Dual signaling by innate and adaptive immune receptors is required for TLR7-induced B-cell–mediated autoimmunity

Toll-like receptor 7 (Tlr7) has been linked to systemic lupus disease incidence in humans and mice, but how TLR7 potentiates autoimmunity is unclear. We used a Tlr7 transgenic (tg) mouse model to investigate the cellular and molecular events required to induce spontaneous autoimmunity through increased TLR7 activity. We determined that Tlr7 exerts B-cell–intrinsic effects in promoting spontaneous germinal center (GC) and plasmablast B-cell development, and that these B-cell subsets are dependent on T-cell–derived signals through CD40L and SLAM-associated protein (SAP), but not IL-17. Antigen specificity also factored into TLR7-induced disease, as both a restricted T cell receptor (TCR) specificity and MHC haplotype H2^k/k protected Tlr7tg mice from spontaneous lymphocyte activation and autoantibody production. Inflammatory myeloid cell expansion and autoimmunity did not develop in Tlr7tgIgH^−/− mice, suggesting either that spontaneous TLR7 activation does not occur in dendritic cells, or, if it does occur, cannot drive these events in the absence of B-cell aid. These data indicate that autoimmune disease in Tlr7tg mice is contingent upon B cells receiving stimulation both through innate pathways and T-cell–derived signals and suggest a codependent relationship between B cells and T cells in the development of autoimmunity.     

Global phosphorylation analysis of β-arrestin–mediated signaling downstream of a seven transmembrane receptor (7TMR)

β-Arrestin–mediated signaling downstream of seven transmembrane receptors (7TMRs) is a relatively new paradigm for signaling by these receptors. We examined changes in protein phosphorylation occurring when HEK293 cells expressing the angiotensin II type 1A receptor (AT1aR) were stimulated with the β-arrestin–biased ligand Sar¹, Ile⁴, Ile⁸-angiotensin (SII), a ligand previously found to signal through β-arrestin–dependent, G protein-independent mechanisms. Using a phospho-antibody array containing 46 antibodies against signaling molecules, we found that phosphorylation of 35 proteins increased upon SII stimulation. These SII-mediated phosphorylation events were abrogated after depletion of β-arrestin 2 through siRNA-mediated knockdown. We also performed an MS-based quantitative phosphoproteome analysis after SII stimulation using a strategy of stable isotope labeling of amino acids in cell culture (SILAC). We identified 1,555 phosphoproteins (4,552 unique phosphopeptides), of which 171 proteins (222 phosphopeptides) showed increased phosphorylation, and 53 (66 phosphopeptides) showed decreased phosphorylation upon SII stimulation of the AT1aR. This study identified 38 protein kinases and three phosphatases whose phosphorylation status changed upon SII treatment. Using computational approaches, we performed system-based analyses examining the β-arrestin–mediated phosphoproteome including construction of a kinase-substrate network for β-arrestin–mediated AT1aR signaling. Our analysis demonstrates that β-arrestin–dependent signaling processes are more diverse than previously appreciated. Notably, our analysis identifies an AT1aR-mediated cytoskeletal reorganization network whereby β-arrestin regulates phosphorylation of several key proteins, including cofilin and slingshot. This study provides a system-based view of β-arrestin–mediated phosphorylation events downstream of a 7TMR and opens avenues for research in a rapidly evolving area of 7TMR signaling.     

Structure of the trypanosome haptoglobin–hemoglobin receptor and implications for nutrient uptake and innate immunity

