Performance and Health Decrements Associated With Relative Energy Deficiency in Sport for Division I Women Athletes During a Collegiate Cross-Country Season: A Case Series.

The purpose of this case series was to evaluate the presence of low Energy Availability (EA) and its impact on components of Relative Energy Deficiency in Sport (RED-S) in a population of female collegiate runners. Seven female NCAA Division I athletes (age: 22.3 ± 1.5 yrs; height: 169.7 ± 5.7 cm; weight: 58.3 ± 4.1 kg) were tracked from August until February, covering the beginning (Pre XC), end (Post XC) of their competitive cross country season, and beginning of the following track season (Pre Track). The athletes were assessed for female athlete triad (Triad) risk, energy availability, body composition, resting metabolic rate (RMR), nutritional intake, and blood markers (including vitamin D, ferritin, and triiodothyronine (T3)). From Pre XC to Post XC there were no significant differences in body mass, fat free mass or body fat percentage. At Pre XC, mean EA was 31.6 ± 13.3 kcal/kg FFM∙d-1. From Post XC to Pre Track, there was a significant increase in body mass (59.1 ± 5.1 to 60.6 ± 5.7 kg, p<0.001,d=0.27). From Post XC to Pre Track, there was a significant increase in RMR (1466 ± 123.6 to 1614.6 ± 89.1 kcal·d-1, p<0.001,d=2.6). For 25(OH) vitamin D, there was a significant reduction from Pre XC to Post XC (44.1 ± 10.6 vs 39.5 ± 12.2 ng·mL-1, p=0.047,d=-0.4), and a significant increase from Post XC to Pre Track (39.5 ± 12.2 vs. 48.1 ± 10.4 ng·mL-1, p=0.014,d=0.75). For ferritin, there was a trend towards a decrease from Pre XC to Post XC (24.2 ± 13.2 vs. 15.7 ± 8.8 ng·mL-1, p=0.07, d=-0.75), as well as a trend toward an increase from Post XC to Pre Track (15.7 ± 8.8 vs. 34.1 ± 18.0 ng·mL-1, p=0.08, d=1.3). No differences in T3 were observed across time points. Average Triad risk score was 2.3 ± 1.4. Notably, 5 of 7 athletes met criteria for moderate risk. Despite many athletes meeting criteria for low EA and having elevated Triad risk assessment scores, most were able to maintain body mass and RMR. One athlete suffered severe performance decline and a reduced RMR. Surprisingly, she was the only athlete above the recommended value for ferritin. Following increased nutritional intake and reduced training volume, her performance and RMR recovered. Changes in body mass and body composition were not indicative of the presence of other concerns associated with RED-S. This exploratory work serves as a guide for future, larger studies for tracking athletes, using RMR and nutritional biomarkers to assess RED-S.

Cell Type- and Stimulation-Dependent Transcriptional Programs Regulated by Atg16L1 and Its Crohn's Disease Risk Variant T300A.

Genome-wide association studies have identified common genetic variants impacting human diseases; however, there are indications that the functional consequences of genetic polymorphisms can be distinct depending on cell type-specific contexts, which produce divergent phenotypic outcomes. Thus, the functional impact of genetic variation and the underlying mechanisms of disease risk are modified by cell type-specific effects of genotype on pathological phenotypes. In this study, we extend these concepts to interrogate the interdependence of cell type- and stimulation-specific programs influenced by the core autophagy gene Atg16L1 and its T300A coding polymorphism identified by genome-wide association studies as linked with increased risk of Crohn's disease. We applied a stimulation-based perturbational profiling approach to define Atg16L1 T300A phenotypes in dendritic cells and T lymphocytes. Accordingly, we identified stimulus-specific transcriptional signatures revealing T300A-dependent functional phenotypes that mechanistically link inflammatory cytokines, IFN response genes, steroid biosynthesis, and lipid metabolism in dendritic cells and iron homeostasis and lysosomal biogenesis in T lymphocytes. Collectively, these studies highlight the combined effects of Atg16L1 genetic variation and stimulatory context on immune function.

Efficacy of fenugreek seed extract on men's psychological and physical health: a randomized placebo-controlled double-blind clinical trial.

