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Bacillus anthracispeptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern (PAMP) contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic lymphocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. Here, we tested the hypothesis thatB. anthracisPGN inhibits the capacity of human monocyte-derived, tissue-like macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD206+CD163+MΦ to PGN for 24h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the pro-efferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVβ5, CD36 and TIM-3, whereas TIM-1, αVβ5, CD300b, CD300f, STABILIN-1 and STABILIN-2 were unaffected. Soluble forms of MERTK, TYRO3, AXL, CD36, and TIM-3 were increased in PGN-treated supernatants, suggesting involvement of proteases. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. ADAM17 inhibitors TAPI-0 and Marimastat abolished TNF release, indicating effective protease inhibition, modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and thatB. anthracisPGN inhibits efferocytosis in part by reducing cell surface expression of efferocytic receptors.

ObjectiveIdentify autoantibodies in anti-Ro/SS-A negative primary Sjögren’s syndrome (SS).MethodsThis is a proof-of-concept, case-control study of SS, healthy (HC) and other disease (OD) controls. A discovery dataset of plasma samples (n=30 SS, n=15 HC) was tested on human proteome arrays containing 19 500 proteins. A validation dataset of plasma and stimulated parotid saliva from additional SS cases (n=46 anti-Ro+, n=50 anti-Ro–), HC (n=42) and OD (n=54) was tested on custom arrays containing 74 proteins. For each protein, the mean+3 SD of the HC value defined the positivity threshold. Differences from HC were determined by Fisher’s exact test and random forest machine learning using 2/3 of the validation dataset for training and 1/3 for testing. Applicability of the results was explored in an independent rheumatology practice cohort (n=38 Ro+, n=36 Ro–, n=10 HC). Relationships among antigens were explored using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) interactome analysis.ResultsRo+SS parotid saliva contained autoantibodies binding to Ro60, Ro52, La/SS-B and muscarinic receptor 5. SS plasma contained 12 novel autoantibody specificities, 11 of which were detected in both the discovery and validation datasets. Binding to ≥1 of the novel antigens identified 54% of Ro–SS and 37% of Ro+SS cases, with 100% specificity in both groups. Machine learning identified 30 novel specificities showing receiver operating characteristic area under the curve of 0.79 (95% CI 0.64 to 0.93) for identifying Ro–SS. Sera from Ro–cases of an independent cohort bound 17 of the non-canonical antigens. Antigenic targets in both Ro+and Ro–SS were part of leukaemia cell, ubiquitin conjugation and antiviral defence pathways.ConclusionWe identified antigenic targets of the autoantibody response in SS that may be useful for identifying up to half of Ro seronegative SS cases.

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen associated molecular pattern contributing to anthrax pathology including coagulopathy in late stage disease. Lymphocyte apoptosis is an established phenomenon in bacterial sepsis, including fulminant anthrax. Clearance of apoptotic cells (efferocytosis) is thought to be mediated by tissue-resident macrophages (MF), modeled in vitro as M2-like MF. Circulating innate cells need serum opsonization of PGN for cellular activation, but opsonization requirements for human MF activation have not been reported. Herein, we tested the hypothesis that PGN requires human serum opsonization for MF activation and inhibits human M2-like MF efferocytosis of apoptotic human neutrophils. MF were differentiated in vitro with M-CSF and polarized to M2 phenotype with dexamethasone. Cytokine and soluble receptor levels were assessed by ELISA and Luminex assays. Cell surface markers and fraction of MF containing fluorescently-labeled apoptotic human neutrophils were assessed by flow cytometry. M2-like MF recognition of PGN was enhanced in the presence of human but not bovine serum, noted by increased secretion of TNF-a and IL-10 in PGN-treated culture supernatants. Efferocytosis, and cell surface expression of MerTK, Tyro3, Axl, αVβ5, CD36 and Tim-3, all pro-efferocytic receptors, was significantly downregulated by pre-treatment of MF with PGN. Increases in soluble pro-efferocytic receptors were detected in PGN-treated MF culture supernatants. We conclude that PGN impairs efferocytosis by human M2-like macrophages, likely by down-regulating the expression of cell surface receptors known to signal apoptotic cell engulfment. Supported by NIH (U19AI062629).

