Lupus Pathogenesis Research Articles

Synthesis of peptidomimetics and chemo-biological tools for CD95/PLCγ1 interaction analysis.

The death receptor CD95 (also known as Fas) induces apoptosis through protein/protein association and the formation of the death-inducing signaling complex. On the other hand, in certain biological conditions, this receptor recruits different proteins and triggers the formation of another complex designated motility-inducing signaling complex, which promotes cell migration and inflammation. This pathway relies on a short sequence of CD95, called calcium-inducing domain (CID), which interacts with the phospholipase PLCγ1. To better understand how CID/PLCγ1 interaction occurs, we synthesized different α-AA peptides mimicking CID. Some of these peptidomimetics are as potent as the natural peptide to disrupt the CID/PLCγ1 interaction and cell migration, and showed improved pharmacokinetic properties. We also generated biotinyl- and palmitoyl-labelled peptidomimetics, useful chemico-biological tools to further explore the pro-inflammatory signal of CD95, which plays an important role in the pathogenesis of lupus and other autoimmune diseases.

Lupus nephritis is linked to disease-activity associated expansions and immunity to a gut commensal

Background/PurposeTo search for a transmissible agent involved in lupus pathogenesis, we investigated the faecal microbiota of patients with systemic lupus erythematosus (SLE) for candidate pathobiont(s) and evaluated them for special...

Genomic Identification of Low-Density Granulocytes and Analysis of Their Role in the Pathogenesis of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of low-density granulocytes (LDGs) with a heightened capacity for spontaneous NETosis, but the contribution of LDGs to SLE pathogenesis remains unclear. To characterize LDGs in human SLE, gene expression profiles derived from isolated LDGs were characterized by weighted gene coexpression network analysis, and a 92-gene module was identified. The LDG gene signature was enriched in genes related to neutrophil degranulation and cell cycle regulation. This signature was assessed in gene expression datasets from two large-scale SLE clinical trials to study associations between LDG enrichment, SLE manifestations, and treatment regimens. LDG enrichment in the blood was associated with corticosteroid treatment as well as anti-dsDNA, low serum complement, renal manifestations, and vasculitis, but the latter two of these associations were dependent on concomitant corticosteroid treatment. In addition, LDG enrichment was associated with enrichment of gene signatures induced by type I IFN and TNF irrespective of corticosteroid treatment. Notably, LDG enrichment was not found in numerous tissues affected by SLE. Comparison with relevant reference datasets indicated that LDG enrichment is likely reflective of increased granulopoiesis in the bone marrow and not peripheral neutrophil activation. The results have uncovered important determinants of the appearance of LDGs in SLE and have emphasized the likely role of LDGs in specific aspects of lupus pathogenesis.

In vivo depletion of microRNA-183-96-182 cluster alleviates autoimmunity in B6-lpr lupus mice

Abstract We have previously reported a significant upregulation of microRNA-183-96-182 cluster (miR-183C) in splenocytes of several murine lupus models. In this study, we found that the upregulation of miR-183C is inversely related to the expression of Foxo1 and Foxo3a, which play important roles in the regulation of T lymphocyte homeostasis and immune responses. Importantly, we found that inhibition of miR-183C with specific antagomirs significantly reduces lupus-related inflammatory cytokines expression such as IFNg, IL-6, and IL-17 in in vitro activated splenocytes from both MRL and MRL-lpr mice. We further showed that inhibition of miR-183C increases expression of Foxo1 and that inhibition of Foxo1 with siRNA increases IFNg and IL-6 in activated splenocytes from MRL mice. These data suggest that the miR-183C may regulate inflammatory response by targeting Foxo1. To further characterize the role of miR-183C in lupus pathogenesis, we developed B6-lpr mice with conditional depletion of miR-183C in T and B lymphocyte (miR-183C−/− B6-lpr) by serial crossbreeding of B6-lpr with miR-183Cfl/fl and hCD2-iCre mice. To our surprise, there was no significant difference in the production of IFNg and IL-6 in in vitro activated splenocytes from knock out (miR-183C−/− B6-lpr) mice when compared to that from control (miR-183Cfl/fl B6-lpr) mice. Impressively, we found that miR-183C−/− B6-lpr had reduced serum anti-dsDNA levels, spleen and lymphoid node weight when compared to controls. The histopathological analysis also revealed reduced inflammation in kidney in miR-183C−/− B6-lpr mice. Together, our results suggest a potential role of miR-183C in lupus pathogenesis and that miR-183C might be a new potential target for lupus therapy.

