Natural isoaspartyl protein modification of ZAP70 alters T cell responses in lupus.

Protein posttranslational modifications (PTMs) arise in a number of normal cellular biological pathways and in response to pathology caused by inflammation and/or infection. Indeed, a number of PTMs have been identified and linked to specific autoimmune responses and metabolic pathways. One particular PTM, termed isoaspartyl (isoAsp or isoD) modification, is among the most common spontaneous PTM occurring at physiological pH and temperature. Herein, we demonstrate that isoAsp modifications arise within the ZAP70 protein tyrosine kinase upon T-cell antigen receptor (TCR) engagement. The enzyme protein L-isoaspartate O-methyltransferase (PCMT1, or PIMT, EC 2.1.1.77) evolved to repair isoaspartyl modifications in cells. In this regard, we observe that increased levels of isoAsp modification that arise under oxidative stress are correlated with reduced PIMT activity in patients with systemic lupus erythematosus (SLE). PIMT deficiency leads to T cell hyper-proliferation and hyper-phosphorylation through ZAP70 signaling. We demonstrate that inducing the overexpression of PIMT can correct the hyper-responsive phenotype in lupus T cells. Our studies reveal a phenotypic role of isoAsp modification and phosphorylation of ZAP70 in lupus T cell autoimmunity and provide a potential therapeutic target through the repair of isoAsp modification.

Autoantibody and T cell responses to oxidative post-translationally modified insulin neoantigenic peptides in type 1 diabetes.

AIMS/HYPOTHESIS: Antibodies specific to oxidative post-translational modifications (oxPTM) of insulin (oxPTM-INS) are present in most individuals with type 1 diabetes, even before the clinical onset. However, the antigenic determinants of such response are still unknown. In this study, we investigated the antibody response to oxPTM-INS neoepitope peptides (oxPTM-INSPs) and evaluated their ability to stimulate humoral and T cell responses in type 1 diabetes. We also assessed the concordance between antibody and T cell responses to the oxPTM-INS neoantigenic peptides. METHODS: oxPTM-INS was generated by exposing insulin to various reactive oxidants. The insulin fragments resulting from oxPTM were fractionated by size-exclusion chromatography further to ELISA and LC-MS/MS analysis to identify the oxidised peptide neoepitopes. Immunogenic peptide candidates were produced and then modified in house or designed to incorporate in silico-oxidised amino acids during synthesis. Autoantibodies to the oxPTM-INSPs were tested by ELISA using sera from 63 participants with new-onset type 1 diabetes and 30 control participants. An additional 18 fresh blood samples from participants with recently diagnosed type 1 diabetes, five with established disease, and from 11 control participants were used to evaluate, in parallel, CD4+ and CD8+ T cell activation by oxPTM-INSPs. RESULTS: We observed antibody and T cell responses to three out of six LC-MS/MS-identified insulin peptide candidates: A:12-21 (SLYQLENYCN, native insulin peptide 3 [Nt-INSP-3]), B:11-30 (LVEALYLVCGERGFFYTPKT, Nt-INSP-4) and B:21-30 (ERGFFYTPKT, Nt-INSP-6). For Nt-INSP-4 and Nt-INSP-6, serum antibody binding was stronger in type 1 diabetes compared with healthy control participants (p≤0.02), with oxidised forms of ERGFFYTPKT, oxPTM-INSP-6 conferring the highest antibody binding (83% binders to peptide modified in house by hydroxyl radical [●OH] and >88% to in silico-oxidised peptide; p≤0.001 vs control participants). Nt-INSP-4 induced the strongest T cell stimulation in type 1 diabetes compared with control participants for both CD4+ (p<0.001) and CD8+ (p=0.049). CD4+ response to oxPTM-INSP-6 was also commoner in type 1 diabetes than in control participants (66.7% vs 27.3%; p=0.039). Among individuals with type 1 diabetes, the CD4+ response to oxPTM-INSP-6 was more frequent than to Nt-INSP-6 (66.7% vs 27.8%; p=0.045). Overall, 44.4% of patients showed a concordant autoimmune response to oxPTM-INSP involving simultaneously CD4+ and CD8+ T cells and autoantibodies. CONCLUSIONS/INTERPRETATION: Our findings support the concept that oxidative stress, and neoantigenic epitopes of insulin, may be involved in the immunopathogenesis of type 1 diabetes.

