Neetu Gupta

Neutrophil Expression of Fas Ligand and Perforin Directs Effector CD8 T Cell Infiltration into Antigen-Challenged Skin

Contact hypersensitivity (CHS) is a T cell response to hapten skin challenge of sensitized individuals proposed to be mediated by hapten-primed CD8 cytolytic T cells. Effector CD8 T cell recruitment into hapten challenge sites to elicit CHS requires prior CXCL1- and CXCL2-mediated neutrophil infiltration into the site. We investigated whether neutrophil activities directing hapten-primed CD8 T cell skin infiltration in response to 2,4-dinitro-1-fluorobenzene (DNFB) required Fas ligand (FasL) and perforin expression. Although DNFB sensitization of gld/perforin−/− mice induced hapten-specific CD8 T cells producing IFN-γ and IL-17, these T cells did not infiltrate the DNFB challenge site to elicit CHS but did infiltrate the challenge site and elicit CHS when transferred to hapten-challenged naive wild-type recipients. Hapten-primed wild-type CD8 T cells, however, did not elicit CHS when transferred to naive gld/perforin−/− recipients. Wild-type bone marrow neutrophils expressed FasL and perforin, and when transferred to sensitized gld/perforin−/− mice, they restored hapten-primed CD8 T cell infiltration into the challenge site and CHS. The FasL/perforin-mediated activity of wild-type neutrophils induced the expression of T cell chemoattractants, CCL1, CCL2, and CCL5, within the hapten-challenged skin. These results indicate FasL/perforin-independent functions of hapten-primed CD8 T cells in CHS and identify new functions for neutrophils in regulating effector CD8 T cell recruitment and immune responses in the skin.

Spatial Coupling of JNK Activation to the B Cell Antigen Receptor by Tyrosine-Phosphorylated Ezrin

The ezrin-radixin-moesin proteins regulate B lymphocyte activation via their effect on BCR diffusion and microclustering. This relies on their ability to dynamically tether the plasma membrane with actin filaments that is in turn facilitated by phosphorylation of the conserved threonine residue in the actin-binding domain. In this study, we describe a novel function of ezrin in regulating JNK activation that is mediated by phosphorylation of a tyrosine (Y353) residue that is unconserved with moesin and radixin. BCR, but not CD40, TLR4, or CXCR5 stimulation, induced phosphorylation of ezrin at Y353 in mouse splenic B cells. Ezrin existed in a preformed complex with Syk in unstimulated B cells and underwent Syk-dependent phosphorylation upon anti-IgM stimulation. Y353-phosphorylated ezrin colocalized with the BCR within minutes of stimulation and cotrafficked with the endocytosed BCRs through the early and late endosomes. The T567 residue of ezrin was rephosphorylated in late endosomes and at the plasma membrane at later times of BCR stimulation. Expression of a nonphosphorylatable Y353F mutant of ezrin specifically impaired JNK activation. BCR crosslinking induced the association of Y353-phosphorylated ezrin with JNK and its kinase MAPKK7, as well as spatial colocalization with phosphorylated JNK in the endosomes. The yellow fluorescent protein–tagged Y353F mutant displayed reduced colocalization with the endocytosed BCR as compared with wild-type ezrin-yellow fluorescent protein. Taken together, our data identify a novel role for ezrin as a spatial adaptor that couples JNK signaling components to the BCR signalosome, thus facilitating JNK activation.

Re-defining ERM function in lymphocyte activation and migration

Lymphocyte activation and migration involve large‐scale actin cytoskeletal remodeling. The Ezrin–Radixin–Moesin (ERM) family proteins reversibly link the plasma membrane and cortical actin meshwork and mediate the dynamic nature of the membrane‐cytoskeletal interface to facilitate remodeling. The reversibility of this linkage is controlled by the conformation of ERM proteins and depends on the phosphorylation of a conserved threonine residue in the actin‐binding domain. Disruption of the phospho‐cycling nature of ERM proteins through dominant negative and constitutively active mutants results in impaired lymphocyte migration and activation. In recent years, a novel role has emerged for ERM proteins as signaling scaffolds that can modulate B and T‐cell activation through additional posttranslational modifications at tyrosine residues. Here, we highlight recent studies that have redefined the role of ERM proteins in lymphocyte activation and migration. We discuss how lymphocyte‐specific knockouts of ERM proteins and high resolution imaging techniques have identified a novel function for them as rheostats that modulate the strength of antigen receptor signaling in B cells. Finally, we describe scenarios in which ERM protein function is coopted by pathogens for their own transmission and speculate on the potential of ERM proteins for regulating undesirable lymphocyte behaviors such as autoimmunity and malignancy.