Improved antimalarial delivery to inflammatory cells driving lupus pathogenesis using shape-based targeting nanoparticles

Abstract Systemic lupus erythematosus (SLE) is a chronic disease that attacks healthy organs via self antigen-autoantibody immune complexes. These complexes activate plasmacytoid dendritic cells (pDCs) via (toll-like receptor) TLR7 and 9, driving inflammatory type I interferon (IFN). Antimalarials, hydroxychloroquine (HCQ) and chloroquine (CQ), are TLR antagonists used to alleviate inflammation in SLE patients, but require ~3+ months before therapeutic efficacy. Recent work reports 7.5% patients treated with HCQ are at risk of irreversible retinopathy after 5 years, which increases to 1 in 5 patients with duration of therapy. We hypothesized that PEG-b-PPS filomicelles (FMs) will improve drug activity and reduce toxicity by directly delivering HCQ or CQ to pDCs via passive, shape-based targeting. Human peripheral blood mononuclear cells (hPBMCs) were treated with soluble CQ or CQ in FMs, stimulated with CpG-A ODN 2216 and quantified for type I IFN via myxovirus-resistance 1 (MX1) gene expression using real-time RT-PCR. Our results show that 50 μg CQ/mg FM decreased MX1 expression 20 fold to soluble CQ and 10 fold to CpG-A ODN 2216 control, while CQ in control polymer PLGA spheres had no effect. Additionally, cellular-level distribution of FMs in hPBMCs show that FMs accumulate in pDCs more than B and T cells over a 48 hour time course. These results show polymer shape impacts drug delivery, CQ-FMs inhibit SLE-relevant pathways and CQ-FMs may be effective and more targeted than CQ.

A rationally engineered DNase1-Fc fusion protein ameliorates autoimmune glomerulonephritis

Abstract Loss of B cell tolerance and generation of autoreactive anti-nuclear antibodies are hallmarks of systemic lupus erythematosus and lupus nephritis. The mechanism of anti-nuclear immunoglobulin accumulation and clearance in lupus nephritis pathogenesis remains largely uncharacterized. Here, we show that innate immune activation in the NZB/W F1 mouse model and human lupus nephritis biopsies rapidly reduces expression of DNase1 (also called DNase I) in renal cortex proximal tubular cells. Unexpectedly, kidney DNase1 levels were no longer affected by TLR ligand agonism upon depletion of macrophages, indicating that local macrophage activation is required for the downmodulation of DNase1 in kidney epithelial cells. To overcome the loss of endogenous DNase1, we rationally designed a hyperactive actin-resistant variant of DNase1 with improved catalytic activity and acceptable in vivo pharmacokinetics. The engineered DNase1-Fc fusion protein ameliorates nephritis in a murine model of lupus and reduces immune complex deposition/complement fixation. Our data suggest that the loss of renal DNase1 through TLR signaling or other innate immune activation impairs clearance of autoreactive anti-nuclear immune complex deposits in the kidney to promote nephritis progression. While autoantibodies and autoreactive B cell activation are central to lupus pathogenesis, we hypothesize that renal cortical epithelial cells expressing high levels of DNase1 serve as gatekeepers to prevent detrimental nuclear immune complex deposition. Our findings provide a therapeutic rationale for using an engineered hyperactive actin-resistant DNase1-Fc for lupus nephritis.