African trypanosomes are protected by a densely packed surface monolayer of variant surface glycoprotein (VSG). A haptoglobin–hemoglobin receptor (HpHbR) within this VSG coat mediates heme acquisition. HpHbR is also exploited by the human host to mediate endocytosis of trypanolytic factor (TLF)1 from serum, contributing to innate immunity. Here, the crystal structure of HpHbR from Trypanosoma congolense has been solved, revealing an elongated three α-helical bundle with a small membrane distal head. To understand the receptor in the context of the VSG layer, the dimensions of Trypanosoma brucei HpHbR and VSG have been determined by small-angle X-ray scattering, revealing the receptor to be more elongated than VSG. It is, therefore, likely that the receptor protrudes above the VSG layer and unlikely that the VSG coat can prevent immunoglobulin binding to the receptor. The HpHb-binding site has been mapped by single-residue mutagenesis and surface plasmon resonance. This site is located where it is readily accessible above the VSG layer. A single HbHpR polymorphism unique to human infective T. brucei gambiense has been shown to be sufficient to reduce binding of both HpHb and TLF1, modulating ligand affinity in a delicate balancing act that allows nutrient acquisition but avoids TLF1 uptake.African trypanosomes infect humans and domestic and game animals, causing disease and placing a large constraint on the agricultural productivity of rural sub-Saharan Africa (1). Infection is transmitted by tsetse flies, and, once established in the mammalian host, the trypanosomes multiply in the bloodstream and tissue spaces. Infection can persist for years because of a population-survival strategy based on autoregulation of parasitaemia and a sophisticated system of antigenic variation that produces novel variants at a frequency sufficient to avoid complete clearance by the immune response (2, 3). This antigenic variation is based on a single protein, the variant surface glycoprotein (VSG). Only one VSG is expressed at any one time and an antigenic switch follows either a gene conversion from the genomic reservoir of VSG genes or an epigenetic switch that activates a VSG gene in a different expression site (4). In addition to its role in antigenic variation, VSG also protects the underlying plasma membrane as it forms a coat that covers the entire external surface with a packing density approaching the maximum possible (5) and sufficient to shield epitopes adjacent to the plasma membrane (6).Receptors within the VSG coat mediate uptake of large ligands from the host, the two best-characterized being the transferrin receptor for iron (7, 8) and the haptoglobin–hemoglobin receptor (HpHbR) for heme (9). VSG is an elongated homodimer attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (10, 11), and any receptor must be able to bind ligand in the context of the VSG coat. The structure of the transferrin receptor has not been determined, but there is evidence that it has a GPI-anchor, is structurally related to VSGs (13, 14), and that the ligand-binding site is distal to the plasma membrane (14). Modeling has suggested that this location and the number, size, and position of N-linked oligosaccharides facilitate ligand access (15). The HpHbR shows little apparent sequence similarity to VSGs and is less well characterized but is also linked to the plasma membrane through a C-terminal GPI anchor.HpHbR also plays a central role in determining whether humans can be infected by trypanosomes. Most African trypanosomes, such as Trypanosoma brucei brucei and Trypanosoma congolense, cannot infect humans because of innate immunity (16). For T. brucei brucei, this is mediated by trypanolytic factors 1 and 2 (TLF1 and TLF2) (17–19). TLFs are characterized by the presence of apolipoprotein (Apo)L1 and haptoglobin-related protein (Hpr) (9, 20). In TLF1, Hpr is complexed with hemoglobin (HprHb) (21), and TLF1 uptake occurs via binding of the HprHb component to the receptor (22–24). After endocytosis of TLF1, ApoL1 is trafficked to the lysosome, where it causes swelling and rupture, resulting in cell death (25–27). Human infective trypanosomes have overcome this innate immunity. In East Africa, T. brucei rhodesiense expresses the serum resistance-associated (SRA) protein (28–30), which binds to and inactivates ApoL1 (23). T. brucei gambiense, the human infective form present in West Africa, does not contain the SRA gene, and it has been proposed that resistance to TLF1 results from reduced uptake caused by reduced expression and sequence polymorphisms in HpHbR (31). Indeed, HpHbR deletion in T. brucei brucei disrupts TLF1 uptake, and expression of the receptor from T. brucei gambiense cannot restore this (31).Here, the structure of a trypanosome receptor, HpHbR, from T. congolense is reported, and the HpHb-binding site is identified. HpHbR is an elongated three α-helical bundle with a small head structure that is distal to the C-terminal GPI-anchor attachment site. This head structure contains the ligand-binding site. The relative dimensions of HpHbR and VSG suggest that the receptor protrudes above the VSG layer, rendering the binding site accessible to ligand but also making it unlikely that the VSG coat can prevent immunoglobulin binding to the receptor. A single HbHpR polymorphism unique to human infective T. brucei gambiense is sufficient to reduce binding of both HpHb and TLF1, altering ligand affinity in a delicate balancing act that retains nutrient acquisition but avoids uptake of TLF1.     

Involvement of the Toll-like receptor 4 pathway and use of TNF-α antagonists for treatment of the mucopolysaccharidoses

Enzyme replacement therapy is currently available for three of the mucopolysaccharidoses (MPSs) but has limited effects on the skeletal lesions. We investigated the involvement of the Toll-like receptor 4 (TLR4) signaling pathway in the pathogenesis of MPS bone and joint disease, and the use of the anti-TNF-α drug, Remicade (Centocor, Inc.), for treatment. TLR4 KO (TLR4^(lps−/−)) mice were interbred with MPS VII mice to produce double-KO (DKO) animals. The DKO mice had longer and thinner faces and longer femora as revealed by micro-computed tomography analysis compared with MPS VII mice. Histological analyses also revealed more organized and thinner growth plates. The serum levels of TNF-α were normalized in the DKO animals, and the levels of phosphorylated STAT1 and STAT3 in articular chondrocytes were corrected. These findings led us to evaluate the effects of Remicade in MPS VI rats. When initiated at 1 month of age, i.v. treatment prevented the elevation of TNF-α, receptor activator of NF-κB, and other inflammatory molecules not only in the blood but in articular chondrocytes and fibroblast-like synoviocytes (FLSs). Treatment of 6-month-old animals also reduced the levels of these molecules to normal. The number of apoptotic articular chondrocytes in MPS VI rats was similarly reduced, with less infiltration of synovial tissue into the underlying bone. These studies revealed the important role of TLR4 signaling in MPS bone and joint disease and suggest that targeting TNF-α may have positive therapeutic effects.     

