling in a subset of these patients might explain heightened Th1-mediated inflammation at the level of the intestinal mucosa.

Acquisition of a multifunctional IgA + plasma cell phenotype in the gut

The largest mucosal surface in the body is in the gastrointestinal tract, a location that is heavily colonized by microbes that are normally harmless. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the gastrointestinal tract is the production and transepithelial transport of poly-reactive IgA (ref. 1). Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T-cell help, undergo class switch recombination of their immunoglobulin receptor to IgA, and differentiate to become plasma cells. However, IgA-secreting plasma cells probably have additional attributes that are needed for coping with the tremendous bacterial load in the gastrointestinal tract. Here we report that mouse IgA+ plasma cells also produce the antimicrobial mediators tumour-necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), and express many molecules that are commonly associated with monocyte/granulocytic cell types. The development of iNOS-producing IgA+ plasma cells can be recapitulated in vitro in the presence of gut stroma, and the acquisition of this multifunctional phenotype in vivo and in vitro relies on microbial co-stimulation. Deletion of TNF-α and iNOS in B-lineage cells resulted in a reduction in IgA production, altered diversification of the gut microbiota and poor clearance of a gut-tropic pathogen. These findings reveal a novel adaptation to maintaining homeostasis in the gut, and extend the repertoire of protective responses exhibited by some B-lineage cells.

Proinflammatory GM-CSF–producing B cells in multiple sclerosis and B cell depletion therapy

GM-CSF–producing B cells contribute to multiple sclerosis pathogenesis and the therapeutic action of B cell depletion. Eclipsing multiple sclerosis B cell depletion therapy (BCDT) has been shown to limit inflammation in some cases of multiple sclerosis (MS); however, how exactly BCDT works has remained unclear. Now, Li et al. report that a subset of B cells that produce the cytokine granulocyte macrophage–colony stimulating factor (GM-CSF) contributes to MS pathogenesis. These cells are more frequent in MS patients than in healthy controls and increase proinflammatory myeloid responses. Moreover, production of these cells counterbalances the generation of interleukin-10 (IL-10)–producing regulatory B cells, which are thought to be protective in disease. After BCDT, the ratio of GM-CSF/IL-10–producing B cells is normalized, suggesting that BCDT may work in part by decreasing the number of pathogenic GM-CSF–producing B cells. B cells are not limited to producing protective antibodies; they also perform additional functions relevant to both health and disease. However, the relative contribution of functionally distinct B cell subsets in human disease, the signals that regulate the balance between such subsets, and which of these subsets underlie the benefits of B cell depletion therapy (BCDT) are only partially elucidated. We describe a proinflammatory, granulocyte macrophage–colony stimulating factor (GM-CSF)–expressing human memory B cell subset that is increased in frequency and more readily induced in multiple sclerosis (MS) patients compared to healthy controls. In vitro, GM-CSF–expressing B cells efficiently activated myeloid cells in a GM-CSF–dependent manner, and in vivo, BCDT resulted in a GM-CSF–dependent decrease in proinflammatory myeloid responses of MS patients. A signal transducer and activator of transcription 5 (STAT5)– and STAT6-dependent mechanism was required for B cell GM-CSF production and reciprocally regulated the generation of regulatory IL-10–expressing B cells. STAT5/6 signaling was enhanced in B cells of untreated MS patients compared with healthy controls, and B cells reemerging in patients after BCDT normalized their STAT5/6 signaling as well as their GM-CSF/IL-10 cytokine secretion ratios. The diminished proinflammatory myeloid cell responses observed after BCDT persisted even as new B cells reconstituted. These data implicate a proinflammatory B cell/myeloid cell axis in disease and underscore the rationale for selective targeting of distinct B cell populations in MS and other human autoimmune diseases.

Mice overexpressing BAFF develop a commensal flora–dependent, IgA-associated nephropathy

B cell activation factor of the TNF family (BAFF) is a potent B cell survival factor. BAFF overexpressing transgenic mice (BAFF-Tg mice) exhibit features of autoimmune disease, including B cell hyperplasia and hypergammaglobulinemia, and develop fatal nephritis with age. However, basal serum IgA levels are also elevated, suggesting that the pathology in these mice may be more complex than initially appreciated. Consistent with this, we demonstrate here that BAFF-Tg mice have mesangial deposits of IgA along with high circulating levels of polymeric IgA that is aberrantly glycosylated. Renal disease in BAFF-Tg mice was associated with IgA, because serum IgA was highly elevated in nephritic mice and BAFF-Tg mice with genetic deletion of IgA exhibited less renal pathology. The presence of commensal flora was essential for the elevated serum IgA phenotype, and, unexpectedly, commensal bacteria-reactive IgA antibodies were found in the blood. These data illustrate how excess B cell survival signaling perturbs the normal balance with the microbiota, leading to a breach in the normal mucosal-peripheral compartmentalization. Such breaches may predispose the nonmucosal system to certain immune diseases. Indeed, we found that a subset of patients with IgA nephropathy had elevated serum levels of a proliferation inducing ligand (APRIL), a cytokine related to BAFF. These parallels between BAFF-Tg mice and human IgA nephropathy may provide a new framework to explore connections between mucosal environments and renal pathology.