A Variant of the Histone-Binding Protein sNASP Contributes to Mouse Lupus.

The Sle2c1rec1c (rec1c) sublocus is derived from the mouse lupus susceptibility 2 (Sle2) locus identified in the NZM2410 model. Our current study dissected the functional characters and the genetic basis of the rec1c locus relative to lupus when co-expressed with the Faslpr mutation, an established inducer of autoimmunity. The rec1c.lpr mice exhibited mild expansion of lymph nodes and had a normal T cell cellularity, but developed significantly kidney and lung inflammation, indicating that the rec1c amplifies lpr-induced autoimmune pathogenesis. A variant of somatic nuclear autoantigenic sperm protein (sNASP) was identified from the rec1c interval as a substitution of two consecutive amino acid residues in the histone-binding domain, resulting in an increased binding affinity to histone H4 and H3.1/H4 tetramer. To determine the role of the sNASP rec1c allele in mouse lupus, a novel strain was generated by introducing the rec1c mutations into the B6 genome. In this transgenic model, the sNASP allele synergized with the lpr mutation leading to moderate autoimmune phenotypes and aggravating inflammatory pathology alterations in kidney and lung that were similar to those observed in the rec1c.lpr mice. These results establish that the sNASP allele is a pathogenic genetic element in the rec1c sublocus, which not only promotes autoimmunity, but also exacerbates the inflammation reaction of end organs in mouse lupus pathogenesis. It also shows the complexity of the Sle2c locus, initially mapped as the major locus associated with B1a cell expansion. In addition to Cdkn2c, which regulates this expansion, we have now identified in the same locus a protective allele of Csf3r, a variant of Skint6 associated with T cell activation, and now a variant of sNASP that amplifies autoimmunity and tissue damage.

The Role of Human Fc Gamma Receptors in the Pathophysiology of Lupus Syndrome

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune syndrome, which currently has no curative treatment. The main drivers of this inflammatory disease are the IgG family autoantibodies that are produced against self‐antigens resulting from dysregulated apoptosis. These pathogenic autoantibodies form immune complexes (ICs), which become deposited in various tissues and cause organ damage. IgG ICs mainly interact with target cells by engaging with a family of receptors known as Fc gamma receptors (FcγRs). FcγRs are thought to be important contributors to inflammation in autoimmune diseases such as SLE. These receptors cooperate in modulating antigen processing and presentation, autoantibody production, and the promotion of inflammatory processes. Current mouse models do not adequately produce translatable data regarding the crucial role of FcγRs in SLE pathogenesis because the repertoire of FcγRs in mice is significantly different to that of humans. The human FcγR family consists of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb whereas mice express FcγRI, FcγRIIb, FcγRIII and FcγRIV. Furthermore, FcγRIIa, which is absent in wild type (WT) mice, is the only IgG receptor present on human platelets. The present study used the pristane method of lupus induction in WT mice and in mice in which all murine FcγRs have been replaced by human FcγRs (hu‐FcγR mice) to study the role of individual human FcγRs in the initiation and progression of lupus‐like autoimmune syndrome. After chemically inducing SLE in hu‐FcγR mice, we have found that both the initiation and progression of SLE is significantly accelerated compared to WT mice, suggesting that the combination of human FcγRs play a dominant role in the pathogenesis of lupus. Symptoms such as hypothermia, chopped breathing and weight loss was present in 61% of hu‐FcγR mice after 15 days or less after injection compared to only 33% in WT mice (p<0.05). hu‐FcγR mice presented splenomegaly, glomerulonephritis, and lung vasculitis as early as 10 days after injection. Antinuclear antibodies were found in 47% of injected hu‐FcγR mice compared to 28% in WT mice (p<0.05). These results suggest that FcγRs can significantly affect the development of a lupus‐like syndrome in mice. Furthermore, mice with human FcγRs should be considered as a more suitable model to study autoimmune disorders such as SLE. The present study and other necessary investigations in this topic may not only profoundly impact our knowledge of inflammation in SLE, but also identify specific FcγRs as novel therapeutic targets. Ultimately, because of the involvement of FcγRs in modulation of the inflammatory response, our findings have the potential to impact therapeutic approaches for other inflammatory autoimmune disorders, such as rheumatoid arthritis and antiphospholipid syndrome.Support or Funding InformationThis study was supported by Florida Hospital and the Adventist University of Health Sciences.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Full high-throughput sequencing analysis of differences in expression profiles of long noncoding RNAs and their mechanisms of action in systemic lupus erythematosus