To assess the types of salivary gland (SG) T cells contributing to Sj&ouml, gren&rsquo, s syndrome (SS), we evaluated SG T cell subtypes for association with disease features and compared the SG CD4+ memory T cell transcriptomes of subjects with either primary SS (pSS) or non-SS sicca (nSS). SG biopsies were evaluated for proportions and absolute numbers of CD4+ and CD8+ T cells. SG memory CD4+ T cells were evaluated for gene expression by microarray. Differentially-expressed genes were identified, and gene set enrichment and pathways analyses were performed. CD4+CD45RA&minus, T cells were increased in pSS compared to nSS subjects (33.2% vs. 22.2%, p &lt, 0.0001), while CD8+CD45RA&minus, T cells were decreased (38.5% vs. 46.0%, p = 0.0014). SG fibrosis positively correlated with numbers of memory T cells. Proportions of SG CD4+CD45RA&minus, T cells correlated with focus score (r = 0.43, p &lt, 0.0001), corneal damage (r = 0.43, p &lt, 0.0001), and serum Ro antibodies (r = 0.40, p &lt, 0.0001). Differentially-expressed genes in CD4+CD45RA&minus, cells indicated a T follicular helper (Tfh) profile, increased homing and increased cellular interactions. Predicted upstream drivers of the Tfh signature included TCR, TNF, TGF-&beta, 1, IL-4, and IL-21. In conclusion, the proportions and numbers of SG memory CD4+ T cells associate with key SS features, consistent with a central role in disease pathogenesis.

There are few studies of animal communities in coastal zones, especially those of herpetofauna. We conducted herpetofaunal surveys in open shoreline habitats for 3 yr in three large protected areas in the Mixedwoods Plains Ecozone of the southern Laurentian Great Lakes. These locations are isolated preserves in one of the most heavily human-modified areas of North America. We found 17 species with low to moderate similarity of species membership and highly uneven relative abundance and biomass. Common Five-Lined Skinks dominated communities in relative abundance, dominated biomass at one location, and codominated biomass with Common Gartersnakes and Eastern Foxsnakes or Milksnakes at the other two locations. Five other species also regularly inhabited the coastal zone, whereas others were more transient users. Review of species' dietary requirements suggest that consumption of arthropods and other invertebrates may play an important role in structuring the community. Both the legacy of historical...

Summary RNA-binding nuclear antigens are a major class of self-antigen to which immune tolerance is lost in rheumatic diseases. Serological tolerance to one such antigen, La/Sjögren's syndrome (SS)-B (La), is controlled by CD4+ T cells. This study investigated peripheral tolerance to human La (hLa) by tracking the fate of hLa-specific CD4+ T cells expressing the transgenic (Tg) 3B5.8 T cell receptor (TCR) after adoptive transfer into lymphocyte-replete recipient mice expressing hLa as a neo-self-antigen. After initial antigen-specific cell division, hLa-specific donor CD4+ T cells expressed forkhead box protein 3 (FoxP3). Donor cells retrieved from hLa Tg recipients displayed impaired proliferation and secreted interleukin (IL)−10 in vitro in response to antigenic stimulation. Transfer of highly purified FoxP3-negative donor cells demonstrated that accumulation of hLa-specific regulatory T cells (Treg) was due primarily to expansion of small numbers of donor Treg. Depletion of recipient plasmacytoid dendritic cells (pDC), but not B cells, severely hampered the accumulation of FoxP3+ donor Treg in hLa Tg recipients. Recipient pDC expressed tolerogenic markers and higher levels of co-stimulatory and co-inhibitory molecules than B cells. Adoptive transfer of hLa peptide-loaded pDC into mice lacking expression of hLa recapitulated the accumulation of hLa-specific Treg. Blockade of the type 1 interferon (IFN) receptor in hLa Tg recipients of hLa-specific T cells impaired FoxP3+ donor T cell accumulation. Therefore, peripheral expansion of Treg specific for an RNA-binding nuclear antigen is mediated by antigen-presenting pDC in a type 1 IFN-dependent manner. These results reveal a regulatory function of pDC in controlling autoreactivity to RNA-binding nuclear antigens.

