Antigen receptor signaling and cell death resistance controls intestinal humoral response zonation.

Immunoglobulin A (IgA) maintains commensal communities in the intestine while preventing dysbiosis. IgA generated against intestinal microbes assures the simultaneous binding to multiple, diverse commensal-derived antigens. However, the exact mechanisms by which B cells mount broadly reactive IgA to the gut microbiome remains elusive. Here, we have shown that IgA B cell receptor (BCR) is required for B cell fitness during the germinal center (GC) reaction in Peyer's patches (PPs) and for generation of gut-homing plasma cells (PCs). We demonstrate that IgA BCR drove heightened intracellular signaling in mouse and human B cells, and as a consequence, IgA+ B cells received stronger positive selection cues. Mechanistically, IgA BCR signaling offset Fas-mediated death, possibly rescuing low-affinity B cells to promote a broad humoral response to commensals. Our findings reveal an additional mechanism linking BCR signaling, B cell fate, and antibody production location, which have implications for how intestinal antigen recognition shapes humoral immunity.

Integrin αvß3 Limits Cytokine Production by Plasmacytoid Dendritic Cells and Restricts TLR-Driven Autoimmunity.

Plasmacytoid dendritic cells (pDCs) are strongly implicated as a major source of IFN-I in systemic lupus erythematosus (SLE), triggered through TLR-mediated recognition of nucleic acids released from dying cells. However, relatively little is known about how TLR signaling and IFN-I production are regulated in pDCs. In this article, we describe a role for integrin αvß3 in regulating TLR responses and IFN-I production by pDCs in mouse models. We show that αv and ß3-knockout pDCs produce more IFN-I and inflammatory cytokines than controls when stimulated through TLR7 and TLR9 in vitro and in vivo. Increased cytokine production was associated with delayed acidification of endosomes containing TLR ligands, reduced LC3 conjugation, and increased TLR signaling. This dysregulated TLR signaling results in activation of B cells and promotes germinal center (GC) B cell and plasma cell expansion. Furthermore, in a mouse model of TLR7-driven lupus-like disease, deletion of αvß3 from pDCs causes accelerated autoantibody production and pathology. We therefore identify a pDC-intrinsic role for αvß3 in regulating TLR signaling and preventing activation of autoreactive B cells. Because αvß3 serves as a receptor for apoptotic cells and cell debris, we hypothesize that this regulatory mechanism provides important contextual cues to pDCs and functions to limit responses to self-derived nucleic acids.

Regulatory strategies limiting endosomal Toll-like receptor activation in B cells.

The recognition of pathogen-associated nucleic acid (NA) promotes effective immunity against invading pathogens. However, endosomal Toll-like receptor (TLR) activation by self-NA also underlies the pathogenesis of systemic autoimmune diseases, such as systemic lupus erythematosus (SLE). For this reason, the activation thresholds of NA-sensing TLRs must be tightly regulated to balance protective and pathogenic immune responses. In this study, we will provide an overview of the evolutionary mechanisms designed to limit the aberrant activation of endosomal TLRs by self-ligands, focusing on four broad strategies. These include the following: 1) the production of nucleases able to degrade self-DNA and RNA; 2) the cell-specific regulation of endosomal TLR expression; 3) the spatial and temporal control of TLR positioning at a sub-cellular level; and 4) the modulation of downstream TLR signaling cascades. Given the critical role of B cells in lupus pathogenesis, where possible, we will describe evidence for B cell-specific induction of these regulatory mechanisms. We will also highlight our own work showing how modulation of B cell endolysosomal flux tunes NA-sensing TLR activation signals. In the face of inevitable generation of self-NA during normal cellular turnover, these parallel mechanisms are vital to protect against pathogenic inflammation.

Inflammation-induced interstitial migration of effector CD4⁺ T cells is dependent on integrin αV.

