Activation-Induced Cytidine Deaminase Impacts the Primary Antibody Repertoire in Naive Mice.

Genetic and environmental cues shape the evolution of the B cell Ig repertoire. Activation-induced cytidine deaminase (AID) is essential to generating Ig diversity through isotype class switching and somatic mutations, which then directly influence clonal selection. Impaired B cell development in AID-knockout mice has made it difficult to study Ig diversification in an aging repertoire. Therefore, in this report, we used a novel inducible AID-knockout mouse model and discovered that deleting AID in adult mice caused spontaneous germinal center formation. Deep sequencing of the IgH repertoire revealed that Ab diversification begins early in life and evolves over time. Our data suggest that activated B cells form germinal centers at steady state and facilitate continuous diversification of the B cell repertoire. In support, we identified shared B cell lineages that were class switched and showed age-dependent rates of mutation. Our data provide novel context to the genesis of the B cell repertoire that may benefit the understanding of autoimmunity and the strength of an immune response to infection.

The kinase IRAK4 promotes endosomal TLR and immune complex signaling in B cells and plasmacytoid dendritic cells.

The dysregulation of multiple signaling pathways, including those through endosomal Toll-like receptors (TLRs), Fc gamma receptors (FcγR), and antigen receptors in B cells (BCR), promote an autoinflammatory loop in systemic lupus erythematosus (SLE). Here, we used selective small-molecule inhibitors to assess the regulatory roles of interleukin-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Bruton's tyrosine kinase (BTK) in these pathways. The inhibition of IRAK4 repressed SLE immune complex- and TLR7-mediated activation of human plasmacytoid dendritic cells (pDCs). Correspondingly, the expression of interferon (IFN)-responsive genes (IRGs) in cells and in mice was positively regulated by the kinase activity of IRAK4. Both IRAK4 and BTK inhibition reduced the TLR7-mediated differentiation of human memory B cells into plasmablasts. TLR7-dependent inflammatory responses were differentially regulated by IRAK4 and BTK by cell type: In pDCs, IRAK4 positively regulated NF-κB and MAPK signaling, whereas in B cells, NF-κB and MAPK pathways were regulated by both BTK and IRAK4. In the pristane-induced lupus mouse model, inhibition of IRAK4 reduced the expression of IRGs during disease onset. Mice engineered to express kinase-deficient IRAK4 were protected from both chemical (pristane-induced) and genetic (NZB/W_F1 hybrid) models of lupus development. Our findings suggest that kinase inhibitors of IRAK4 might be a therapeutic in patients with SLE.

Critical role of activation induced cytidine deaminase in experimental autoimmune encephalomyelitis.

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE.

Inducible nitric oxide synthase and estradiol exhibit complementary neuroprotective roles after ischemic brain injury.

Estradiol-17beta exerts profound neuroprotective actions following cerebral ischemia through multiple molecular mechanisms. To examine the putative anti-inflammatory mechanisms employed by estradiol during stroke, we explored the interactions between estradiol and inducible nitric oxide synthase (iNOS) in both wildtype and iNOS knockout (iNOSKO) female mice following permanent middle cerebral artery occlusion (MCAO). Female mice were ovariectomized and treated with estradiol. One week later, they were subjected to MCAO, and then killed 24 h later. Analysis of total, cortical and striatal infarct volumes confirmed that estradiol is neuroprotective in wildtype mice. Infarct volumes were also significantly smaller in female iNOSKO female mice, but estradiol did not further decrease injury. We found that one mechanism by which estradiol may act is by decreasing nitric oxide synthase 2 gene expression in the cortex and in the striatum of wildtype mice. These results show that the pro-inflammatory actions of iNOS exacerbate stroke-induced injury within the cortex and striatum, and that iNOS deletion is neuroprotective in ovariectomized and estrogen-replaced female mice.

Timing of estrogen therapy after ovariectomy dictates the efficacy of its neuroprotective and antiinflammatory actions.

Recent studies describing the seemingly contradictory actions of estrogens in ischemic stroke injury have led us to reevaluate the circumstances under which estrogen therapy (ET) provides benefits against cerebral stroke and decipher its mechanisms of action. One prominent feature that follows stroke injury is massive central and peripheral inflammatory responses. Evidence now suggests that postischemic inflammatory responses strongly contribute to the extent of brain injury, and 17beta-estradiol (E(2)) may protect the ischemic brain by exerting antiinflammatory actions. In an attempt to explain recently reported dichotomous effects of E(2) in stroke injury, we tested the hypothesis that an extended period of hypoestrogenicity both prevents E(2) from protecting the brain against ischemia and simultaneously suppresses its antiinflammatory actions. We report that E(2) exerts profound neuroprotective action when administered immediately upon ovariectomy, but not when administered after 10 weeks of hypoestrogenicity. Consistently, E(2) treatment given immediately at the time of ovariectomy attenuated central and peripheral production of proinflammatory cytokines after ischemic stroke. In contrast, E(2) did not suppress production of proinflammatory molecules when it was administered after 10 weeks postovariectomy. These results demonstrate that a prolonged period of hypoestrogenicity disrupts both neuroprotective and antiinflammatory actions of E(2). Our findings may help to explain the results of the Women's Health initiative that reported no beneficial effect of ET against stroke because the majority of the subjects initiated ET after an extended period of hypoestrogenicity.

Estradiol enhances neurogenesis following ischemic stroke through estrogen receptors alpha and beta.

Neurogenesis persists throughout life under normal and degenerative conditions. The adult subventricular zone (SVZ) generates neural stem cells capable of differentiating to neuroblasts and migrating to the site of injury in response to brain insults. In the present study, we investigated whether estradiol increases neurogenesis in the SVZ in an animal model of stroke to potentially promote the ability of the brain to undergo repair. Ovariectomized C57BL/6J mice were implanted with capsules containing either vehicle or 17beta-estradiol, and 1 week later they underwent experimental ischemia. We utilized double-label immunocytochemistry to identify the phenotype of newborn cells (5-bromo-2'-deoxyuridine-labeled) with various cellular markers; doublecortin and PSA-NCAM as the early neuronal marker, NeuN to identify mature neurons, and glial fibrillary acidic protein to identify astrocytes. We report that low physiological levels of estradiol treatment, which exert no effect in the uninjured state, significantly increase the number of newborn neurons in the SVZ following stroke injury. This effect of estradiol is limited to the dorsal region of the SVZ and is absent from the ventral SVZ. The proliferative actions of estradiol are confined to neuronal precursors and do not influence gliosis. Furthermore, we show that both estrogen receptors alpha and beta play pivotal functional roles, insofar as knocking out either of these receptors blocks the ability of estradiol to increase neurogenesis. These findings clearly demonstrate that estradiol stimulates neurogenesis in the adult SVZ, thus potentially facilitating the brain to remodel and repair after injury.