Immune sensing of food allergens promotes avoidance behaviour.

In addition to its canonical function of protection from pathogens, the immune system can also alter behaviour1,2. The scope and mechanisms of behavioural modifications by the immune system are not yet well understood. Here, using mouse models of food allergy, we show that allergic sensitization drives antigen-specific avoidance behaviour. Allergen ingestion activates brain areas involved in the response to aversive stimuli, including the nucleus of tractus solitarius, parabrachial nucleus and central amygdala. Allergen avoidance requires immunoglobulin E (IgE) antibodies and mast cells but precedes the development of gut allergic inflammation. The ability of allergen-specific IgE and mast cells to promote avoidance requires cysteinyl leukotrienes and growth and differentiation factor 15. Finally, a comparison of C57BL/6 and BALB/c mouse strains revealed a strong effect of the genetic background on the avoidance behaviour. These findings thus point to antigen-specific behavioural modifications that probably evolved to promote niche selection to avoid unfavourable environments.

Dendritic Cells Regulate Extrafollicular Autoreactive B Cells via T Cells Expressing Fas and Fas Ligand.

The extrafollicular (EF) plasmablast response to self-antigens that contain Toll-like receptor (TLR) ligands is prominent in murine lupus models and some bacterial infections, but the inhibitors and activators involved have not been fully delineated. Here, we used two conventional dendritic cell (cDC) depletion systems to investigate the role of cDCs on a classical TLR-dependent autoreactive EF response elicited in rheumatoid-factor B cells by DNA-containing immune complexes. Contrary to our hypothesis, cDC depletion amplified rather than dampened the EF response in Fas-intact but not Fas-deficient mice. Further, we demonstrated that cDC-dependent regulation requires Fas and Fas ligand (FasL) expression by T cells, but not Fas expression by B cells. Thus, cDCs activate FasL-expressing T cells that regulate Fas-expressing extrafollicular helper T (Tefh) cells. These studies reveal a regulatory role for cDCs in B cell plasmablast responses and provide a mechanistic explanation for the excess autoantibody production observed in Fas deficiency.

Local triggering of the ICOS coreceptor by CD11c(+) myeloid cells drives organ inflammation in lupus.

The inducible T cell costimulator (ICOS) is a potent promoter of organ inflammation in murine lupus. ICOS stimulates T follicular helper cell differentiation in lymphoid tissue, suggesting that it might drive autoimmunity by enhancing autoantibody production. Yet the pathogenic relevance of this mechanism remains unclear. It is also unknown whether other ICOS-induced processes might contribute to lupus pathology. Here we show that selective ablation of ICOS ligand (ICOSL) in CD11c(+) cells, but not in B cells, dramatically ameliorates kidney and lung inflammation in lupus-prone MRL.Fas(lpr) mice. Autoantibody formation was largely unaffected by ICOSL deficiency in CD11c(+) cells. However, ICOSL display by CD11c(+) cells in inflamed organs had a nonredundant role in protecting invading T cells from apoptosis by elevating activity of the PI3K-Akt signaling pathway, thereby facilitating T cell accrual. These findings reveal a mechanism that locally sustains organ inflammation in lupus.

Cross-Reactive Antigen Expressed by B6 Splenocytes Drives Receptor Editing and Marginal Zone Differentiation of IgG2a-Reactive AM14 Vκ8 B Cells.

The AM14 BCR, derived from an autoimmune MRL/lpr mouse, binds autologous IgG2aa/j with low affinity, and as a result, AM14 B cells only proliferate in response to IgG2a immune complexes that incorporate DNA, RNA, or nucleic acid-binding proteins that serve as autoadjuvants. As such, AM14 B cells have served as a useful model for demonstrating the importance of BCR/TLR coengagement in the activation of autoreactive B cells. We now show that the same receptor recognizes an additional murine-encoded Ag, expressed by B6 splenocytes, with sufficient avidity to induce a TLR-independent proliferative response of BALB/c AM14 Vκ8 B cells both in vivo and in vitro. Moreover, detection of this cross-reactive Ag by B6 AM14 Vκ8 B cells promotes an anergic phenotype as reflected by suboptimal responses to BCR cross-linking and the absence of mature B cells in the bone marrow. The B6 Ag further impacts B cell development as shown by a dramatically expanded marginal zone compartment and extensive receptor editing in B6 AM14 Vκ8 mice but not BALB/c AM14 Vκ8 mice. Despite their anergic phenotypes, B6 AM14 Vκ8 B cells can respond robustly to autoantigen/autoadjuvant immune complexes and could therefore participate in both autoimmune responses and host defense.