Rapid signaling actions of environmental estrogens in developing granule cell neurons are mediated by estrogen receptor ß

Estrogenic endocrine disrupting chemicals (EDCs) constitute a diverse group of man-made chemicals and natural compounds derived from plants and microbial metabolism. Estrogen-like actions are mediated via the nuclear hormone receptor activity of estrogen receptor (ER)α and ERβ and rapid regulation of intracellular signaling cascades. Previous study defined cerebellar granule cell neurons as estrogen responsive and that granule cell precursor viability was developmentally sensitive to estrogens. In this study experiments using Western blot analysis and pharmacological approaches have characterized the receptor and signaling modes of action of selective and nonselective estrogen ligands in developing cerebellar granule cells. Estrogen treatments were found to briefly increase ERK1/2-phosphorylation and then cause prolonged depression of ERK1/2 activity. The sensitivity of granule cell precursors to estrogen-induced cell death was found to require the integrated activation of membrane and intracellular ER signaling pathways. The sensitivity of granule cells to selective and nonselective ER agonists and a variety of estrogenic and nonestrogenic EDCs was also examined. The ERβ selective agonist DPN, but not the ERα selective agonist 4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol or other ERα-specific ligands, stimulated cell death. Only EDCs with selective or nonselective ERβ activities like daidzein, equol, diethylstilbestrol, and bisphenol A were observed to induce E2-like neurotoxicity supporting the conclusion that estrogen sensitivity in granule cells is mediated via ERβ. The presented results also demonstrate the utility of estrogen sensitive developing granule cells as an in vitro assay for elucidating rapid estrogen-signaling mechanisms and to detect EDCs that act at ERβ to rapidly regulate intracellular signaling.     

Homeostatic IL-23 receptor signaling limits Th17 response through IL-22–mediated containment of commensal microbiota

Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, including the intestinal tract. In mice, the presence of segmented filamentous bacteria (SFB) promotes Th17 differentiation and the development of autoimmune disease. Here, we demonstrate that the IL-23 pathway dynamically regulates the abundance of SFB as well as mucosal barrier function in the adult animal. Genetic or pharmacological inactivation of the pathway selectively perturbs the abundance of a small group of commensals, including SFB, and results in an impaired mucosal barrier. Defective barrier function leads to systemic dissemination of microbial products, provoking induction of the IL-23 pathway with dual consequences: IL-23 drives IL-22 production to reinforce mucosal barrier function and elicit antimicrobial activities, and it also drives the differentiation of Th17 cells in an attempt to combat escaped microbes in the lamina propria and in distal tissues. Thus, barrier defects generate a systemic environment that facilitates Th17 development.The human gastrointestinal tract harbors an estimated 500–1,000 distinct bacterial species, and the total numbers of intestinal bacteria are in the trillions (1–3). Although the presence of microbiota is required for immune system development, the host depends on an intact barrier to contain the microbiota in their assigned niches, and on an effective immune system to destroy bacteria that may have escaped and invaded host tissues. It is thought that diseases such as inflammatory bowel disease and atopic dermatitis result, in part, from barrier defects, leading to bacterial invasion, breakdown of tolerance, and inflammation. Interestingly, germ-free mice or antibiotic-treated mice are resistant to intestinal as well as extraintestinal autoimmune diseases and defective in the generation of Th17 cells, a lineage of CD4⁺ T cells contributing to autoimmune pathologies (4–6), suggesting that immune cell education takes place in colonized tissues and may have consequences elsewhere in the organism.Genetic modification of the host was reported to result in altered composition of the intestinal microbial community (7, 8). However, it remains largely unknown to what extent commensal homeostasis can be modulated by host factors in the adult WT animal. Furthermore, previous literature strongly implicates IL-22 in the maintenance of gut homeostasis (9, 10). IL-22 production by T cells and innate lymphoid cells is potently induced by IL-23, an antigen-presenting cell–derived cytokine that is induced upon sensing of pathogen-associated molecular patterns or damage-associated molecular patterns (11–13). Thus, we hypothesized that IL-23 induction in response to segmented filamentous bacteria (SFB) colonization and/or systemic dissemination of microbial components following barrier disruption has two main consequences. First, IL-23 triggers IL-22 production to elicit antimicrobial peptide production, which reestablishes microbial containment. Second, IL-23 drives Th17 cell differentiation, which is critical to neutralize escaped commensal microbes in the lamina propria, and perhaps even in distal tissues. Here, we present data that are consistent with this model and suggest that microbial induction of the IL-23 pathway, while protecting the host through IL-22 activity, also generates a systemic environment supportive of Th17 cell development.     