A discovery-based proteomics approach identifies protein disulphide isomerase (PDIA1) as a biomarker of ß cell stress in type 1 diabetes.

BACKGROUND: Stress responses within the ß cell have been linked with both increased ß cell death and accelerated immune activation in type 1 diabetes (T1D). At present, information on the timing and scope of these responses as well as disease-related changes in islet ß cell protein expression during T1D development is lacking. METHODS: Data independent acquisition-mass spectrometry was performed on islets collected longitudinally from NOD mice and NOD-SCID mice rendered diabetic through T cell adoptive transfer. FINDINGS: In islets collected from female NOD mice at 10, 12, and 14 weeks of age, we found a time-restricted upregulation of proteins involved in stress mitigation and maintenance of ß cell function, followed by loss of expression of protective proteins that heralded diabetes onset. EIF2 signalling and the unfolded protein response, mTOR signalling, mitochondrial function, and oxidative phosphorylation were commonly modulated pathways in both NOD mice and NOD-SCID mice rendered acutely diabetic by T cell adoptive transfer. Protein disulphide isomerase A1 (PDIA1) was upregulated in NOD islets and pancreatic sections from human organ donors with autoantibody positivity or T1D. Moreover, PDIA1 plasma levels were increased in pre-diabetic NOD mice and in the serum of children with recent-onset T1D compared to non-diabetic controls. INTERPRETATION: We identified a core set of modulated pathways across distinct mouse models of T1D and identified PDIA1 as a potential human biomarker of ß cell stress in T1D. FUNDING: NIH (R01DK093954, DK127308, U01DK127786, UC4DK104166, R01DK060581, R01GM118470, and 5T32DK101001-09). VA Merit Award I01BX001733. JDRF (2-SRA-2019-834-S-B, 2-SRA-2018-493-A-B, 3-PDF-20016-199-A-N, 5-CDA-2022-1176-A-N, and 3-PDF-2017-385-A-N).

Biomarkers of autoimmunity and beta cell metabolism in type 1 diabetes.

Posttranslational protein modifications (PTMs) are an inherent response to physiological changes causing altered protein structure and potentially modulating important biological functions of the modified protein. Besides cellular metabolic pathways that may be dictated by PTMs, the subtle change of proteins also may provoke immune attack in numerous autoimmune diseases. Type 1 diabetes (T1D) is a chronic autoimmune disease destroying insulin-producing beta cells within the pancreatic islets, a result of tissue inflammation to specific autoantigens. This review summarizes how PTMs arise and the potential pathological consequence of PTMs, with particular focus on specific autoimmunity to pancreatic beta cells and cellular metabolic dysfunction in T1D. Moreover, we review PTM-associated biomarkers in the prediction, diagnosis and in monitoring disease activity in T1D. Finally, we will discuss potential preventive and therapeutic approaches of targeting PTMs in repairing or restoring normal metabolic pathways in pancreatic islets.

Immune cells and their inflammatory mediators modify ß cells and cause checkpoint inhibitor-induced diabetes.