BackgroundThe specific function of long noncoding RNAs (lncRNAs) in systemic lupus erythematosus (SLE) and the mechanism of their involvement in related pathological changes remain to be elucidated, so, in this study, we analyzed the differences in the expression profiles of lncRNAs and their mechanisms of action in SLE using full high-throughput sequencing, bioinformatics, etc. methods.MethodsWe used high-throughput sequencing to detect differences in the expression profiles of lncRNAs, miRNAs, and mRNAs in PBMCs from patients with SLE at the genome-wide level. Next, we predicted target genes of 30 lincRNAs (long intergenic noncoding RNAs) by constructing a coexpression network of differential lincRNAs and mRNAs and identified the role of lincRNAs. Then, we analyzed the coexpression network of 23 optimized lincRNAs and their corresponding 353 miRNAs, evaluated the cis- and trans-effects of these lincRNAs, and performed GO and KEGG analyses of target genes. We also selected 8 lincRNAs and 2 newly discovered lncRNAs for q-PCR validation and lncRNA–miRNA–mRNA analysis. Finally, we also analyzed respectively the relation between lncRNAs and gender bias in SLE patients using RT-qPCR, the relation between Systemic Lupus Erythematosus Disease Activity Index score and the “IFN signature” using ELISA, and the relation between the differential expression of lncRNAs and a change in the number of a cell type of PBMCs in SLE patients using RT-qPCR.ResultsThe profiles of 1087 lncRNAs, 102 miRNAs, and 4101 mRNAs in PBMCs significantly differed between patients with SLE and healthy controls. The coexpression network analysis showed that the network contained 23 lincRNAs and 353 mRNAs. The evaluation of the cis- and trans-effects showed that the 23 lincRNAs acted on 704 target genes. GO and KEGG analyses of the target genes predicted the biological functions of the 23 lincRNAs. q-PCR validation showed 7 lincRNAs and 2 novel lncRNAs were identical to the sequencing results. The ceRNA network contained 7 validated lincRNAs, 15 miRNAs, and 155 mRNAs. In addition, the differential expression of lncRNAs may be gender dependent in SLE patients, SLE patients also exhibit a robust “IFN signature,” and PBMCs exhibiting differential expression of lncRNAs may be due to a change in the number of a cell type.ConclusionThis work determined specific lncRNAs that play important biological functions in the pathogenesis of lupus and provided a new direction for diagnosis and treatment of disease.

The Lupus Susceptibility Locus Sgp3 Encodes the Suppressor of Endogenous Retrovirus Expression SNERV

Elevated endogenous retrovirus (ERV) transcription and anti-ERV antibody reactivity are implicated in lupus pathogenesis. Overproduction of non-ecotropic ERV (NEERV) envelope glycoprotein gp70 and resultant nephritis occur in lupus-prone mice, but whether NEERV mis-expression contributes to lupus etiology is unclear. Here we identified suppressor of NEERV (Snerv) 1 and 2, Krüppel-associated box zinc-finger proteins (KRAB-ZFPs) that repressed NEERV by binding the NEERV long terminal repeat to recruit the transcriptional regulator KAP1. Germline Snerv1/Snerv2 deletion increased activating chromatin modifications, transcription, and gp70 expression from NEERV loci. F1 crosses of lupus-prone New Zealand Black (NZB) and 129 mice to Snerv1/Snerv2-/- mice failed to restore NEERV repression, demonstrating that loss of SNERV underlies the lupus autoantigen gp70 overproduction that promotes nephritis in susceptible mice and that SNERV encodes for Sgp3 (in NZB mice) and Gv-1 loci (in 129 mice). Increased ERV expression in lupus patients inversely correlated with three putative ERV-suppressing KRAB-ZFPs, suggesting that loss of KRAB-ZFP-mediated ERV control may contribute to human lupus pathogenesis.

