Background GWAS have linked loss-of-function mutations in phagocytic NADPH oxidase complex (NOX2) genes, including NCF1 and NCF2, to lupus pathogenesis. The prevailing model holds that reduced NOX2 ...promotes SLE via defective efferocytosis, the immunologically silent clearance of apoptotic cells. We previously showed that defects in endolysosomal flux increase B cell TLR signals, resulting in humoral autoimmunity.1–3 Since NCF1/NCF2 are known regulators of myeloid endosomal trafficking, we hypothesized that a parallel B cell-intrinsic mechanism contributes to lupus risk. Methods We tested the impact of NOX2 family gene deletion on B cell TLR signaling using in vivo animal models, primary murine B cells, NCF1-null human B cell lymphoma lines, and CRISPR-edited primary human B cells. Results NOX2-deficient mice exhibited increased humoral responses to nucleic acid-containing antigens, findings which correlate with enhanced B cell signals downstream of endosomal TLRs. In keeping with important roles for B cell TLRs in lupus pathogenesis, B cell-intrinsic NADPH oxidase deletion promoted humoral autoimmunity in mice. To understand the underlying mechanisms, we quantified TLR-induced intracellular trafficking in B cells using live cell microscopy. Following CpG stimulation, NADPH oxidase activation promoted trafficking of TLR-containing endosomes to lysosomes, resulting in TLR signal termination. Whereas initial uptake and aggregation of fluorescent CpG in early endosomes was preserved in NCF1-null B cell lymphomas, loss of NADPH oxidase activity limited signal degradation resulting in enhanced downstream NFkB activation. Finally, CRISPR-mediated disruption of NCF1 in primary human B cells confirmed a direct role for NOX2 in regulating endosomal TLR signaling in B cells. Following TLR9 activation in vitro, NCF1-deficient human B cells exhibited increased differentiation into IgM- and IgG-producing plasma cells, supporting a mechanistic link between B cell NADPH oxidase activity and human lupus pathogenesis. Conclusions Loss of function NCF1/NCF2 variants exert a B cell-intrinsic contribution to lupus pathogenesis. References Acharya M., F. Raso, S. Sagadiev, E. Gilbertson, L. Kadavy, Q.Z. Li, M. Yan, L.M. Stuart, J.A. Hamerman, A. Lacy-Hulbert. B Cell alphav Integrins Regulate TLR-Driven Autoimmunity. J Immunol 2020;205:1810–1818. Acharya M., A. Sokolovska, J.M. Tam, K.L. Conway, C. Stefani, F. Raso, S. Mukhopadhyay, M. Feliu, E. Paul, J. Savill, R.O. Hynes, R.J. Xavier, J.M. Vyas, L.M. Stuart, A. Lacy-Hulbert. alphav Integrins combine with LC3 and atg5 to regulate Toll-like receptor signalling in B cells. Nat Commun 2016;7:10917. Raso F., S. Sagadiev, S. Du, E. Gage, T. Arkatkar, G. Metzler, L.M. Stuart, M.T. Orr, D.J. Rawlings, S.W. Jackson, A. Lacy-Hulbert, M. Acharya. alphav Integrins regulate germinal center B cell responses through noncanonical autophagy. J Clin Invest 2018;128:4163–4178.
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 β3 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.
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.
BackgroundUnderstanding key signals that control the differentiation, function, and survival of plasma cells (PCs) is critical for development of improved therapeutic approaches to attenuate ...pathogenic antibody responses in SLE. While phosphatidylinositide 3-kinase delta (PI3Kδ) plays an essential role in humoral immune responses, its role(s) in PC function remains poorly understood.MethodsWe utilized a conditional mouse model of Activated PI3Kδ Syndrome (APDS), to interrogate the role of this key signaling program.ResultsMice expressing a gain-of-function mutation in PIK3CD in B cells, referred to as activated (a) PIK3CD, generated increased numbers of memory B cells, mounted enhanced secondary response, yet exhibited a rapid decay of antibody levels over time. Consistent with these findings, aPIK3CD expression markedly impaired plasma cell generation. Remarkably, PC specific aPIK3CD expression was sufficient to diminish humoral responses in vivo. Mechanistically, aPIK3CD disrupted endoplasmic reticulum proteostasis and autophagy, leading to increased PC death. Notably, this defect was driven primarily by elevated mTORC1 signaling and modulated by treatment with PI3Kδ-specific inhibitors.ConclusionsTaken together, these data demonstrate an unexpected requirement to down-regulate PI3Kδ activity to balance autophagy and the unfolded protein response, events essential to modulate ER stress and ensure PC survival. Thus, enhancing PI3Kδ activity may provide a novel means to trigger early PC death and dampen autoantibody responses.