Checkpoint inhibitors (CPIs) targeting programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) have revolutionized cancer treatment but can trigger autoimmune complications, including CPI-induced diabetes mellitus (CPI-DM), which occurs preferentially with PD-1 blockade. We found evidence of pancreatic inflammation in patients with CPI-DM with shrinkage of pancreases, increased pancreatic enzymes, and in a case from a patient who died with CPI-DM, peri-islet lymphocytic infiltration. In the NOD mouse model, anti-PD-L1 but not anti-CTLA-4 induced diabetes rapidly. RNA sequencing revealed that cytolytic IFN-γ+CD8+ T cells infiltrated islets with anti-PD-L1. Changes in ß cells were predominantly driven by IFN-γ and TNF-α and included induction of a potentially novel ß cell population with transcriptional changes suggesting dedifferentiation. IFN-γ increased checkpoint ligand expression and activated apoptosis pathways in human ß cells in vitro. Treatment with anti-IFN-γ and anti-TNF-α prevented CPI-DM in anti-PD-L1-treated NOD mice. CPIs targeting the PD-1/PD-L1 pathway resulted in transcriptional changes in ß cells and immune infiltrates that may lead to the development of diabetes. Inhibition of inflammatory cytokines can prevent CPI-DM, suggesting a strategy for clinical application to prevent this complication.

Borrelia miyamotoi Meningoencephalitis in an Immunocompetent Patient.

Borrelia miyamotoi is an underdiagnosed cause of tick-borne illness in endemic regions and, in rare cases, causes neurological disease in immunocompetent patients. Here, we present a case of serologically confirmed Borrelia miyamotoi meningoencephalitis in an otherwise healthy patient who rapidly improved following initiation of antibiotic therapy.

Defining Clinical and Immunological Predictors of Poor Immune Responses to COVID-19 mRNA Vaccines in Patients with Primary Antibody Deficiency.

Immune responses to coronavirus disease 2019 (COVID-19) mRNA vaccines in primary antibody deficiencies (PADs) are largely unknown. We investigated antibody and CD4+ T-cell responses specific for SARS-CoV-2 spike protein (S) before and after vaccination and associations between vaccine response and patients' clinical and immunological characteristics in PADs. The PAD cohort consisted of common variable immune deficiency (CVID) and other PADs, not meeting the criteria for CVID diagnosis (oPADs). Anti-S IgG, IgA, and IgG subclasses 1 and 3 increased after vaccination and correlated with neutralization activity in HCs and patients with oPADs. However, 42% of CVID patients developed such responses after the 2nd dose. A similar pattern was also observed with S-specific CD4+ T-cells as determined by OX40 and 4-1BB expression. Patients with poor anti-S IgG response had significantly lower levels of baseline IgG, IgA, CD19+ B-cells, switched memory B-cells, naïve CD8+ T-cells, and a higher frequency of EM CD8+ T-cells and autoimmunity compared to patients with adequate anti-S IgG responses. Patients with oPADs can develop humoral and cellular immune responses to vaccines similar to HCs. However, a subset of CVID patients exhibit impairment in developing such responses, which can be predicted by the baseline immune profile and history of autoimmunity.

Carbonyl Posttranslational Modification Associated With Early-Onset Type 1 Diabetes Autoimmunity.

Inflammation and oxidative stress in pancreatic islets amplify the appearance of various posttranslational modifications to self-proteins. In this study, we identified a select group of carbonylated islet proteins arising before the onset of hyperglycemia in NOD mice. Of interest, we identified carbonyl modification of the prolyl-4-hydroxylase ß subunit (P4Hb) that is responsible for proinsulin folding and trafficking as an autoantigen in both human and murine type 1 diabetes. We found that carbonylated P4Hb is amplified in stressed islets coincident with decreased glucose-stimulated insulin secretion and altered proinsulin-to-insulin ratios. Autoantibodies against P4Hb were detected in prediabetic NOD mice and in early human type 1 diabetes prior to the onset of anti-insulin autoimmunity. Moreover, we identify autoreactive CD4+ T-cell responses toward carbonyl-P4Hb epitopes in the circulation of patients with type 1 diabetes. Our studies provide mechanistic insight into the pathways of proinsulin metabolism and in creating autoantigenic forms of insulin in type 1 diabetes.

Citrullination of glucokinase is linked to autoimmune diabetes.