Prognostic role of plasma cell free DNA in a cohort of Egyptian systemic lupus erythematosus patients

Background: Cell death has an important event in lupus pathogenesis as it leads to release of antigens as nucleic acids for immune complex formation. DNA-antibody complexes in the circulation are one of the hallmarks of Systemic Lupus Erythematosus (SLE) that leads to the clinical manifestations of SLE. Fluctuation in circulating DNA level might be one of the driving factors behind flare-ups. The objective of this study is to estimate the prevalence of plasma circulating cell-free deoxyribonucleic acid (cf DNA) in SLE patients and evaluate it as a prognostic marker in SLE and its relation to drug therapy. Methods: A case control study conducted on 75 Egyptian SLE patients divided into 2 groups; 45 SLE patients on therapy and 30 SLE patients recently diagnosed (without therapy) and 25 matched healthy control group. All patients are subjected to detailed medical history and clinical examination. Disease activity was done using SLEDAI-2k, related laboratory investigations in addition to estimation of cf-DNA concentration by real-time PCR technique. Findings: On comparing the 2 SLE groups, there was significant increase in nephritis, neuropsychiatric manifestations, vasculitis, fever, SLEDAI -2k, ESR, ANA, Anti-DNA titre and cf-DNA concentration and significant decrease in TLC, Hb, PLT, C3 and C4 in SLE without therapy than those on therapy (P<0.001). On comparing the 3 studied groups there was significant increase in cf-DNA concentration in SLE patients without therapy than those on therapy than in the control group (p <0.001). There was a significant correlation between cf-DNA concentration and neuropsychiatric manifestations, nephritis, fever, ESR, lymphopenia, anemia, thrombocytopenia, ANA titre, Anti-DNA titre, C3, C4 levels and SLEDAI -2k in all SLE patients (p <0.001). Conclusion: Plasma level of cf-DNA is significantly increased in SLE patients especially before starting therapy, so cf-DNA can be used as a marker of disease activity and treatment follow-up.

Immunometabolic disorders in the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus is a chronic autoimmune disease connected with complex and unclear disorders of the immune system, which causes inflammation of body tissues and internal organs. It leads to the formation of anti-nuclear antibodies (ANA) and immune complexes. Numerous immune system disorders and dysfunctions in the biochemical processes can occur in the course of the disease, and a wide range of abnormalities associated with cellular respiratory processes and mitochondrial function have been documented. The following paper presents the current understanding of the contribution of these disorders to the pathogenesis of lupus.

A New Driver for Lupus Pathogenesis is conserved in Humans and Mice.

Lupus is experienced as an “aggressive and expansive” disease affecting patients’ activities of daily living, sense of self, and social functioning [1]. Currently there is no cure. Despite substantial efforts, the lupus drug development field has witnessed only one FDA-approved therapy in the last 50 years. There is an urgent need to better our understanding of the pathogenic mechanisms for lupus, and to develop novel therapeutic strategies for both cutaneous and systemic lupus erythematosus.

Collagen triple helix repeat containing-1: a novel biomarker associated with disease activity in Systemic lupus erythematosus.