B Cell-Extrinsic Myd88 and Fcer1g Negatively Regulate Autoreactive and Normal B Cell Immune Responses.

MyD88 and FcR common γ-chain (Fcer1g, FcRγ) elicit proinflammatory responses to exogenous Ags. Deletion of these receptors in autoimmune models has generally led to reduced overall disease. In B cells, Myd88 is required for anti-DNA and anti-RNA autoantibody responses, whereas Fcer1g is not expressed in these cells. The roles of these receptors in myeloid cells during B cell autoimmune activation remain less clear. To investigate the roles of Myd88 and Fcer1g in non-B cells, we transferred anti-self-IgG (rheumatoid factor) B cells and their physiologic target Ag, anti-chromatin Ab, into mice lacking Fcer1g, Myd88, or both and studied the extrafollicular plasmablast response. Surprisingly, we found a markedly higher and more prolonged response in the absence of either molecule; this effect was accentuated in doubly deficient recipients, with a 40-fold increase compared with wild-type recipients at day 10. This enhancement was dependent on CD40L, indicating that Myd88 and FcRγ, presumably on myeloid APCs, were required to downregulate T cell help for the extrafollicular response. To extend the generality, we then investigated a classic T cell-dependent response to (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken γ globulin and found a similar effect. Thus, these results reveal novel regulatory roles in the B cell response for receptors that are typically proinflammatory.

Rheumatoid factor B cell memory leads to rapid, switched antibody-forming cell responses.

B cells are critical in the initiation and maintenance of lupus. Autoreactive B cells clonally expand, isotype switch, and mutate--properties associated with memory B cells (MBCs), which are typically generated via germinal centers. The development and functions of autoreactive MBCs in lupus are poorly understood. Moreover, mounting evidence implicates the extrafollicular (EF) response in the generation of switched and mutated autoantibodies that are driven by BCR and TLR corecognition, raising the question of whether MBCs are generated in this context. In this study, we investigated autoreactive MBC generation associated with this type of response. We transferred B cells from AM14 site-directed BCR transgenic mice into nontransgenic normal recipients and elicited an EF response with anti-chromatin Ab, as in prior studies. By following the fate of the stimulated cells at late time points, we found that AM14 B cells persisted at increased frequency for up to 7 wk. Furthermore, these cells had divided in response to Ag but were subsequently quiescent, with a subset expressing the memory marker CD73. These cells engendered rapid, isotype-switched secondary plasmablast responses upon restimulation. Both memory and rapid secondary responses required T cell help to develop, emphasizing the need for T-B collaboration for long-term self-reactivity. Thus, using this model system, we show that the EF response generated persistent and functional MBCs that share some, but not all, of the characteristics of traditional MBCs. Such cells could play a role in chronic or flaring autoimmune disease.

Exacerbated autoimmunity in the absence of TLR9 in MRL.Fas(lpr) mice depends on Ifnar1.

TLR9 suppresses TLR7-driven pathogenesis in the MRL.Fas(lpr) murine model of systemic lupus erythematosus, but the mechanisms by which TLR7 promotes and TLR9 prevents disease in this and other lupus models remain unclear. Type I IFNs (IFN-I) have also been implicated in the pathogenesis of lupus both in patients and in several murine models of disease, but their role in MRL.Fas(lpr) mice is controversial. Using MRL.Fas(lpr) mice genetically deficient in a subunit of the receptor for IFN-I, Ifnar1, we show that IFN-I contribute significantly to renal disease in this model. Ifnar1 had no effect on anti-nucleosome or anti-Sm autoantibody titers, but instead regulated anticytoplasmic and anti-RNA specificities. Moreover, Ifnar1 deficiency prevented the exacerbation of clinical disease observed in Tlr9-deficient animals in this lupus model. Thus, IFN-I signaling is an important mediator of lupus pathogenesis and anti-RNA Ab production that is dysregulated in the absence of Tlr9.

TLR9 promotes tolerance by restricting survival of anergic anti-DNA B cells, yet is also required for their activation.