INTRODUCTION Sjögren’s syndrome (SS) is a rheumatic autoimmune disease characterized by focal lymphocytic infiltrates in the lacrimal and salivary glands, severe dry mouth and eyes, pain and debilitation. Diagnosis requires autoantibodies to ubiquitous Ro antigens or a lip biopsy positive for focal lymphocytic infiltrates. Here we used human proteome arrays to identify novel antibodies in plasma from Ro positive and Ro/La antibody negative SS patients compared with healthy controls. METHODS Anti-Ro positive (n=15) and anti-Ro negative (n=15) cases meeting 2016 ACR/EULAR classification criteria for SS were age, race, and sex matched with each other and healthy controls (n=15). Plasma IgG binding to human proteome arrays containing >19,500 recombinant human proteins representing >80% of the human proteome (HuProt v3.2 arrays, CDI Laboratories) was assessed. Data were normalized by the Robust Linear Model using the PAA Bioconductor Package in R and log intensity values for each protein generated. Thresholds of mean + 4SD were established using the controls. Antigens bound by IgG more frequently in cases compared to controls (p RESULTS IgG from Ro positive SS cases significantly bound 18 proteins, including the canonical SS antigens Ro60 and Ro52. IgG from Ro negative SS cases significantly bound 4 proteins compared to controls, 3 of which were shared with the Ro positive group. Binding to any one of 4 novel proteins identified 73% of the Ro negative SS cases. CONCLUSION A total of 17 novel antigen specificities were identified in SS, with 4 antigens being bound by plasma IgG from Ro/La negative SS cases. These antigens may be useful for diagnosing SS without a lip biopsy.

Objective To assess disease features in Sle1.Yaa mice with genetic interleukin-6 (IL-6) deficiency. Methods Sera and tissues were collected from C57BL/6 (B6), Sle1.Yaa, and Sle1.Yaa.IL-6−/− mice and analyzed for various features of disease. Using serum samples, autoantibody specificities were determined by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence, cytokine production was analyzed by Luminex and ELISA, and levels of blood urea nitrogen were determined by ELISA. Renal, lung, and salivary gland tissue sections were evaluated for pathologic changes. Lymphocyte phenotypes, including CD4+ T cell cytokine production, and those of follicular and extrafollicular T helper subsets, germinal center B cells, and plasma cells, were determined using flow cytometry. Results IL-6 deficiency not only ameliorated autoantibody production and renal disease in this model, but also effectively reduced inflammation of lungs and salivary glands. Furthermore, IL-6 deficiency abrogated differentiation of Th1 and extrafollicular T helper cells, germinal center B cells, and plasma cells in the spleen and eliminated renal T cells with IL-17, interferon-γ, and IL-21 production potential. Conclusion Our findings highlight IL-6–mediated T cell aberrations in Yaa-driven autoimmunity and support the concept of therapeutic IL-6/IL-6 receptor blockade in systemic lupus erythematosus and Sjogren's syndrome by impairing the production of autoantibodies and lymphocytic infiltration of the kidneys, lungs, and salivary glands.

The Bacillus anthracis Edema Toxin (ET), composed of a Protective Antigen (PA) and the Edema Factor (EF), is a cellular adenylate cyclase that alters host responses by elevating cyclic adenosine monophosphate (cAMP) to supraphysiologic levels. However, the role of ET in systemic anthrax is unclear. Efferocytosis is a cAMP-sensitive, anti-inflammatory process of apoptotic cell engulfment, the inhibition of which may promote sepsis in systemic anthrax. Here, we tested the hypothesis that ET inhibits efferocytosis by primary human macrophages and evaluated the mechanisms of altered efferocytic signaling. ET, but not PA or EF alone, inhibited the efferocytosis of early apoptotic neutrophils (PMN) by primary human M2 macrophages (polarized with IL-4, IL-10, and/or dexamethasone) at concentrations relevant to those encountered in systemic infection. ET inhibited Protein S- and MFGE8-dependent efferocytosis initiated by signaling through MerTK and &alpha, V&beta, 5 receptors, respectively. ET inhibited Rac1 activation as well as the phosphorylation of Rac1 and key activating sites of calcium calmodulin-dependent kinases CamK1&alpha, CamK4, and vasodilator-stimulated phosphoprotein, that were induced by the exposure of M2(Dex) macrophages to Protein S-opsonized apoptotic PMN. These results show that ET impairs macrophage efferocytosis and alters efferocytic receptor signaling.