Inflammation, including reactive oxygen species and inflammatory cytokines in tissues amplify various post-translational modifications of self-proteins. A number of post-translational modifications have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes. Here we show the citrullination of pancreatic glucokinase as a result of inflammation, triggering autoimmunity and affecting glucokinase biological functions. Glucokinase is expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells as a glucose sensor to initiate glycolysis and insulin signaling. We identify autoantibodies and autoreactive CD4+ T cells to glucokinase epitopes in the circulation of Type 1 diabetes patients and NOD mice. Finally, citrullination alters glucokinase biologic activity and suppresses glucose-stimulated insulin secretion. Our study define glucokinase as a Type 1 diabetes biomarker, providing new insights of how inflammation drives post-translational modifications to create both neoautoantigens and affect beta cell metabolism.

CD8+ T Cells Variably Recognize Native Versus Citrullinated GRP78 Epitopes in Type 1 Diabetes.

In type 1 diabetes, autoimmune ß-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8+ T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8+ T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8+ T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8+ T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Vaccine-induced ErbB (EGFR/HER2)-specific immunity in spontaneous canine cancer.

Epidermal Growth Factor Receptor (EGFR) is overexpressed on a number of human cancers, and often is indicative of a poor outcome. Treatment of EGFR/HER2 overexpressing cancers includes monoclonal antibody therapy (cetuximab/trastuzumab) either alone or in conjunction with other standard cancer therapies. While monoclonal antibody therapy has been proven to be efficacious in the treatment of EGFR/HER2 overexpressing tumors, drawbacks include the lack of long-lasting immunity and acquired resistance to monoclonal therapy. An alternative approach is to induce a polyclonal anti-EGFR/HER2 tumor antigen response by vaccine therapy. In this phase I/II open-label study, we examined anti-tumor immunity in companion dogs with spontaneous EGFR expressing tumors. Canine cancers represent an outbred population in which the initiation, progression of disease, mutations and growth factors closely resemble that of human cancers. Dogs with EGFR expressing tumors were immunized with a short peptide of the EGFR extracellular domain with sequence homology to HER2. Serial serum analyses demonstrated high titers of EGFR/HER2 binding antibodies with biological activity similar to that of cetuximab and trastuzumab. Canine antibodies bound both canine and human EGFR on tumor cell lines and tumor tissue. CD8 T cells and IgG deposition were evident in tumors from immunized dogs. The antibodies inhibited EGFR intracellular signaling and inhibited tumor growth in vitro. Additionally, we illustrate objective responses in reducing tumors at metastatic sites in host animals. The data support the approach of amplifying anti-tumor immunity that may be relevant in combination with other immune modifying therapies such as checkpoint inhibitors.

Citrullination and PAD Enzyme Biology in Type 1 Diabetes - Regulators of Inflammation, Autoimmunity, and Pathology.

The generation of post-translational modifications (PTMs) in human proteins is a physiological process leading to structural and immunologic variety in proteins, with potentially altered biological functions. PTMs often arise through normal responses to cellular stress, including general oxidative changes in the tissue microenvironment and intracellular stress to the endoplasmic reticulum or immune-mediated inflammatory stresses. Many studies have now illustrated the presence of 'neoepitopes' consisting of PTM self-proteins that induce robust autoimmune responses. These pathways of inflammatory neoepitope generation are commonly observed in many autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes (T1D), among others. This review will focus on one specific PTM to self-proteins known as citrullination. Citrullination is mediated by calcium-dependent peptidylarginine deiminase (PAD) enzymes, which catalyze deimination, the conversion of arginine into the non-classical amino acid citrulline. PADs and citrullinated peptides have been associated with different autoimmune diseases, notably with a prominent role in the diagnosis and pathology of rheumatoid arthritis. More recently, an important role for PADs and citrullinated self-proteins has emerged in T1D. In this review we will provide a comprehensive overview on the pathogenic role for PADs and citrullination in inflammation and autoimmunity, with specific focus on evidence for their role in T1D. The general role of PADs in epigenetic and transcriptional processes, as well as their crucial role in histone citrullination, neutrophil biology and neutrophil extracellular trap (NET) formation will be discussed. The latter is important in view of increasing evidence for a role of neutrophils and NETosis in the pathogenesis of T1D. Further, we will discuss the underlying processes leading to citrullination, the genetic susceptibility factors for increased recognition of citrullinated epitopes by T1D HLA-susceptibility types and provide an overview of reported autoreactive responses against citrullinated epitopes, both of T cells and autoantibodies in T1D patients. Finally, we will discuss recent observations obtained in NOD mice, pointing to prevention of diabetes development through PAD inhibition, and the potential role of PAD inhibitors as novel therapeutic strategy in autoimmunity and in T1D in particular.