Leukocytes must traverse inflamed tissues to effectively control local infection. Although motility in dense tissues seems to be integrin independent and based on actomyosin-mediated protrusion and contraction, during inflammation, changes to the extracellular matrix (ECM) may necessitate distinct motility requirements. Indeed, we found that the interstitial motility of T cells was critically dependent on Arg-Gly-Asp (RGD)-binding integrins in the inflamed dermis. Inflammation-induced deposition of fibronectin was functionally linked to higher expression of integrin αV on effector CD4⁺ T cells. By intravital multiphoton imaging, we found that the motility of CD4⁺ T cells was dependent on αV expression. Selective blockade or knockdown of αV arrested T helper type 1 (TH1) cells in the inflamed tissue and attenuated local effector function. Our data demonstrate context-dependent specificity of lymphocyte movement in inflamed tissues that is essential for protective immunity.

B Cell αv Integrins Regulate TLR-Driven Autoimmunity.

Systemic lupus erythematosus (SLE) is defined by loss of B cell tolerance, resulting in production of autoantibodies against nucleic acids and other cellular Ags. Aberrant activation of TLRs by self-derived RNA and DNA is strongly associated with SLE in patients and in mouse models, but the mechanism by which TLR signaling to self-ligands is regulated remains poorly understood. In this study, we show that αv integrin plays a critical role in regulating B cell TLR signaling to self-antigens in mice. We show that deletion of αv from B cells accelerates autoantibody production and autoimmune kidney disease in the Tlr7.1 transgenic mouse model of SLE. Increased autoimmunity was associated with specific expansion of transitional B cells, extrafollicular IgG2c-producing plasma cells, and activation of CD4 and CD8 T cells. Our data show that αv-mediated regulation of TLR signaling in B cells is critical for preventing autoimmunity and indicate that loss of αv promotes escape from tolerance. Thus, we identify a new regulatory pathway in autoimmunity and elucidate upstream signals that adjust B cell activation to prevent development of autoimmunity in a mouse model.

Tetraspanin CD82 Organizes Dectin-1 into Signaling Domains to Mediate Cellular Responses to Candida albicans.

Tetraspanins are a family of proteins possessing four transmembrane domains that help in lateral organization of plasma membrane proteins. These proteins interact with each other as well as other receptors and signaling proteins, resulting in functional complexes called "tetraspanin microdomains." Tetraspanins, including CD82, play an essential role in the pathogenesis of fungal infections. Dectin-1, a receptor for the fungal cell wall carbohydrate ß-1,3-glucan, is vital to host defense against fungal infections. The current study identifies a novel association between tetraspanin CD82 and Dectin-1 on the plasma membrane of Candida albicans-containing phagosomes independent of phagocytic ability. Deletion of CD82 in mice resulted in diminished fungicidal activity, increased C. albicans viability within macrophages, and decreased cytokine production (TNF-α, IL-1ß) at both mRNA and protein level in macrophages. Additionally, CD82 organized Dectin-1 clustering in the phagocytic cup. Deletion of CD82 modulates Dectin-1 signaling, resulting in a reduction of Src and Syk phosphorylation and reactive oxygen species production. CD82 knockout mice were more susceptible to C. albicans as compared with wild-type mice. Furthermore, patient C. albicans-induced cytokine production was influenced by two human CD82 single nucleotide polymorphisms, whereas an additional CD82 single nucleotide polymorphism increased the risk for candidemia independent of cytokine production. Together, these data demonstrate that CD82 organizes the proper assembly of Dectin-1 signaling machinery in response to C. albicans.

Dectin-1-dependent LC3 recruitment to phagosomes enhances fungicidal activity in macrophages.

Autophagy has been postulated to play role in mammalian host defense against fungal pathogens, although the molecular details remain unclear. Here, we show that primary macrophages deficient in the autophagic factor LC3 demonstrate diminished fungicidal activity but increased cytokine production in response to Candida albicans stimulation. LC3 recruitment to fungal phagosomes requires activation of the fungal pattern receptor dectin-1. LC3 recruitment to the phagosome also requires Syk signaling but is independent of all activity by Toll-like receptors and does not require the presence of the adaptor protein Card9. We further demonstrate that reactive oxygen species generation by NADPH oxidase is required for LC3 recruitment to the fungal phagosome. These observations directly link LC3 to the inflammatory pathway against C. albicans in macrophages.