OBJECTIVES: Limited research has examined the effects of fenugreek (Trigonella foenum-graecum L.) supplementation to improve healthy younger men's aging male symptoms. The study objective was to examine whether a fenugreek seed extract would improve healthy men's aging male symptoms, health-related quality of life (HRQoL), grip strength, and anxiety. METHODS: Randomized double-blind placebo-controlled trial was employed, using a parallel design, with assessments at baseline, Day 30, and Day 60. Healthy male volunteers (n = 57, mean age = 26.1 years) were randomized to: fenugreek 400 mg/d (n = 19), fenugreek 500 mg/d (n = 19), or placebo group (n = 19). RESULTS: The fenugreek groups reported significant improvements in aging male symptoms, anxiety levels, grip strength, and indicators of HRQoL compared to the placebo group, p's < 0.05. No adverse events were reported. CONCLUSION: Fenugreek supplementation is an effective nutritional intervention for improving aging male symptoms, anxiety levels, grip strength, and aspects of HRQoL in healthy recreationally active men. Future researchers are encouraged to examine the health and ergogenic effects of fenugreek supplementation in hypogonadal and older populations. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT03528538.

Distinct Tissue-Specific Roles for the Disease-Associated Autophagy Genes ATG16L2 and ATG16L1.

The clear role of autophagy in human inflammatory diseases such as Crohn disease was first identified by genome-wide association studies and subsequently dissected in multiple mechanistic studies. ATG16L1 has been particularly well studied in knockout and hypomorph settings as well as models recapitulating the Crohn disease-associated T300A polymorphism. Interestingly, ATG16L1 has a single homolog, ATG16L2, which is independently implicated in diseases, including Crohn disease and systemic lupus erythematosus. However, the contribution of ATG16L2 to canonical autophagy pathways and other cellular functions is poorly understood. To better understand its role, we generated and analyzed the first, to our knowledge, ATG16L2 knockout mouse. Our results show that ATG16L1 and ATG16L2 contribute very distinctly to autophagy and cellular ontogeny in myeloid, lymphoid, and epithelial lineages. Dysregulation of any of these lineages could contribute to complex diseases like Crohn disease and systemic lupus erythematosus, highlighting the value of examining cell-specific effects. We also identify a novel genetic interaction between ATG16L2 and epithelial ATG16L1. These findings are discussed in the context of how these genes may contribute distinctly to human disease.

The Crohn's disease polymorphism, ATG16L1 T300A, alters the gut microbiota and enhances the local Th1/Th17 response.

Inflammatory bowel disease (IBD) is driven by dysfunction between host genetics, the microbiota, and immune system. Knowledge gaps remain regarding how IBD genetic risk loci drive gut microbiota changes. The Crohn's disease risk allele ATG16L1 T300A results in abnormal Paneth cells due to decreased selective autophagy, increased cytokine release, and decreased intracellular bacterial clearance. To unravel the effects of ATG16L1 T300A on the microbiota and immune system, we employed a gnotobiotic model using human fecal transfers into ATG16L1 T300A knock-in mice. We observed increases in Bacteroides ovatus and Th1 and Th17 cells in ATG16L1 T300A mice. Association of altered Schaedler flora mice with B. ovatus specifically increased Th17 cells selectively in ATG16L1 T300A knock-in mice. Changes occur before disease onset, suggesting that ATG16L1 T300A contributes to dysbiosis and immune infiltration prior to disease symptoms. Our work provides insight for future studies on IBD subtypes, IBD patient treatment and diagnostics.

Antigen discovery and specification of immunodominance hierarchies for MHCII-restricted epitopes.

Identifying immunodominant T cell epitopes remains a significant challenge in the context of infectious disease, autoimmunity, and immuno-oncology. To address the challenge of antigen discovery, we developed a quantitative proteomic approach that enabled unbiased identification of major histocompatibility complex class II (MHCII)-associated peptide epitopes and biochemical features of antigenicity. On the basis of these data, we trained a deep neural network model for genome-scale predictions of immunodominant MHCII-restricted epitopes. We named this model bacteria originated T cell antigen (BOTA) predictor. In validation studies, BOTA accurately predicted novel CD4 T cell epitopes derived from the model pathogen Listeria monocytogenes and the commensal microorganism Muribaculum intestinale. To conclusively define immunodominant T cell epitopes predicted by BOTA, we developed a high-throughput approach to screen DNA-encoded peptide-MHCII libraries for functional recognition by T cell receptors identified from single-cell RNA sequencing. Collectively, these studies provide a framework for defining the immunodominance landscape across a broad range of immune pathologies.