The Role of ß Cell Stress and Neo-Epitopes in the Immunopathology of Type 1 Diabetes.

Due to their secretory function, ß cells are predisposed to higher levels of endoplasmic reticulum (ER) stress and greater sensitivity to inflammation than other cell types. These stresses elicit changes in ß cells that alter their function and immunogenicity, including defective ribosomal initiation, post-translational modifications (PTMs) of endogenous ß cell proteins, and alternative splicing. Multiple published reports confirm the presence of not only CD8+ T cells, but also autoreactive CD4+ T cells within pancreatic islets. Although the specificities of T cells that infiltrate human islets are incompletely characterized, they have been confirmed to include neo-epitopes that are formed through stress-related enzymatic modifications of ß cell proteins. This article summarizes emerging knowledge about stress-induced changes in ß cells and data supporting a role for neo-antigen formation and cross-talk between immune cells and ß cells that provokes autoimmune attack - leading to a breakdown in tissue-specific tolerance in subjects who develop type 1 diabetes.

Macrophage Migration Inhibitory Factor Regulates U1 Small Nuclear RNP Immune Complex-Mediated Activation of the NLRP3 Inflammasome.

OBJECTIVE: High-expression alleles of macrophage migration inhibitory factor (MIF) are linked genetically to the severity of systemic lupus erythematosus (SLE). The U1 small nuclear RNP (snRNP) immune complex containing U1 snRNP and anti-U1 snRNP antibodies, which are found in patients with SLE, activates the NLRP3 inflammasome, comprising NLRP3, ASC, and procaspase 1, in human monocytes, leading to the production of interleukin-1ß (IL-1ß). This study was undertaken to investigate the role of the snRNP immune complex in up-regulating the expression of MIF and its interface with the NLRP3 inflammasome. METHODS: MIF, IL-1ß, NLRP3, caspase 1, ASC, and MIF receptors were analyzed by enzyme-linked immunosorbent assay, Western blotting, quantitative polymerase chain reaction, and cytometry by time-of-flight mass spectrometry (CytoF) in human monocytes incubated with or without the snRNP immune complex. MIF pathway responses were probed with the novel small molecule antagonist MIF098. RESULTS: The snRNP immune complex induced the production of MIF and IL-1ß from human monocytes. High-dimensional, single-cell CytoF analysis established that MIF regulates activation of the NLRP3 inflammasome, including findings of a quantitative relationship between MIF and its receptors and IL-1ß levels in the monocytes. MIF098, which blocks MIF binding to its cognate receptor, suppressed the production of IL-1ß, the up-regulation of NLRP3, which is a rate-limiting step in NLRP3 inflammasome activation, and the activation of caspase 1 in snRNP immune complex-stimulated human monocytes. CONCLUSION: The U1 snRNP immune complex is a specific stimulus of MIF production in human monocytes, with MIF having an upstream role in defining the inflammatory characteristics of activated monocytes by regulating NLRP3 inflammasome activation and downstream IL-1ß production. These findings provide mechanistic insight and a therapeutic rationale for targeting MIF in subgroups of lupus patients, such as those classified as high genotypic MIF expressers or those with anti-snRNP antibodies.

Inflammation-Induced Citrullinated Glucose-Regulated Protein 78 Elicits Immune Responses in Human Type 1 Diabetes.