NADPH oxidase exerts a B cell-intrinsic contribution to lupus risk by modulating endosomal TLR signals.

Genome-wide association studies in systemic lupus erythematosus (SLE) have linked loss-of-function mutations in phagocytic NADPH oxidase complex (NOX2) genes, including NCF1 and NCF2, to disease pathogenesis. The prevailing model holds that reduced NOX2 activity promotes SLE via defective efferocytosis, the immunologically silent clearance of apoptotic cells. Here, we describe a parallel B cell-intrinsic mechanism contributing to breaks in tolerance. In keeping with an important role for B cell Toll-like receptor (TLR) pathways in lupus pathogenesis, NOX2-deficient B cells exhibit enhanced signaling downstream of endosomal TLRs, increased humoral responses to nucleic acid-containing antigens, and the propensity toward humoral autoimmunity. Mechanistically, TLR-dependent NOX2 activation promotes LC3-mediated maturation of TLR-containing endosomes, resulting in signal termination. CRISPR-mediated disruption of NCF1 confirmed a direct role for NOX2 in regulating endosomal TLR signaling in primary human B cells. Together, these data highlight a new B cell-specific mechanism contributing to autoimmune risk in NCF1 and NCF2 variant carriers.

Transcriptomic analysis of pathways associated with ITGAV/alpha(v) integrin-dependent autophagy in human B cells.

Macroautophagy/autophagy proteins have been linked with the development of immune-mediated diseases including lupus, but the mechanisms for this are unclear due to the complex roles of these proteins in multiple immune cell types. We have previously shown that a form of noncanonical autophagy induced by ITGAV/alpha(v) integrins regulates B cell activation by viral and self-antigens, in mice. Here, we investigate the involvement of this pathway in B cells from human tissues. Our data reveal that autophagy is specifically induced in the germinal center and memory B cell subpopulations of human tonsils and spleens. Transcriptomic analysis show that the induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of ITGAV/alpha(v) integrin-dependent autophagy in human B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells, we found that ITGAV/alpha(v)-dependent autophagy limits activation of specific pathways related to B cell responses, while promoting others. These data provide new mechanistic links for autophagy and B-cell-mediated immune dysregulation in diseases such as lupus.

Differential regulation of monocyte cytokine release by αV and ß(2) integrins that bind CD23.

The human soluble CD23 (sCD23) protein displays highly pleiotropic cytokine-like activity. Monocytic cells express the sCD23-binding integrins αVß(3), αVß(5), αMß(2) and αXß(2), but it is unclear which of these four integrins most acutely regulates sCD23-driven cytokine release. The hypothesis that ligation of different sCD23-binding integrins promoted release of distinct subsets of cytokines was tested. Lipopolysaccharide (LPS) and sCD23 promoted release of distinct groups of cytokines from the THP-1 model cell line. The sCD23-driven cytokine release signature was characterized by elevated amounts of RANTES (CCL5) and a striking increase in interleukin-8 (IL-8; CXCL8) secretion, but little release of macrophage inflammatory protein 1ß (MIP-1ß; CCL4). Antibodies to αVß(3) or αXß(2) both promoted IL-8 release, consistent with the sCD23-driven pattern, but both also evoked strong MIP-1ß secretion; simultaneous ligation of these two integrins further increased cytokine secretion but did not alter the pattern of cytokine output. In both model cell lines and primary tissue, integrin-mediated cytokine release was more pronounced in immature monocyte cells than in mature cells. The capacity of anti-integrin monoclonal antibodies to elicit a cytokine release response is epitope-dependent and also reflects the differentiation state of the cell. Although a pattern of cytokine release identical to that provoked by sCD23 could not be elicited with any individual anti-integrin monoclonal antibody, αXß(2) and αVß(3) appear to regulate IL-8 release, a hallmark feature of sCD23-driven cytokine secretion, more acutely than αMß(2) or αVß(5).