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.
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•Screen for inhibitors disrupting bacterial AB toxins vesicular trafficking pathways•C910 affects EEA1/Rab5-positive early endosome morphology and sorting functions•C910 protects cells against eight AB toxins, SARS-CoV-2 and influenza A virus•C910 accumulates in lung tissues and protects mice against influenza A virus
Biological sciences; Microbiology
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.
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.
Abstract
B cell activation by self-antigen and signaling through Toll-like receptors (TLR) such as TLR7 and TLR9 has been implicated in the development of autoimmunity. We have recently identified a ...mechanism by which αvβ3 integrin limit excessive B cell responses to antigens containing TLR ligands, including self-antigens. We have previously reported that mice in which αv integrins are selectively deleted from B cells develop increased levels of autoantibodies to RNA and double stranded DNA.
Based on these findings, we hypothesized that deletion of αv integrins would accelerate TLR dependent autoimmunity. To study this further, we crossed mice lacking αv integrins on B cells (αv-CD19 mice) with mice expressing increased levels of TLR7 (TLR7.1tg mice) that develop Lupus-like autoimmunity. We find that the resulting αv-CD19 TLR7tg mice had increased spleen weight in comparison with age-matched TLR7tg littermates. αv-CD19 TLR7tg mice also had increased number of IgG2c+ plasma cells and increased autoantibodies to RNA, all indicative of enhanced B Cell activation and development of autoimmunity. In addition, deletion of αv integrin on B Cells in this model leads to enhanced activation of T Cells. Currently, we are investigating the role of αv integrins on B Cell subpopulations in driving T Cell activation and antigen presentation in this model of autoimmunity.
B Cells contribute to autoimmunity by activating T Cells in addition to producing autoantibodies and cytokines. These studies allow us to understand how altered B Cell TLR signaling can lead to enhanced immune activation in autoimmunity and highlight the importance of αv integrins in regulating this process.
Abstract
Immunoglobulin A (IgA) is the main antibody isotype found in the intestine: deficiency in IgA alters microbiome composition, leads to increased susceptibility to enteric pathogens and ...reduces tissue response to oral vaccine. IgA can bind multiple unrelated bacterial taxa, but mechanistic insights into the generation of such cross-species reactivity are lacking. IgA+ B cells arise during germinal center (GC) reaction in Peyer’s patches: however, the role of IgA B cell receptor (BCR) in shaping the humoral response at the intestinal interface remains unknown.
Here we showed that GC B cells lacking IgA are rapidly outcompeted by IgA+ B cells during GC reaction in Peyer’s patches and they are unable to contribute to memory B cell and intestinal-homing plasma cell compartments. IgA BCR did not impact cell proliferation in dark zone, and ectopic Bcl-2 expression was unable to rescue IgA-deficient GC B cells in mixed bone marrow chimeras. In contrast, IgA-deficient GC B cells underwent increased apoptosis in the light zone. In line with these findings, we observed that IgA BCR mediated faster and stronger intracellular Ca2+ signaling and led to increase BCR-dependent phosphorylation events. Mechanistically, IgA BCR signaling conferred resistance to Fas ligand (FasL)-dependent cell death in vitro, and genetic inhibition of Fas-FasL pathway restored IgA-deficient B cell fitness during GC reaction in vivo.
Thus, our results showed that IgA-expressing GC B cells are protected from FasL counterselection in the light zone via enhanced BCR intracellular signaling. This process might allow low affinity B cell clones to participate to the GC reaction and assure a comprehensive humoral response to poorly immunogenic commensal species.
Supported by grants from NIH (T32 AI132152, T32 AI007349 and R01 AI155727-02)
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