p40 phox -Deficient Mice Exhibit Impaired Bacterial Clearance and Enhanced Pro-inflammatory Responses during Salmonella enterica serovar Typhimurium Infection.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of acute gastroenteritis in humans. During infection, reactive oxygen species (ROS), generated from NADPH oxidase (a multisubunit enzyme complex), are required for pathogen killing upon phagocytosis and for regulating pro-inflammatory signaling in phagocytic cells. Mutations in subunits forming the NADPH complex may lead to enhanced susceptibility to infection and inflammatory disease. Compared to other NADPH oxidase subunits, the function of p40 phox is relatively understudied, particularly in the context of intestinal bacterial infection. In this study, we utilized genetically engineered mice to determine the role of p40 phox in the response to S. Typhimurium infection. We show that mice lacking p40 phox are more susceptible to oral infection with S. Typhimurium, as demonstrated by significantly enhanced bacterial dissemination to spleen and liver, and development of exacerbated bacterial colitis. Moreover, we demonstrate that the increased infection and disease severity are correlated with markedly increased F4/80+ macrophage and Ly6G+ neutrophil infiltration in the infected tissues, coincident with significantly elevated pro-inflammatory cytokines (IL-1ß and TNF-α) and chemoattractant molecules in the infected tissues. Functional analysis of macrophages and neutrophils further shows that p40 phox deficiency impairs bacteria- or PMA-induced intracellular ROS production as well as intracellular killing of Salmonella. These observations indicate that the p40 phox subunit of NADPH oxidase plays an essential role in suppressing intracellular multiplication of Salmonella in macrophages and in the regulation of both systemic and mucosal inflammatory responses to bacterial infection.

αv Integrins combine with LC3 and atg5 to regulate Toll-like receptor signalling in B cells.

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 ß3 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.

Ubiquitin Ligase TRIM62 Regulates CARD9-Mediated Anti-fungal Immunity and Intestinal Inflammation.

CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation.

Small-molecule enhancers of autophagy modulate cellular disease phenotypes suggested by human genetics.

Studies of human genetics and pathophysiology have implicated the regulation of autophagy in inflammation, neurodegeneration, infection, and autoimmunity. These findings have motivated the use of small-molecule probes to study how modulation of autophagy affects disease-associated phenotypes. Here, we describe the discovery of the small-molecule probe BRD5631 that is derived from diversity-oriented synthesis and enhances autophagy through an mTOR-independent pathway. We demonstrate that BRD5631 affects several cellular disease phenotypes previously linked to autophagy, including protein aggregation, cell survival, bacterial replication, and inflammatory cytokine production. BRD5631 can serve as a valuable tool for studying the role of autophagy in the context of cellular homeostasis and disease.

The kinase DYRK1A reciprocally regulates the differentiation of Th17 and regulatory T cells.

The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity.

Multivariate inference of pathway activity in host immunity and response to therapeutics.

Developing a quantitative view of how biological pathways are regulated in response to environmental factors is central for understanding of disease phenotypes. We present a computational framework, named Multivariate Inference of Pathway Activity (MIPA), which quantifies degree of activity induced in a biological pathway by computing five distinct measures from transcriptomic profiles of its member genes. Statistical significance of inferred activity is examined using multiple independent self-contained tests followed by a competitive analysis. The method incorporates a new algorithm to identify a subset of genes that may regulate the extent of activity induced in a pathway. We present an in-depth evaluation of specificity, robustness, and reproducibility of our method. We benchmarked MIPA's false positive rate at less than 1%. Using transcriptomic profiles representing distinct physiological and disease states, we illustrate applicability of our method in (i) identifying gene-gene interactions in autophagy-dependent response to Salmonella infection, (ii) uncovering gene-environment interactions in host response to bacterial and viral pathogens and (iii) identifying driver genes and processes that contribute to wound healing and response to anti-TNFα therapy. We provide relevant experimental validation that corroborates the accuracy and advantage of our method.

Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense.

A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1ß production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1-dependent antibacterial autophagy or IL-1ß production. These findings demonstrate how the T300A polymorphism leads to cell type- and pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.

IL-1 receptor blockade restores autophagy and reduces inflammation in chronic granulomatous disease in mice and in humans.