Preferential expression of integrin αvß8 promotes generation of regulatory T cells by mouse CD103+ dendritic cells.

BACKGROUND & AIMS: Immune responses in the intestine are controlled by regulatory T cells (Tregs), which prevent inflammation in response to commensal bacteria. A specific population of intestinal dendritic cells (DCs), marked by expression of CD103, generate Tregs more efficiently than other DC populations through mechanisms that involve retinoic acid and transforming growth factor (TGF)-ß. However, it is not clear how CD103(+) DCs are specialized for this function. We investigated the ability of CD103(+) DCs to promote Treg generation through activation of TGF-ß and the role of integrins with the αv subunit in this process. METHODS: Naïve T cells were cultured with purified DCs from mesenteric lymph nodes (MLNs) or intestines of wild-type and αv conditional knockout mice to assess generation of Tregs. Antigens were administered orally to mice, and antigen-specific generation of Tregs was measured in intestinal tissues. Expression of the integrin αv subunit was measured in purified subpopulations of DCs by quantitative polymerase chain reaction and immunoblot analyses. RESULTS: In vitro, CD103(+) DCs generated more Tregs in the presence of latent TGF-ß than other MLN DCs. Efficient generation of Tregs required expression of the integrin αv subunit by DCs; mice that lacked αv in immune cells did not convert naïve T cells to intestinal Tregs in response to oral antigen. CD103(+) DCs derived from the MLNs selectively expressed high levels of integrin αvß8 compared with other populations of DCs. CONCLUSIONS: Expression of αvß8 is required for CD103(+) DCs to become specialized and activate latent TGF-ß and generate Tregs during the induction of tolerance to intestinal antigens in mice.

Autophagy Induced by Toll-like Receptor Ligands Regulates Antigen Extraction and Presentation by B Cells.

The engagement of B cells with surface-tethered antigens triggers the formation of an immune synapse (IS), where the local secretion of lysosomes can facilitate antigen uptake. Lysosomes intersect with other intracellular processes, such as Toll-like Receptor (TLR) signaling and autophagy coordinating immune responses. However, the crosstalk between these processes and antigen presentation remains unclear. Here, we show that TLR stimulation induces autophagy in B cells and decreases their capacity to extract and present immobilized antigens. We reveal that TLR stimulation restricts lysosome repositioning to the IS by triggering autophagy-dependent degradation of GEF-H1, a Rho GTP exchange factor required for stable lysosome recruitment at the synaptic membrane. GEF-H1 degradation is not observed in B cells that lack αV integrins and are deficient in TLR-induced autophagy. Accordingly, these cells show efficient antigen extraction in the presence of TLR stimulation, confirming the role of TLR-induced autophagy in limiting antigen extraction. Overall, our results suggest that resources associated with autophagy regulate TLR and BCR-dependent functions, which can finetune antigen uptake by B cells. This work helps to understand the mechanisms by which B cells are activated by surface-tethered antigens in contexts of subjacent inflammation before antigen recognition, such as sepsis.

C910 chemical compound inhibits the traffiking of several bacterial AB toxins with cross-protection against influenza virus.

The development of anti-infectives against a large range of AB-like toxin-producing bacteria includes the identification of compounds disrupting toxin transport through both the endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of compounds blocking Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, which was followed by orthogonal screens against two toxins that hijack the endolysosomal (diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule C910 that induces the enlargement of EEA1-positive early endosomes associated with sorting defects of CNF1 and Shiga toxins to their trafficking pathways. C910 protects cells against eight bacterial AB toxins and the CNF1-mediated pathogenic Escherichia coli invasion. Interestingly, C910 reduces influenza A H1N1 and SARS-CoV-2 viral infection in vitro. Moreover, parenteral administration of C910 to mice resulted in its accumulation in lung tissues and a reduction in lethal influenza infection.