Clonidine ameliorates cerebral ischemia–reperfusion injury by up-regulating the GluN3 subunits of NMDA receptor

Metabolic Brain Disease - This study aimed to investigate the protective effects of the alpha-2 adrenergic receptor (α2-AR) agonist, clonidine, on the cerebral ischemia–reperfusion (I/R)...     

Alterations of autophagic and innate immune responses by the Crohn’s disease-associated ATG16L1 mutation

Crohn’s disease (CD) is driven by the loss of tolerance to intestinal microbiota and excessive production of pro-inflammatory cytokines. These pro-inflammatory cytokines are produced by macrophages and dendritic cells (DCs) upon sensing the intestinal microbiota by the pattern recognition receptors (PRRs). Impaired activation of PRR-mediated signaling pathways is associated with chronic gastrointestinal inflammation, as shown by the fact that loss-of-function mutations in the nucleotide-binding oligomerization domain 2 gene increase the risk of CD development. Autophagy is an intracellular degradation process, during which cytoplasmic nutrients and intracellular pathogens are digested. Given that impaired reaction to intestinal microbiota alters signaling pathways mediated by PRRs, it is likely that dysfunction of the autophagic machinery is involved in the development of CD. Indeed, the loss-of-function mutation T300A in the autophagy related 16 like 1 (ATG16L1) protein, a critical regulator of autophagy, increases susceptibility to CD. Recent studies have provided evidence that ATG16L1 is involved not only in autophagy, but also in PRR-mediated signaling pathways. ATG16L1 negatively regulates pro-inflammatory cytokine responses of macrophages and DCs after these cells sense the intestinal microbiota by PRRs. Here, we discuss the molecular mechanisms underlying the development of CD in the T300A ATG16L1 mutation by focusing on PRR-mediated signaling pathways.     

Fas-associated death domain (FADD) is a negative regulator of T-cell receptor–mediated necroptosis

Cell death is an important mechanism to limit uncontrolled T-cell expansion during immune responses. Given the role of death-receptor adapter protein Fas-associated death domain (FADD) in apoptosis, it is intriguing that T-cell receptor (TCR)–induced proliferation is blocked in FADD-defective T cells. Necroptosis is an alternate form of death that can be induced by death receptors and is linked to autophagy. It requires the death domain-containing kinase RIP1 and, in certain instances, RIP3. FADD and its apoptotic partner, Caspase-8, have also been implicated in necroptosis. To accurately assess the role of FADD in mature T-cell proliferation and death, we generated a conditional T-cell–specific FADD knockout mouse strain. The T cells of these mice develop normally, but lack FADD at the mature stage. FADD-deficient T cells respond poorly to TCR triggering, exhibit slow cell cycle entry, and fail to expand over time. We find that programmed necrosis occurs during the late stage of normal T-cell proliferation and that this process is greatly amplified in FADD-deficient T cells. Inhibition of necroptosis using an inhibitor of RIP1 kinase activity rescues the FADD knockout proliferative defect. However, TCR-induced necroptosis did not appear to require autophagy or involve RIP3. Consistent with their defective CD8 T-cell response, these mice succumb to Toxoplasma gondii infection more readily than wild-type mice. We conclude that FADD constitutes a mechanism to keep TCR-induced programmed necrotic signaling in check during early phases of T-cell clonal expansion.     

Molecular basis for SH3 domain regulation of F-BAR–mediated membrane deformation

Members of the Bin/amphiphysin/Rvs (BAR) domain protein superfamily are involved in membrane remodeling in various cellular pathways ranging from endocytic vesicle and T-tubule formation to cell migration and neuromorphogenesis. Membrane curvature induction and stabilization are encoded within the BAR or Fer-CIP4 homology-BAR (F-BAR) domains, α-helical coiled coils that dimerize into membrane-binding modules. BAR/F-BAR domain proteins often contain an SH3 domain, which recruits binding partners such as the oligomeric membrane-fissioning GTPase dynamin. How precisely BAR/F-BAR domain-mediated membrane deformation is regulated at the cellular level is unknown. Here we present the crystal structures of full-length syndapin 1 and its F-BAR domain. Our data show that syndapin 1 F-BAR-mediated membrane deformation is subject to autoinhibition by its SH3 domain. Release from the clamped conformation is driven by association of syndapin 1 SH3 with the proline-rich domain of dynamin 1, thereby unlocking its potent membrane-bending activity. We hypothesize that this mechanism might be commonly used to regulate BAR/F-BAR domain-induced membrane deformation and to potentially couple this process to dynamin-mediated fission. Our data thus suggest a structure-based model for SH3-mediated regulation of BAR/F-BAR domain function.