Patients with chronic granulomatous disease (CGD) have a mutated NADPH complex resulting in defective production of reactive oxygen species; these patients can develop severe colitis and are highly susceptible to invasive fungal infection. In NADPH oxidase-deficient mice, autophagy is defective but inflammasome activation is present despite lack of reactive oxygen species production. However, whether these processes are mutually regulated in CGD and whether defective autophagy is clinically relevant in patients with CGD is unknown. Here, we demonstrate that macrophages from CGD mice and blood monocytes from CGD patients display minimal recruitment of microtubule-associated protein 1 light chain 3 (LC3) to phagosomes. This defect in autophagy results in increased IL-1ß release. Blocking IL-1 with the receptor antagonist (anakinra) decreases neutrophil recruitment and T helper 17 responses and protects CGD mice from colitis and also from invasive aspergillosis. In addition to decreased inflammasome activation, anakinra restored autophagy in CGD mice in vivo, with increased Aspergillus-induced LC3 recruitment and increased expression of autophagy genes. Anakinra also increased Aspergillus-induced LC3 recruitment from 23% to 51% (P < 0.01) in vitro in monocytes from CGD patients. The clinical relevance of these findings was assessed by treating CGD patients who had severe colitis with IL-1 receptor blockade using anakinra. Anakinra treatment resulted in a rapid and sustained improvement in colitis. Thus, inflammation in CGD is due to IL-1-dependent mechanisms, such as decreased autophagy and increased inflammasome activation, which are linked pathological conditions in CGD that can be restored by IL-1 receptor blockade.

Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection.

BACKGROUND & AIMS: Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. METHODS: We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) × Villin-cre) or CD11c(+) cells (Atg16l1(f/f) × CD11c-cre); these mice were used to assess cell type-specific antibacterial autophagy. All responses were compared with Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells. RESULTS: Autophagy was induced in small intestine and cecum after infection with S typhimurium, and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3ß (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) × Villin-cre mice. Atg16l1(f/f) × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1(f/f) × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. CONCLUSIONS: Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria.

Card9 mediates intestinal epithelial cell restitution, T-helper 17 responses, and control of bacterial infection in mice.

BACKGROUND & AIMS: Caspase recruitment domain 9 (CARD9) is an adaptor protein that integrates signals downstream of pattern recognition receptors. CARD9 has been associated with autoinflammatory disorders, and loss-of-function mutations have been associated with chronic mucocutaneous candidiasis, but the role of CARD9 in intestinal inflammation is unknown. We characterized the role of Card9 in mucosal immune responses to intestinal epithelial injury and infection. METHODS: We induced intestinal inflammation in Card9-null mice by administration of dextran sulfate sodium (DSS) or Citrobacter rodentium. We analyzed body weight, assessed inflammation by histology, and measured levels of cytokines and chemokines using quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Cell populations were compared between wild-type and Card9-null mice by flow cytometry analysis. RESULTS: Colon tissues and mesenteric lymph nodes of Card9-null mice had reduced levels of interleukin (IL)-6, interferon-γ, and T-helper (Th)17 cytokines after administration of DSS, compared with wild-type mice. IL-17A and IL-22 expression were reduced in the recovery phase after DSS administration, coincident with decreased expression of antimicrobial peptides and the chemokine (C-C motif) ligand 20 (Ccl20). Although Card9-null mice had more intestinal fungi based on 18S analysis, their Th17 responses remained defective even when an antifungal agent was administered throughout DSS exposure. Moreover, Card9-null mice had impaired immune responses to C rodentium, characterized by decreased levels of colonic IL-6, IL-17A, IL-22, and regenerating islet-derived 3 gamma (RegIIIγ), as well as fewer IL-22-producing innate lymphoid cells (ILCs) in colon lamina propria. CONCLUSIONS: The adaptor protein CARD9 coordinates Th17- and innate lymphoid cell-mediated intestinal immune responses after epithelial injury in mice.

ATG5 regulates plasma cell differentiation.