The objective of this article is to investigate whether the aberrant expression of collagen triple helix repeat containing-1 (CTHRC1) from patients with systemic lupus erythematosus (SLE) could contribute to the pathogenesis of lupus. We divided SLE patients into active groups (Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score ≥ 6) and inactive groups (SLEDAI score < 6). Serum concentrations of CTHRC1, interferon alpha, interleukin (IL)-28A and IL-28B were determined using an enzyme-linked immunosorbent assay in a group of 40 patients with SLE. Results were compared with those from 23 healthy controls. Serum CTHRC1 protein levels were higher in patients with SLE compared with healthy controls. Patients with active disease displayed higher CTHRC1 levels compared with those with inactive disease as well. There was a positive association between serum CTHRC1 levels and SLEDAI and erythrocyte sedimentation rate, and a negative correlation with complement 3 and 4. Moreover, serum CTHRC1 levels were higher in SLE patients with arthritis and anemia compared with patients without the above-mentioned manifestations. These findings indicate CTHRC1 probably plays an important part in the pathogenesis of SLE, and is positively associated with disease activity, while it also likely refers to the development of arthritis and anemia in SLE. Therefore, CTHRC1 may provide a novel research target and shed new light on the pathogenesis and therapy of SLE.

Epigenome-wide association study of peripheral blood mononuclear cells in systemic lupus erythematosus: Identifying DNA methylation signatures associated with interferon-related genes based on ethnicity and SLEDAI

Systemic lupus erythematosus (SLE or lupus) is a heterogeneous autoimmune disease characterized by the involvement of multiple organs and the production of antinuclear antibodies. DNA methylation plays an important role in the pathogenesis of lupus. We have performed an epigenome-wide DNA methylation study in lupus and healthy control (non-lupus) subjects to identify epigenetic patterns in lupus characterized ethnicity and SLE disease activity index (SLEDAI). A total of fifty-seven lupus patients (39 African American (AA) and 18 European American (EA)) and 33 healthy controls (17 AA and 16 EA) were studied. Differential DNA methylation between lupus patients and controls was assessed for approximately 485,000 CpG sites across the genome. We identified 41 differentially methylated sites (associated with 30 genes) between lupus and control s subjects, 85% of which were hypomethylated. Significant hypomethylation of differentially methylated sites was associated with several interferon-related genes, including MX1, IFI44L, PARP9, DT3XL, IFIT1, IFI44, RSAD2, PLSCR1, and IRF7. Several of these associated genes were also hypomethylated in comparisons between AA lupus and AA non-lupus subjects and between lupus patients with SLEDAI>6 and non-lupus subjects. Our analysis of gene expression data through RT-PCR confirmed these findings. Thus, the results indicate epigenetics susceptibility in lupus, which may be associated with SLEDAI score and ethnicity. In addition, our findings support the importance of the Type 1 interferon pathway in lupus pathogenesis.

B cell responses to apoptotic cells in MFG-E8-/- mice

Defective clearance of apoptotic cells in MFG-E8 deficient mice results in lupus-like disease in the mixed B6x129, but not pure B6 background. The lack of overt autoimmunity in MFG-E8-/- B6 mice suggests that accumulation of apoptotic cells is not sufficient to break central tolerance. However, the delayed clearance of apoptotic cells in the follicles of MFG-E8-/- B6 mice provides an excellent opportunity to investigate how B cells respond to excessive apoptotic cells in the periphery under relatively non-inflammatory conditions. In MFG-E8-/- B6 mice, we found increased IgG2c production against apoptotic cells and oxidized LDL. Apoptotic cell induced antibody responses depended on MyD88 signal and T cell help. In addition, MFG-E8-/- B6 mice had enlarged MZ B cell compartments as well as an enhanced antibody response to NP-Ficoll. Moreover, a significant percentage of MZ B cells in aged MFG-E8-/- B6 mice migrated into follicles. Injecting apoptotic cells or oxidized LDL into wild type mice as well as physiological accumulation of LDL in ApoE-/- mice recapitulated the translocation of MZ B cells. To determine how MFG-E8 deficiency affects the functions of autoreactive B cells specific for nucleic acids in the periphery under non-inflammatory conditions, we utilized BCR transgenic mice to bypass central selection and compared the differentiation of TLR9 dependent anti-dsDNA 56R B cells and TLR7 dependent anti-ssRNA H564 B cells in MFG-E8-/- mice. In MFG-E8-/- 56R mice, anti-dsDNA specific 56R/Vκ38c B cells differentiated into MZ B cells but not AFCs. On the contrary, in MFG-E8-/-H564 mice, anti-ssRNA specific H564 B cells further differentiated into GC B cells and AFCs. Adoptive transfer of activated autoreactive B cells confirmed that H564 B cells were more sensitive to apoptotic cell antigens than 56R B cells. Our observations provide new insights about the MZ B cell translocation in lupus patients as well as the dichotomy of TLR9 and TLR7 signals in the pathogenesis of lupus.