Nucleic acid-reactive B cells frequently arise in the bone marrow but are tolerized by mechanisms including receptor editing, functional anergy, and/or deletion. TLR9, a sensor of endosomal dsDNA, both promotes and regulates systemic autoimmunity in vivo, but the precise nature of its apparently contradictory roles in autoimmunity remained unclear. In this study, using the 3H9 anti-DNA BCR transgene in the autoimmune-prone MRL.Fas(lpr) mouse model of systemic lupus erythematosus, we identify the stages at which TLR9 contributes to establishing and breaking B cell tolerance. Although TLR9 is dispensable for L chain editing during B cell development in the bone marrow, TLR9 limits anti-DNA B cell life span in the periphery and is thus tolerogenic. In the absence of TLR9, anti-DNA B cells have much longer life spans and accumulate in the follicle, neither activated nor deleted. These cells retain some characteristics of anergic cells, in that they have elevated basal BCR signaling but impaired induced responses and downregulate their cell-surface BCR expression. In contrast, whereas TLR9-intact anergic B cells accumulate near the T/B border, TLR9-deficient anti-DNA B cells are somewhat more dispersed throughout the follicle. Nonetheless, in older autoimmune-prone animals, TLR9 expression specifically within the B cell compartment is required for spontaneous peripheral activation of anti-DNA B cells and their differentiation into Ab-forming cells via an extrafollicular pathway. Thus, TLR9 has paradoxical roles in regulating anti-DNA B cells: it helps purge the peripheral repertoire of autoreactive cells, yet is also required for their activation.

Spontaneous loss of tolerance of autoreactive B cells in Act1-deficient rheumatoid factor transgenic mice.

Self-reactive B cells in BALB/c AM14 transgenic (Tg) rheumatoid factor mice are not subject to central or peripheral tolerization. Instead, they remain at a stage of "clonal ignorance"; that is, they do not proliferate and differentiate into Ab-forming cells. However, the immunoregulatory mechanisms that prevent autoantibody production in these mice remain unclear. In this study, we show that crossing AM14 Tg mice to a mouse strain deficient in Act1, a molecule involved in the regulation of BAFF-R and CD40-signaling in B cells, results in spontaneous activation of AM14 Tg B cells and production of AM14-specific Abs. Three- to 5-mo-old AM14 Tg Act1(-/-) mice showed significant expansion of AM14 Tg B cells, including a 2- to 3-fold increase in the spleen and cervical lymph nodes compared with AM14 Tg Act1(+/+) mice. Furthermore, in the presence of endogenous self-Ag (IgH(a) congenic background), AM14 Tg Act1(-/-) B cells were spontaneously activated and differentiated into Ab-forming cells. In contrast with previous studies using AM14 Tg MLR.Fas(lpr) mice, we found that a significant number of AM14 Tg cells AM14 Tg Act1(-/-) mice displayed phenotypic characteristics of germinal center B cells. Anti-CD40L treatment significantly limited the expansion and activation of AM14 Tg Act1(-/-) B cells, suggesting that CD40L-mediated signals are required for the retention of these cells. Our results support the important role of Act1 in the regulation of self-reactive B cells and reveal how Act1 functions to prevent the production of autoantibodies.

Facultative role for T cells in extrafollicular Toll-like receptor-dependent autoreactive B-cell responses in vivo.

Extrafollicular (EF) B-cell responses are increasingly being recognized as an alternative pathway of B-cell activation, particularly in autoimmunity. Critical cellular interactions required for the EF B-cell response are unclear. A key question in autoimmunity, in which Toll-like receptor (TLR) signals are costimulatory and could be sufficient for B-cell activation, is whether T cells are required for the response. This is pivotal, because autoreactive B cells are considered antigen-presenting cells for autoreactive T cells, but where such interactions occur has not been identified. Here, using AM14 site-directed transgenic rheumatoid factor (RF) mice, we report that B cells can be activated, differentiate, and isotype-switch independent of antigen-specific T-cell help, αß T cells, CD40L signaling, and IL-21 signaling to B cells. However, T cells do dramatically enhance the response, and this occurs via CD40L and IL-21 signals. Surprisingly, the response is completely inducible T-cell costimulator ligand independent. These results establish that, although not required, T cells substantially amplify EF autoantibody production and thereby implicate T-independent autoreactive B cells as a potential vector for breaking T-cell tolerance. We suggest that these findings explain why autoreactivity first focuses on self-components for which B cells carry TLR ligands, because these will uniquely be able to activate B cells independently of T cells, with subsequent T-B interactions activating autoreactive T cells, resulting in chronic autoimmunity.