Autophagy is a conserved homeostatic process in which cytoplasmic contents are degraded and recycled. Two ubiquitin-like conjugation pathways are required for the generation of autophagosomes, and ATG5 is necessary for both of these processes. Studies of mice deficient in ATG5 reveal a key role for autophagy in T lymphocyte function, as well as in B cell development and B-1a B cell maintenance. However, the role of autophagy genes in B cell function and antibody production has not been described. Using mice in which Atg5 is conditionally deleted in B lymphocytes, we showed here that this autophagy gene is essential for plasma cell homeostasis. In the absence of B cell ATG5 expression, antibody responses were significantly diminished during antigen-specific immunization, parasitic infection and mucosal inflammation. Atg5-deficient B cells maintained the ability to produce immunoglobulin and undergo class-switch recombination, yet had impaired SDC1 expression, significantly decreased antibody secretion in response to toll-like receptor ligands, and an inability to upregulate plasma cell transcription factors. These results build upon previous data demonstrating a role for ATG5 in early B cell development, illustrating its importance in late B cell activation and subsequent plasma cell differentiation.

p40phox expression regulates neutrophil recruitment and function during the resolution phase of intestinal inflammation.

NADPH oxidase is a multisubunit complex that assembles during phagocytosis to generate reactive oxygen species. Several components of this complex have been implicated in chronic granulomatous disease and Crohn's disease, highlighting the importance of reactive oxygen species in regulating host immune response. In this study, we use genetically deficient mice to elucidate how p40(phox), one subunit of the NADPH oxidase complex, functions during intestinal inflammation. We show that p40(phox) deficiency enhances inflammation in both dextran sulfate sodium-induced and innate immune-mediated murine colitis models. This inflammation is characterized by severe colonic tissue injury, increased proinflammatory cytokines, and increased neutrophil recruitment. We demonstrate that neutrophils are essential during the recovery phase of intestinal inflammation and that p40(phox) expression is necessary for this restitution. Lastly, using an integrative bioinformatic approach, we show that p40(phox) deficiency leads to upregulation of chemokine receptor 1 and downregulation of enzymes involved in glycan modifications, including fucosyltransferases and sialyltransferases, during inflammation. We propose that p40(phox) deficiency enhances intestinal inflammation through the dysregulation of these two pathways in neutrophils.

EBV latent membrane protein 2A induces autoreactive B cell activation and TLR hypersensitivity.

EBV is associated with systemic lupus erythematosus (SLE), but how it might contribute to the etiology is not clear. Since EBV-encoded latent membrane protein 2A (LMP2A) interferes with normal B cell differentiation and function, we sought to determine its effect on B cell tolerance. Mice transgenic for both LMP2A and the Ig transgene 2-12H specific for the ribonucleoprotein Smith (Sm), a target of the immune system in SLE, develop a spontaneous anti-Sm response. LMP2A allows anti-Sm B cells to overcome the regulatory checkpoint at the early preplasma cell stage by a self-Ag-dependent mechanism. LMP2A induces a heightened sensitivity to TLR ligand stimulation, resulting in increased proliferation or Ab-secreting cell differentiation or both. Thus, we propose a model whereby LMP2A induces hypersensitivity to TLR stimulation, leading to activation of anti-Sm B cells through the BCR/TLR pathway. These data further implicate TLRs in the etiology of SLE and suggest a mechanistic link between EBV infection and SLE.

Autoreactive MZ and B-1 B-cell activation by Faslpr is coincident with an increased frequency of apoptotic lymphocytes and a defect in macrophage clearance.

Murine autoreactive anti-Smith (Sm) B cells are negatively regulated by anergy and developmental arrest, but are also positively selected into the marginal zone (MZ) and B-1 B-cell populations. Despite positive selection, anti-Sm production occurs only in autoimmune-prone mice. To investigate autoreactive B-cell activation, an anti-Sm transgene was combined with the lpr mutation, a mutation of the proapoptotic gene Fas (Fas(lpr)), on both autoimmune (MRL) and nonautoimmune backgrounds. Fas(lpr) induces a progressive and autoantigen-specific loss of anti-Sm MZ and B-1 B cells in young adult Fas(lpr) and MRL/Fas(lpr) mice that does not require that Fas(lpr) be B-cell intrinsic. This loss is accompanied by a bypass of the early pre-plasma cell (PC) tolerance checkpoint. Although the MRL bkg does not lead to a progressive loss of anti-Sm MZ or B-1 B cells, it induces a robust bypass of the early pre-PC tolerance checkpoint. Fas(lpr) mice have a high frequency of apoptotic lymphocytes in secondary lymphoid tissues and a macrophage defect in apoptotic cell phagocytosis. Since Sm is exposed on the surface of apoptotic cells, we propose that anti-Sm MZ and B-1 B-cell activation is the result of a Fas(lpr)-induced defect in apoptotic cell clearance.