Ligand Recognition Determines the Role of Inhibitory B Cell Co-receptors in the Regulation of B Cell Homeostasis and Autoimmunity

B cells express various inhibitory co-receptors including CD22, CD72, and Siglec-G. These receptors contain immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region. Although many of the inhibitory co-receptors negatively regulate BCR signaling by activating SH2-containing protein tyrosine phosphatase 1 (SHP-1), different inhibitory co-receptors have distinct functional properties. CD22, Siglec-G, and CD72 preferentially regulate tonic signaling in conventional B cells, B-1 cell homeostasis, and development of lupus-like disease, respectively. CD72 recognizes RNA-related lupus self-antigen Sm/RNP as a ligand. This ligand recognition recruits CD72 to BCR in Sm/RNP-reactive B cells thereby suppressing production of anti-Sm/RNP autoantibody involved in the pathogenesis of lupus. In contrast, Siglec-G recognizes α2,3 as well as α2,6 sialic acids whereas CD22 recognizes α2,6 sialic acid alone. Because glycoproteins including BCR are dominantly glycosylated with α2,3 sialic acids in B-1 cells, Siglec-G but not CD22 recruits BCR as a ligand specifically in B-1 cells, and regulates B-1 cell homeostasis by suppressing BCR signaling in B-1 cells. Thus, recognition of distinct ligands determines functional properties of different inhibitory B cell co-receptors.

Vitamin D levels in systematic lupus erythematosus patients: association with disease activity index and interferon alpha

Background: In different studies the correlation between systemic lupus erythematosus (SLE) and disease activity with vitamin D has been shown. The role of different interferons especially interferon alpha (IFN-α) in lupus pathogenesis have been previously shown. Considering the role of vitamin D and IFN-α, it is possible that these two could demonstrate the SLE activity. This study aimed to investigate the association between vitamin D levels and IFN-α with disease activity index in Ardabil city SLE patients.Methods: In this case control study, 50 SLE patients and 50 age and sex matched healthy subjects were recruited. Patients had serum 25-OH Vitamin D concentration and disease activity recorded. Vitamin D and IFN-α levels were compared between two groups and also, levels of anti-dsDNA, and SLEDAI in case group was measured. Statistical methods were used to determine the correlation between vitamin D and IFN-α with disease activity index at baseline.Results: Vitamin D deficiency (&lt;40 nmol/L) was detected in 20% of SLE patients. Vitamin D level in case group significantly lower than control group (23.94±11.93 vs. 29.10±11.40 ng/ml, p=0.02). The IFN-α amount in case group significantly upper than control group (396.60±54.73 vs. 200.38±14.42, p=0.001). There was significantly negative correlation between vitamin D with IFN-α (r=-0.413, p=0.003), SLEDAI (r=-0.492, p&lt;0.001) and anti-dsDNA (r=-0.417, p=0.003). There was positive correlation between IFN-α with SLEDAI (r=0.358, p=0.01) and anti-dsDNA (p=0.297, p=0.03).Conclusions: Results showed that the low vitamin D was associated with a higher disease activity in SLE patients. Also, it seems that, the improvement of vitamin D levels by decreasing IFN-α could help in controlling disease activity in future.