The ß-cell has become recognized as a central player in the pathogenesis of type 1 diabetes with the generation of neoantigens as potential triggers for breaking immune tolerance. We report that posttranslationally modified glucose-regulated protein 78 (GRP78) is a novel autoantigen in human type 1 diabetes. When human islets were exposed to inflammatory stress induced by interleukin-1ß, tumor necrosis factor-α, and interferon-γ, arginine residue R510 within GRP78 was converted into citrulline, as evidenced by liquid chromatography-tandem mass spectrometry. This conversion, known as citrullination, led to the generation of neoepitopes, which effectively could be presented by HLA-DRB1*04:01 molecules. With the use of HLA-DRB1*04:01 tetramers and ELISA techniques, we demonstrate enhanced antigenicity of citrullinated GRP78 with significantly increased CD4+ T-cell responses and autoantibody titers in patients with type 1 diabetes compared with healthy control subjects. Of note, patients with type 1 diabetes had a predominantly higher percentage of central memory cells and a lower percentage of effector memory cells directed against citrullinated GRP78 compared with the native epitope. These results strongly suggest that citrullination of ß-cell proteins, exemplified here by the citrullination of GRP78, contributes to loss of self-tolerance toward ß-cells in human type 1 diabetes, indicating that ß-cells actively participate in their own demise.

Epidermal growth factor receptor peptide vaccination induces cross-reactive immunity to human EGFR, HER2, and HER3.

Current treatments for tumors expressing epidermal growth factor receptor (EGFR) include anti-EGFR monoclonal antibodies, often used in conjunction with the standard chemotherapy, radiation therapy, or other EGFR inhibitors. While monoclonal antibody treatment is efficacious in many patients, drawbacks include its high cost of treatment and side effects associated with multiple drug infusions. As an alternative to monoclonal antibody treatments, we have focused on peptide-based vaccination to trigger natural anti-tumor antibodies. Here, we demonstrate that peptides based on a region of the EGFR extracellular domain IV break immune tolerance to EGFR and elicit anti-tumor immunity. Mice immunized with isoforms of EGFR peptide p580-598 generated anti-EGFR antibody and T-cell responses. Iso-aspartyl (iso-Asp)-modified EGFR p580 immune sera inhibit in vitro growth of EGFR overexpressing human A431 tumor cells, as well as promote antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies induced by Asp and iso-Asp p580 bound homologous regions of the EGFR family members HER2 and HER3. EGFR p580 immune sera also inhibited the growth of the human tumor cell line MDA-MB-453 that expresses HER2 but not EGFR. Asp and iso-Asp EGFR p580 induced antibodies were also able to inhibit the in vivo growth of EGFR-expressing tumors. These data demonstrate that EGFR peptides from a region of the EGFR extracellular domain IV promote anti-tumor immunity, tumor cell killing, and antibodies that are cross reactive with ErbB family members.

Immune Recognition of ß-Cells: Neoepitopes as Key Players in the Loss of Tolerance.

Prior to the onset of type 1 diabetes, there is progressive loss of immune self-tolerance, evidenced by the accumulation of islet autoantibodies and emergence of autoreactive T cells. Continued autoimmune activity leads to the destruction of pancreatic ß-cells and loss of insulin secretion. Studies of samples from patients with type 1 diabetes and of murine disease models have generated important insights about genetic and environmental factors that contribute to susceptibility and immune pathways that are important for pathogenesis. However, important unanswered questions remain regarding the events that surround the initial loss of tolerance and subsequent failure of regulatory mechanisms to arrest autoimmunity and preserve functional ß-cells. In this Perspective, we discuss various processes that lead to the generation of neoepitopes in pancreatic ß-cells, their recognition by autoreactive T cells and antibodies, and potential roles for such responses in the pathology of disease. Emerging evidence supports the relevance of neoepitopes generated through processes that are mechanistically linked with ß-cell stress. Together, these observations support a paradigm in which neoepitope generation leads to the activation of pathogenic immune cells that initiate a feed-forward loop that can amplify the antigenic repertoire toward pancreatic ß-cell proteins.

Oxidative Modifications in Tissue Pathology and Autoimmune Disease.