CD4+ T cells predominate in salivary gland (SG) inflammatory lesions in Sjogren's syndrome (SS). However, their antigen specificity, degree of clonal expansion, and relationship to clinical disease features remain unknown. We used multiplex reverse-transcriptase PCR to amplify paired T cell receptor α (TCRα) and β transcripts of single CD4+CD45RA- T cells from SG and peripheral blood (PB) of 10 individuals with primary SS, 9 of whom shared the HLA DR3/DQ2 risk haplotype. TCRα and β sequences were obtained from a median of 91 SG and 107 PB cells per subject. The degree of clonal expansion and frequency of cells expressing two productively rearranged α genes were increased in SG versus PB. Expanded clones from SG exhibited complementary-determining region 3 (CDR3) sequence similarity both within and among subjects, suggesting antigenic selection and shared antigen recognition. CDR3 similarities were shared among expanded clones from individuals discordant for canonical Ro and La autoantibodies, suggesting recognition of alternative SG antigen(s). The extent of SG clonal expansion correlated with reduced saliva production and increased SG fibrosis, linking expanded SG T cells with glandular dysfunction. Knowledge of paired TCRα and β sequences enables further work toward identification of target antigens and development of novel therapies.

A human La/Sjögren’s syndrome-B (hLa)–specific TCR/hLa neo–self-Ag double-transgenic (Tg) mouse model was developed and used to investigate cellular tolerance and autoimmunity to the ubiquitous RNA-binding La Ag often targeted in systemic lupus erythematosus and Sjögren’s syndrome. Extensive thymic clonal deletion of CD4+ T cells occurred in H-2k/k double-Tg mice presenting high levels of the I-Ek–restricted hLa T cell epitope. In contrast, deletion was less extensive in H-2k/b double-Tg mice presenting lower levels of the epitope, and some surviving thymocytes were positively selected as thymic regulatory T cells (tTreg). These mice remained serologically tolerant to hLa and healthy. H-2k/b double-Tg mice deficient of all endogenous Tcra genes, a deficiency known to impair Treg development and function, produced IgG anti-hLa autoantibodies and displayed defective tTreg development. These autoimmune mice had interstitial lung disease characterized by lymphocytic aggregates containing Tg T cells with an activated, effector memory phenotype. Salivary gland infiltrates were notably absent. Thus, expression of nuclear hLa Ag induces thymic clonal deletion and tTreg selection, and lymphocytic infiltration of the lung is a consequence of La-specific CD4+ T cell autoimmunity.

Background Plants contain significant quantities of small RNAs (sRNAs) derived from various sRNA biogenesis pathways. Many of these sRNAs play regulatory roles in plants. Previous analysis revealed that numerous sRNAs in corn, rice and soybean seeds have high sequence similarity to animal genes. However, exogenous RNA is considered to be unstable within the gastrointestinal tract of many animals, thus limiting potential for any adverse effects from consumption of dietary RNA. A recent paper reported that putative plant miRNAs were detected in animal plasma and serum, presumably acquired through ingestion, and may have a functional impact in the consuming organisms. Results To address the question of how common this phenomenon could be, we searched for plant miRNAs sequences in public sRNA datasets from various tissues of mammals, chicken and insects. Our analyses revealed that plant miRNAs were present in the animal sRNA datasets, and significantly miR168 was extremely over-represented. Furthermore, all or nearly all (>96%) miR168 sequences were monocot derived for most datasets, including datasets for two insects reared on dicot plants in their respective experiments. To investigate if plant-derived miRNAs, including miR168, could accumulate and move systemically in insects, we conducted insect feeding studies for three insects including corn rootworm, which has been shown to be responsive to plant-produced long double-stranded RNAs. Conclusions Our analyses suggest that the observed plant miRNAs in animal sRNA datasets can originate in the process of sequencing, and that accumulation of plant miRNAs via dietary exposure is not universal in animals.

