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.
Proliferation in hematopoietic stem cells (HSCs) has been linked to stemness. A variety of mouse transgenic lines with increased HSC proliferation rates have shown concomitant deterioration of their ...function (Dev. Dyn. 2016). While some studies implicated DNA damage accumulation as the underlying reason (Walter D. et al. in Nature 2015), others have maintained that, in fact, quiescence of primitive HSCs might be the reason behind poor resolution of DNA damages (Mohrin M et al. in Cell Stem Cell 2010). In our earlier studies, we demonstrated that outside-in integrin signaling is important to maintain HSC quiescence (Khurana S. et al. in Nat. Comm. 2016). We showed loss of HSC function in mice deficient in interaction between Integrin-αv (Itgav; Vav-Itgav-/- mice) and its ligand Periostin (Postn; Postn-/- mice). Our recent studies show that increase in proliferation of fetal liver (FL) HSCs in these mice results in their efficient expansion without any loss of function. Postn deficient FL tissues showed clear increase in the frequency of primitive HSCs with long-term engraftment potential. They proliferated faster, without any functional decline. However, Vav-Itgav-/- FL HSCs transiting from proliferative to quiescent phenotype showed dependence on Itgav-Postn interaction for quiescence and functional integrity. On the basis of RNASeq based differential pathway analysis using BM and E14.5 FL derived HSCs, we hypothesized that better DNA damage response (DDR) pathways in FL derived HSCs could result in these observations. In deed we observed that HSCs derived from FL show better resistance to DNA damage than adult bone marrow. We went on to show the importance of integrin ligand Postn in the creation of hematopoietic niche in FL tissue. Overall, we propose through our results that proliferation in HSCs affects HSC function in developmental context dependent manner.
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.
The mouse retina is vascularized after birth when angiogenic blood vessels grow and sprout along a pre-formed latticework of astrocytes. How astrocyte-derived cues control patterns of blood vessel ...growth and sprouting, however, remains enigmatic. Here, we have used molecular genetic strategies in mice to demonstrate that αvβ8 integrin expressed in astrocytes is essential for neovascularization of the developing retina. Selective ablation of αv or β8 integrin gene expression in astrocytes leads to impaired blood vessel sprouting and intraretinal hemorrhage, particularly during formation of the secondary vascular plexus. These pathologies correlate, in part, with diminished αvβ8 integrin-mediated activation of extracellular matrix-bound latent transforming growth factor βs (TGFβs) and defective TGFβ signaling in vascular endothelial cells, but not astrocytes. Collectively, our data demonstrate that αvβ8 integrin is a component of a paracrine signaling axis that links astrocytes to blood vessels and is essential for proper regulation of retinal angiogenesis.
The association between acute pancreatitis (AP) and diabetes mellitus (DM) has long been established, with the initial descriptions of AP patients presenting with DM after a bout of AP published in ...the 1940s and 50s. However, the potential mechanisms involved, particularly those components related to the immune system, have not been well defined. The Diabetes RElated to Acute pancreatitis and its Mechanisms (DREAM) study is a multicenter clinical study designed to understand the frequency and phenotype of DM developing after AP. This article describes one objective of the DREAM study: to determine the immunologic mechanisms of DM after AP, including the contribution of β-cell autoimmunity. This component of the study will assess the presence of islet autoimmunity, as well as the magnitude and kinetics of the innate and adaptive immune response at enrollment and during longitudinal follow-up after 1 or more episodes of AP. Finally, DREAM will evaluate the relationship between immune features, DM development, and pancreatitis etiology and severity.
Abstract
Signaling through Toll-like receptors (TLR) such as TLR7 and TLR9 has been implicated in B cell activation by self-antigens and development of autoimmunity. Our recent work based on murine ...models has identified a novel mechanism by which a family of adhesion molecules called αv integrins and the autophagy pathway limit excessive B cell responses to antigens containing TLR ligands and self-derived antigens.
Specifically, we have found that αvβ3 integrin regulates B cell TLR signaling by directing trafficking and maturation of TLR containing endosomes and engaging components of the autophagy pathway. When B cells lack either αv integrins or autophagy components LC3 and Atg5 endosomal trafficking of the TLRs is disrupted and leads to enhanced TLR signaling and increased B cell responses. We have used av conditional knockout mice to determine the consequences of loss of this αvβ3-mediated immune regulation. Mice lacking B cell av show increased antibody responses to antigens containing TLR-ligands and develop increased levels of autoantibodies.