SIGNIFICANCE: Various autoimmune syndromes are characterized by abnormalities found at the level of tissues and cells, as well as by microenvironmental influences, such as reactive oxygen species (ROS), that alter intracellular metabolism and protein expression. Moreover, the convergence of genetic, epigenetic, and even environmental influences can result in B and T lymphocyte autoimmunity and tissue pathology. Recent Advances: This review describes how oxidative stress to cells and tissues may alter post-translational protein modifications, both directly and indirectly, as well as potentially lead to aberrant gene expression. For example, it has been clearly observed in many systems how oxidative stress directly amplifies carbonyl protein modifications. However, ROS also lead to a number of nonenzymatic spontaneous modifications including deamidation and isoaspartate modification as well as to enzyme-mediated citrullination of self-proteins. ROS have direct effects on DNA methylation, leading to influences in gene expression, chromosome inactivation, and the silencing of genetic elements. Finally, ROS can alter many other cellular pathways, including the initiation of apoptosis and NETosis, triggering the release of modified intracellular autoantigens. CRITICAL ISSUES: This review will detail specific post-translational protein modifications, the pathways that control autoimmunity to modified self-proteins, and how products of ROS may be important biomarkers of tissue pathogenesis. FUTURE DIRECTIONS: A clear understanding of the many pathways affected by ROS will lead to potential therapeutic manipulations to alter the onset and/or progression of autoimmune disease.

Microbiota control immune regulation in humanized mice.

The microbiome affects development and activity of the immune system, and may modulate immune therapies, but there is little direct information about this control in vivo. We studied how the microbiome affects regulation of human immune cells in humanized mice. When humanized mice were treated with a cocktail of 4 antibiotics, there was an increase in the frequency of effector T cells in the gut wall, circulating levels of IFN-γ, and appearance of anti-nuclear antibodies. Teplizumab, a non-FcR-binding anti-CD3ε antibody, no longer delayed xenograft rejection. An increase in CD8+ central memory cells and IL-10, markers of efficacy of teplizumab, were not induced. IL-10 levels were only decreased when the mice were treated with all 4 but not individual antibiotics. Antibiotic treatment affected CD11b+CD11c+ cells, which produced less IL-10 and IL-27, and showed increased expression of CD86 and activation of T cells when cocultured with T cells and teplizumab. Soluble products in the pellets appeared to be responsible for the reduced IL-27 expression in DCs. Similar changes in IL-10 induction were seen when human peripheral blood mononuclear cells were cultured with human stool samples. We conclude that changes in the microbiome may impact the efficacy of immunosuppressive medications by altering immune regulatory pathways.

The role for neutrophil extracellular traps in cystic fibrosis autoimmunity.

While respiratory failure in cystic fibrosis (CF) frequently associates with chronic infection by Pseudomonas aeruginosa, no single factor predicts the extent of lung damage in CF. To elucidate other causes, we studied the autoantibody profile in CF and rheumatoid arthritis (RA) patients, given the similar association of airway inflammation and autoimmunity in RA. Even though we observed that bactericidal permeability-increasing protein (BPI), carbamylated proteins, and citrullinated proteins all localized to the neutrophil extracellular traps (NETs), which are implicated in the development of autoimmunity, our study demonstrates striking autoantibody specificity in CF. Particularly, CF patients developed anti-BPI autoantibodies but hardly any anti-citrullinated protein autoantibodies (ACPA). In contrast, ACPA-positive RA patients exhibited no reactivity with BPI. Interestingly, anti-carbamylated protein autoantibodies (ACarPA) were found in both cohorts but did not cross-react with BPI. Contrary to ACPA and ACarPA, anti-BPI autoantibodies recognized the BPI C-terminus in the absence of posttranslational modifications. In fact, we discovered that P. aeruginosa-mediated NET formation results in BPI cleavage by P. aeruginosa elastase, which suggests a novel mechanism in the development of autoimmunity to BPI. In accordance with this model, autoantibodies associated with presence of P. aeruginosa on sputum culture. Finally, our results provide a role for autoimmunity in CF disease severity, as autoantibody levels associate with diminished lung function.