Activated PI3Kδ signals compromise plasma cell survival via limiting autophagy and increasing ER stress.

While phosphatidylinositide 3-kinase delta (PI3Kδ) plays a critical role in humoral immunity, the requirement for PI3Kδ signaling in plasma cells remains poorly understood. Here, we used a conditional mouse model of activated PI3Kδ syndrome (APDS), to interrogate the function of PI3Kδ in plasma cell biology. Mice expressing a PIK3CD gain-of-function mutation (aPIK3CD) in B cells generated increased numbers of memory B cells and mounted an enhanced secondary response but exhibited a rapid decay of antibody levels over time. Consistent with these findings, aPIK3CD expression markedly impaired plasma cell generation, and expression of aPIK3CD intrinsically in plasma cells was sufficient to diminish humoral responses. Mechanistically, aPIK3CD disrupted ER proteostasis and autophagy, which led to increased plasma cell death. Notably, this defect was driven primarily by elevated mTORC1 signaling and modulated by treatment with PI3Kδ-specific inhibitors. Our findings establish an essential role for PI3Kδ in plasma cell homeostasis and suggest that modulating PI3Kδ activity may be useful for promoting and/or thwarting specific immune responses.

αv Integrins regulate germinal center B cell responses through noncanonical autophagy.

Germinal centers (GCs) are major sites of clonal B cell expansion and generation of long-lived, high-affinity antibody responses to pathogens. Signaling through TLRs on B cells promotes many aspects of GC B cell responses, including affinity maturation, class switching, and differentiation into long-lived memory and plasma cells. A major challenge for effective vaccination is identifying strategies to specifically promote GC B cell responses. Here, we have identified a mechanism of regulation of GC B cell TLR signaling, mediated by αv integrins and noncanonical autophagy. Using B cell-specific αv-KO mice, we show that loss of αv-mediated TLR regulation increased GC B cell expansion, somatic hypermutation, class switching, and generation of long-lived plasma cells after immunization with virus-like particles (VLPs) or antigens associated with TLR ligand adjuvants. Furthermore, targeting αv-mediated regulation increased the magnitude and breadth of antibody responses to influenza virus vaccination. These data therefore identify a mechanism of regulation of GC B cells that can be targeted to enhance antibody responses to vaccination.

The Role of Autophagy-Related Proteins in Candida albicans Infections.

Autophagy plays an important role in maintaining cell homeostasis by providing nutrients during periods of starvation and removing damaged organelles from the cytoplasm. A marker in the autophagic process is the reversible conjugation of LC3, a membrane scaffolding protein, to double membrane autophagosomes. Recently, a role for LC3 in the elimination of pathogenic bacteria and fungi, including Candida albicans (C. albicans), was demonstrated, but these organisms reside in single membrane phagosomes. This process is distinct from autophagy and is termed LC3-associated phagocytosis (LAP). This review will detail the hallmarks of LAP that distinguish it from classical autophagy and review the role of autophagy proteins in host response to C. albicans and other pathogenic fungi.

αv Integrins combine with LC3 and atg5 to regulate Toll-like receptor signalling in B cells.

Integrin signalling triggers cytoskeletal rearrangements, including endocytosis and exocytosis of integrins and other membrane proteins. In addition to recycling integrins, this trafficking can also regulate intracellular signalling pathways. Here we describe a role for αv integrins in regulating Toll-like receptor (TLR) signalling by modulating intracellular trafficking. We show that deletion of αv or ß3 causes increased B-cell responses to TLR stimulation in vitro, and αv-conditional knockout mice have elevated antibody responses to TLR-ligand-associated antigens. αv regulates TLR signalling by promoting recruitment of the autophagy component LC3 (microtubule-associated proteins 1 light chain 3) to TLR-containing endosomes, which is essential for progression from NF-κB to IRF signalling, and ultimately for traffic to lysosomes where signalling is terminated. Disruption of LC3 recruitment leads to prolonged NF-κB signalling and increased B-cell proliferation and antibody production. This work identifies a previously unrecognized role for αv and the autophagy components LC3 and atg5 in regulating TLR signalling and B-cell immunity.