Cutting Edge: Deletion of Ezrin in B Cells of Lyn-Deficient Mice Downregulates Lupus Pathology.

Genetic deletion of the Src family tyrosine kinase Lyn in mice recapitulates human systemic lupus erythematosus, characterized by hyperactive BCR signaling, splenomegaly, autoantibody generation, and glomerulonephritis. However, the molecular regulators of autoimmunity in Lyn-deficient mice and in human lupus remain poorly characterized. In this study, we report that conditional deletion of the membrane-cytoskeleton linker protein ezrin in B cells of Lyn-deficient mice (double knockout [DKO] mice) ameliorates B cell activation and lupus pathogenesis. B cells from DKO mice respond poorly to BCR stimulation, with severe downregulation of major signaling pathways. DKO mice exhibit reduced splenomegaly as well as significantly lower levels of autoantibodies against a variety of autoantigens, including dsDNA, histone, and chromatin. Leukocyte infiltration and deposition of IgG and complement component C3 in the kidney glomeruli of DKO mice are markedly reduced. Our data demonstrate that ezrin is a novel molecular regulator of B cell-associated lupus pathology.

Abstract B77: Low-density neutrophils (LDN) in circulating blood of postoperative patients may participate in the development of distant recurrence through the production of neutrophil extracellular traps (NETs)

Abstract Background: Low density neutrophils (LDN) which are co-purified with mononuclear cells by Ficoll-Hypaque density gradient preparations, have been reported to be increased in circulating blood in patients with autoimmune diseases, sepsis and cancer. Recent studies have suggested the LDN contributes to lupus pathogenesis as well as the development of organ damage in sepsis. Method: In patients who underwent curative surgery for gastrointestinal malignancy, peripheral blood was obtained at the timing of laparotomy (preoperative) and just before wound closure (postoperative) and the ratios of LDN were evaluated with flowcytometry. The LDN and mononuclear cells (MNC) in postoperative samples were separated using CD66b microbeads and MACS column and the proliferation of T cells were evaluated by in vitro culture on anti-CD3 coated plate using CFSE dilution method. The LDN were cultured for 2 hours and neutrophil extracellular traps (NETs) were observed with the addition of SYTOX green, dye for extracellular double strand (ds)-DNA. In adhesion experiments, human colon cancer, DLD-1, were stained by PKH-26 and added on the cultured LDN for 5 min. After gentle washing, the remaining cells were counted under fluorescein microscope. Results: 1. Ratios of CD66b(+) LDG in CD45(+) leukocytes were markedly increased in postoperative as compared with preoperative blood samples. 2. Division of CD3(+) T cells were significantly suppressed by the addition autologous CD66b(+) LDN. 3. Short term culture of the LDN produced massive thread-like structures stained with SYTOX green. Immunostaining with specific mAbs revealed that histone and myeloperoxidase (MPO) were colocalized on the fibrous structure, indicating NETs 3. DLD-1, selectively attached to the NETs, which was totally abrogated by the pretreatment with 100u/ml DNAse. Conclusion: During abdominal surgery, immunosuppressive LDN are markedly increased in circulating blood. The LDN extensively produces NETs which support the metastasis formation by trapping the circulating tumor cells in target organ. Functional blockade of the LDN might effectively reduce the recurrence rate after curative surgery. Citation Format: Yuko Kumagai, Rihito Kanamaru, Hideyuki Ohzawa, Hisanaga Horie, Yoshinori Hosoya, Naohiro Sata, Joji Kitayama. Low-density neutrophils (LDN) in circulating blood of postoperative patients may participate in the development of distant recurrence through the production of neutrophil extracellular traps (NETs) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B77.