We therefore propose that αv-mediated TLR regulation serves to maintain the balance between protective immunity to microbes and potential pathological autoimmune responses. We are currently studying the role of αv signaling and autophagy components in development of class switched, high affinity antibodies in response to antigens in the context of infection with a pathogen or in response to self-antigens.
Abstract
Ebola virus causes sporadic outbreaks of severe hemorrhagic fever. Owing to the high mortality rate, it poses a significant health risk and is a potential bioterrorism agent. However, our ...knowledge of how it usurps host cells to complete its life cycle, and more importantly, how this can be combated, is limited.
Here we describe a genome-wide forward genetic approach to identify host genes, known as restriction factors, which confer resistance to viral infection. In this system cells are mutagenized with a transposon that activates genes close to its insertion site. Libraries of mutagenized cells are screened with a cytotoxic virus and resistant cell populations are selected. Illumina sequencing is used to characterize transposon insertion sites and identify candidate resistance genes.
We have performed this screen in five human cell lines to identify genes conferring resistance to either vesicular stomatitis virus (VSV) or recombinant VSV bearing the Ebola virus surface glycoprotein. We have identified genes associated with resistance to only Ebola-VSV, or to both viruses, demonstrating that resistance occurs by targeting either viral entry or viral replication. One of the genes our screen identified is NPC1, the Ebola virus entry receptor, thus confirming that this is a valid method of identifying virus-host interactions. We have also identified a number of novel candidate Ebola restriction factors, which we are characterizing further, and are extending the screen to additional cell lines and viruses. This work provides new insights into host anti-viral defense mechanisms and may identify novel therapeutic targets that can be utilized to promote host-encoded anti-viral immunity.
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-β 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-β during T cell stimulation and thereby promoted differentiation of Th17 cells. Furthermore, pharmacologic inhibition of αv integrins using cyclic RGD peptides blocked TGF-β activation and Th17 cell generation in vitro and protected mice from EAE. These data demonstrate that activation of TGF-β 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.
Most of the squamous cell carcinomas (SCCs) of the skin and head and neck contain p53 mutations. The presence of p53 mutations in premalignant lesions suggests that they represent early events during ...tumor progression and additional alterations may be required for SCC development. Here we show that codeletion of the p53 and αv integrin genes in mouse stratified epithelia induced SCCs in 100% of the mice, more frequently and with much shorter latency than deletion of either gene alone. The SCCs that lacked p53 and αv in the epithelial tumor cells exhibited high Akt activity, lacked multiple types of infiltrating immune cells, contained a defective vasculature and grew slower than tumors that expressed p53 or αv. These results reveal that loss of αv in epithelial cells that lack p53 promotes SCC development, but also prevents remodeling of the tumor microenvironment and delays tumor growth. We observed that Akt inactivation in SCC cells that lack p53 and αv promoted anoikis. Thus, tumors may arise in these mice as a result of the increased cell survival induced by Akt activation triggered by loss of αv and p53, and by the defective recruitment of immune cells to these tumors, which may allow immune evasion. However, the defective vasculature and lack of a supportive stroma create a restrictive microenvironment in these SCCs that slows their growth. These mechanisms may underlie the rapid onset and slow growth of SCCs that lack p53 and αv.
Abstract
Signaling through Toll-like receptors (TLR) has been implicated in activation of immune cell populations such as B lymphocytes. TLR ligands associated with self-antigens can drive increased ...self-reactive B cell responses however TLR ligands present in pathogenic products or vaccines can also enhance protective antibody responses. Our recent work has identified a novel mechanism by which αv integrins, a family of adhesion molecules and the autophagy proteins limit excessive B cell TLR signaling.
Specifically, we have found that αvβ3 integrin regulates TLR signaling (NFKB and IRF7 activation) by directing maturation of TLR containing endosomes, through activation of components of the autophagy pathway (LC3 and Atg5). B cells lacking either αv integrins or autophagy components show enhanced TLR signaling and increased proliferative responses to TLR ligands. Mice with disruption in this pathway develop increased autoantibodies with age and develop accelerated autoimmunity in murine lupus models. Therefore, we propose that αv-mediated regulation of TLR signaling exists to limit excessive B cell responses to self-antigens.
In addition, we find that mice in which αv is specifically deleted from B cells mount stronger antibody responses when immunized with exogenous antigens containing TLR-ligand adjuvants. Immune responses to these antigens are characterized by increased expansion of germinal center cells, increased somatic hyper-mutation and higher levels of high-affinity class-switched antibodies. Therefore this αv mediated regulation of TLR signaling not only regulates B cell responses to self-antigens but it is also important for regulating germinal center B cell responses and production of high-affinity antibodies.
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