Alpha (v) integrins license regulatory T cells to apoptotic cells and self-associated antigens.

Defects in apoptotic cell clearance are thought to contribute to autoimmunity by failure to induce tolerance, coupled with accumulation of immunogenic material. However, little is known about the contribution of apoptosis to immune responses at mucosal sites, where regulatory T cells (T(reg) cells) and other immune cells play an essential active role in maintaining tolerance to self-associated antigens. In recent studies, we have found that α(v) integrins have an important role in apoptotic cell phagocytosis and induction of T(reg) cells in the intestine, and deletion of α(v) from myeloid cells causes colitis associated with failed apoptotic cell removal and loss of T(reg) cells. Our data show that activation of transforming growth factor (TGF)-ß by α(v) ß(8) on dendritic cells (DCs) is essential for generating T(reg) cells and inducing mucosal tolerance. These results provide a mechanism by which tolerance to apoptotic cell-derived and -associated antigens is maintained by DC "licensing" at sites of high TGF-ß expression.

αv Integrin expression by DCs is required for Th17 cell differentiation and development of experimental autoimmune encephalomyelitis in mice.

Th17 cells are a distinct lineage of T helper cells that protect the body from bacterial and fungal infection. However, Th17 cells also contribute to inflammatory and autoimmune disorders such as multiple sclerosis. Th17 cell generation requires exposure of naive T cells to the cytokine TGF-ß in combination with proinflammatory cytokines. Here we show that differentiation of Th17 cells is also critically dependent on αv integrins. In mice, lack of integrin αv in the immune system resulted in loss of Th17 cells in the intestine and lymphoid tissues. It also led to protection from experimental autoimmune encephalomyelitis (EAE). Further analysis indicated that αv integrins on DCs activated latent TGF-ß during T cell stimulation and thereby promoted differentiation of Th17 cells. Furthermore, pharmacologic inhibition of αv integrins using cyclic RGD peptides blocked TGF-ß activation and Th17 cell generation in vitro and protected mice from EAE. These data demonstrate that activation of TGF-ß by αv-expressing myeloid cells may be a critical step in the generation of Th17 cells and suggest that αv integrins could be therapeutic targets in autoimmune disease.

alphavbeta5 integrin sustains growth of human pre-B cells through an RGD-independent interaction with a basic domain of the CD23 protein.

CD23 is a type II transmembrane glycoprotein synthesized by hematopoietic cells that has biological activity in both membrane-bound and freely soluble forms, acting via a number of receptors, including integrins. We demonstrate here that soluble CD23 (sCD23) sustains growth of human B cell precursors via an RGD-independent interaction with the alphavbeta5 integrin. The integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N terminus of the lectin head region of CD23, centered around Arg(172), Lys(173), and Cys(174) (RKC). This RKC motif is present in all forms of sCD23 with cytokine-like activity, and cytokine activity is independent of the lectin head, an "inverse RGD" motif, and the CD21 and IgE binding sites. RKC-containing peptides derived from this region of CD23 bind alphavbeta5 and are biologically active. The binding and activity of these peptides is unaffected by inclusion of a short peptide containing the classic RGD sequence recognized by integrins, and, in far-Western analyses, RKC-containing peptides bind to the beta subunit of the alphavbeta5 integrin. The interaction between alphavbeta5 and sCD23 indicates that integrins deliver to cells important signals initiated by soluble ligands without the requirement for interactions with RGD motifs in their common ligands. This mode of integrin signaling may not be restricted to alphavbeta5.