Targeted Deletion of fgl2 Leads to Impaired Regulatory T Cell Activity and Development of Autoimmune Glomerulonephritis

Mice with targeted deletion of fibrinogen-like protein 2 (fgl2) spontaneously developed autoimmune glomerulonephritis with increasing age, as did wild-type recipients reconstituted with fgl2−/− bone marrow. These data implicate FGL2 as an important immunoregulatory molecule and led us to identify the underlying mechanisms. Deficiency of FGL2, produced by CD4+CD25+ regulatory T cells (Treg), resulted in increased T cell proliferation to lectins and alloantigens, Th 1 polarization, and increased numbers of Ab-producing B cells following immunization with T-independent Ags. Dendritic cells were more abundant in fgl2−/− mice and had increased expression of CD80 and MHCII following LPS stimulation. Treg cells were also more abundant in fgl2−/− mice, but their suppressive activity was significantly impaired. Ab to FGL2 completely inhibited Treg cell activity in vitro. FGL2 inhibited dendritic cell maturation and induced apoptosis of B cells through binding to the low-affinity FcγRIIB receptor. Collectively, these data suggest that FGL2 contributes to Treg cell activity and inhibits the development of autoimmune disease.

A Role for CD21/CD35 and CD19 in Responses to Acute Septic Peritonitis: A Potential Mechanism for Mast Cell Activation

Although it is now appreciated that mast cell-mediated release of TNF-α is critical for resolution of acute septic peritonitis, questions remain as to how mast cells are activated upon peritoneal bacterial infection. Clues to how this may occur have been derived from earlier studies by Prodeus et al. in which complement proteins C3 and C4 were shown to be required for survival following cecal ligation and puncture (CLP), a model for acute septic peritonitis. To evaluate the mechanism for mast cell activation in the CLP model, complement receptor CD21/CD35-deficient mice (Cr2null) were examined in the present study. Along with CD19-deficient (CD19null) mice, these animals exhibit decreased survival following CLP compared with wild-type littermates. Injection of IgM before CLP does not change survival rates for Cr2null mice and only partially improves survival of CD19null mice, implicating CD21/CD35 and CD19 in mast cell activation. Interestingly, early TNF-α release is also impaired in Cr2null and CD19null animals, suggesting that these molecules directly affect mast cell activation. Cr2null and CD19null mice demonstrate an impairment in neutrophil recruitment and a corresponding increase in bacterial load. Examination of peritoneal mast cells by flow cytometry and confocal microscopy reveals the expression and colocalization of CD21/CD35 and CD19. Taken together, these findings suggest that the engagement of complement receptors CD21/CD35 along with CD19 on the mast cell surface by C3 fragments may be necessary for the full expression of mast cell activation in the CLP model.

AID constrains germinal center size by rendering B cells susceptible to apoptosis

The germinal center (GC) is a transient lymphoid tissue microenvironment that fosters T cell-dependent humoral immunity. Within the GC, the B cell-specific enzyme, activation-induced cytidine deaminase (AID), mutates the immunoglobulin locus, thereby altering binding affinity for antigen. In the absence of AID, larger GC structures are observed in both humans and mice, but the reason for this phenomenon is unclear. Because significant apoptosis occurs within the GC niche to cull cells that have acquired nonproductive mutations, we have examined whether a defect in apoptosis could account for the larger GC structures in the absence of AID. In this report, we reveal significantly reduced death of B cells in AID(-/-) mice as well as in B cells derived from AID(-/-) bone marrow in mixed bone marrow chimeric mice. Furthermore, AID-expressing B cells show decreased proliferation and survival compared with AID(-/-) B cells, indicating an AID-mediated effect on cellular viability. The GC is an etiologic site for B-cell autoimmunity and lymphomagenesis, both of which have been linked to aberrant AID activity. We report a link between AID-induced DNA damage and B-cell apoptosis that has implications for the development of B-cell disorders.

Manipulation of lymphoid microenvironments in nonhuman primates by an inhibitor of the lymphotoxin pathway

Reticular networks in lymphoid organs play critical roles in the organization of local microenvironments. A number of these elements are maintained by continual signaling through the lymphotoxin system. Evaluation of the lymphotoxin (LT) pathway in primates using a fusion protein decoy provides a unique opportunity to assess modulation of splenic microenvironments in a species with considerably greater background immunological activity compared with rodents. Within the germinal center microenvironment, treatment resulted in a collapse of follicular dendritic cell (FDC) networks and in the disappearance of a ringlike network of immune complex-carrying cells, although some other attributes of the germinal center appeared to be unaltered. Treatment also resulted in changes in the splenic marginal zone, a microenvironment where the architecture is notably different from that of the rodent. Cessation of treatment and recovery allowed us to monitor reemergence of these cell types and revealed that FDCs rely on LT-dependent signals to recompact into appropriately positioned tight networks. Despite the loss of FDC networks, the primary Ab response to keyhole limpet hemocyanin was unaltered over a 20-day period. Manipulation of these microenvironments may represent a novel approach to modulating immune function in human disease.