To assess changes in functional status in patients with rheumatoid arthritis (RA) receiving the interleukin-1 receptor antagonist anakinra in addition to a disease modifying antirheumatic drug (DMARD).In this large, multicenter, open-label, single-arm study, adult patients with RA receiving methotrexate, sulfasalazine, or hydroxychloroquine foror = 3 months were given anakinra 100 mg once daily for up to 36 weeks. The primary objective was to evaluate changes from baseline to week 36 in the Health Assessment Questionnaire (HAQ) disability index and subscales. Changes in the 28-joint Disease Activity Score (DAS28), proportion of patients meeting European League Against Rheumatism (EULAR) response criteria, and the safety of each combination regimen were also assessed.A total of 1207 patients were enrolled, receivedor = 1 dose of anakinra, and were included in the efficacy and safety analyses. A statistically significant change in the HAQ disability index was observed (p = 0.0001); no significant differences were seen among the 3 DMARD groups. A clinically meaningful improvement in HAQ (0.22) was observed in 51% of patients. Mean improvement in DAS28 was 1.5 (p0.0001), and 64% of patients achieved a good or moderate EULAR response score. Injection site reaction was the most frequently (62%) reported adverse event. The incidence of infections (24%), most commonly respiratory infection, was similar across treatment groups. No notable changes were observed in laboratory findings and vital signs.These findings indicate that anakinra 100 mg/day in combination with DMARD therapy safely improved functional status in patients with active RA.

Sjögren’s syndrome is a systemic rheumatic disorder characterized by dry eyes, dry mouth and T cell infiltration of exocrine gland tissue. To determine the extent of salivary gland (SG) CD4+ memory T cell clonal expansion and whether these expansions are as frequent in peripheral blood (PB), a multiplex PCR method was used to amplify both the α and β T cell receptor (TCR) sequences from memory CD4+ T cells sorted from SG lip biopsy tissue and PB of 10 primary Sjögren’s syndrome patients. Over 3,000 TCR sequences were obtained from 50-115 (median 91) individual SG and 75-121 (median 104) individual PB T cells per patient. The percentage of cells that were part of clonal expansions was significantly higher in SG (median 11%, range 0-28%) compared to PB (median 0.9%, range 0-7%, p=0.003). Sequence analysis revealed: 1) highly homologous CDR3s among different expanded SG T cell clones within single patients, suggesting antigen-driven expansion and 2) unique cases of convergent recombination among unrelated patients, where different V segments/additions/deletions were utilized to make identical CDR3 amino acid sequences. The percentages of clonally expanded SG T cells correlated significantly with degree of SG fibrosis and reduced saliva production but not with systemic features of disease. SG clonal expansions detected in this study likely identify T cells involved in recognition of common antigen(s) and glandular dysfunction.

TLR7 is a single-stranded RNA-recognizing innate immune receptor with roles in anti-viral immunity and systemic lupus erythematosus (SLE) in humans and SLE-like autoimmunity in mice. TLR7 gene duplication drives inflammation and autoimmunity in the BXSB mouse model and other Y autoimmune accelerator (Yaa)-derived models of murine lupus. Additionally, TLR7 SNPs associate with SLE in Eastern Asian populations. In this study, we investigated effects of TLR7 overexpression on CD4+ T cell subsets. In unmanipulated, 5-6 month old TLR7 BAC transgenic mice, we observed amplified activated:naïve T cell ratios and preferential expansion of the Th1 subset compared to wild type controls. We also evaluated the phenotype of TCR transgenic T cells specific for the RNA-binding autoantigen, human La, following adoptive transfer into TLR7 BAC transgenic mice. These human La-specific T cells demonstrated enhanced differentiation to Th1 and PSGL1lowCXCR4+ extrafollicular helper phenotypes after recovery from TLR7-overexpressing human La transgenic mice compared to wild type human La transgenic controls seven days after transfer. Moreover, TLR7 overexpression in recipient mice resulted in significantly reduced expansion of the CD4+ Foxp3+ phenotype among transferred, human La-specific cells. These data indicate that TLR7 overexpression alters CD4+ T cell populations in a manner that potentially contributes to autoinflammatory disease in these animals.