Ezrin Tunes the Magnitude of Humoral Immunity

Ezrin is a member of the ezrin–radixin–moesin family of membrane-actin cytoskeleton cross-linkers that participate in a variety of cellular processes. In B cells, phosphorylation of ezrin at different sites regulates multiple processes, such as lipid raft coalescence, BCR diffusion, microclustering, and endosomal JNK activation. In this study, we generated mice with conditional deletion of ezrin in the B cell lineage to investigate the physiological significance of ezrin’s function in Ag receptor–mediated B cell activation and humoral immunity. B cell development, as well as the proportion and numbers of major B cell subsets in peripheral lymphoid organs, was unaffected by the loss of ezrin. Using superresolution imaging methods, we show that, in the absence of ezrin, BCRs respond to Ag binding by accumulating into larger and more stable signaling microclusters. Loss of ezrin led to delayed BCR capping and accelerated lipid raft coalescence. Although proximal signaling proteins showed stronger activation in the absence of ezrin, components of the distal BCR signaling pathways displayed distinct effects. Ezrin deficiency resulted in increased B cell proliferation and differentiation into Ab-secreting cells ex vivo and stronger T cell–independent and -dependent responses to Ag in vivo. Overall, our data demonstrate that ezrin regulates amplification of BCR signals and tunes the strength of B cell activation and humoral immunity.

CD6 Receptor Regulates Intestinal Ischemia/Reperfusion-induced Injury by Modulating Natural IgM-producing B1a Cell Self-renewal.

Intestinal ischemia/reperfusion (I/R) injury is a relatively common pathological condition that can lead to multi-organ failure and mortality. Regulatory mechanism for this disease is poorly understood, although it is established that circulating pathogenic natural IgM, which is primarily produced by B1a cells outside of the peritoneal cavity, are integrally involved. CD6 was originally identified as a marker for T cells and was later found to be present on some subsets of B cells in humans; however, whether CD6 plays any role in intestinal I/R-induced injury and, if so, the underlying mechanisms, remain unknown. Here we report that CD6−/− mice were significantly protected from intestinal inflammation and mucosal damage compared with WT mice in a model of intestinal I/R-induced injury. Mechanistically, we found that CD6 was selectively expressed on B1 cells outside of the bone marrow and peritoneal cavity and that pathogenic natural IgM titers were reduced in the CD6−/− mice in association with significantly decreased B1a cell population. Our results reveal an unexpected role of CD6 in the pathogenesis of intestinal IR-induced injury by regulating the self-renewal of B1a cells.

Cutting Edge: Ezrin Regulates Inflammation by Limiting B Cell IL-10 Production

IL-10 produced by B cells is important for controlling inflammation, thus underscoring the need to identify mechanisms regulating its production. In this study, we demonstrate that conditional deletion of ezrin in B cells increases IL-10 production induced by TLR4 ligation. The MyD88-independent Toll/IL-1R domain–containing adapter inducing IFN-β–IFN regulatory factor 3 pathway is required for Ezrin-deficient B cells to produce higher IL-10 upon LPS stimulation. Treatment of B cells with a novel small-molecule inhibitor of ezrin induces its dephosphorylation and increases LPS-induced NF-κB and IFN regulatory factor 3 activation and IL-10 secretion, indicating a role for threonine 567 phosphorylation of ezrin in limiting IL-10. Loss of ezrin in B cells results in dampened proinflammatory response to a sublethal dose of LPS in vivo, which is dependent on increased IL-10 production. Taken together, our data yield new insights into molecular and membrane-cytoskeletal regulation of B cell IL-10 production and reveal ezrin as a potential therapeutic target in inflammatory diseases.

Cutting Edge: Deletion of Ezrin in B Cells of Lyn-Deficient Mice Downregulates Lupus Pathology

Genetic deletion of the Src family tyrosine kinase Lyn in mice recapitulates human systemic lupus erythematosus, characterized by hyperactive BCR signaling, splenomegaly, autoantibody generation, and glomerulonephritis. However, the molecular regulators of autoimmunity in Lyn-deficient mice and in human lupus remain poorly characterized. In this study, we report that conditional deletion of the membrane–cytoskeleton linker protein ezrin in B cells of Lyn-deficient mice (double knockout [DKO] mice) ameliorates B cell activation and lupus pathogenesis. B cells from DKO mice respond poorly to BCR stimulation, with severe downregulation of major signaling pathways. DKO mice exhibit reduced splenomegaly as well as significantly lower levels of autoantibodies against a variety of autoantigens, including dsDNA, histone, and chromatin. Leukocyte infiltration and deposition of IgG and complement component C3 in the kidney glomeruli of DKO mice are markedly reduced. Our data demonstrate that ezrin is a novel molecular regulator of B cell–associated lupus pathology.