Cellular Senescence Contributes to the Progression of Hyperoxic Bronchopulmonary Dysplasia.

Oxidative stress, inflammation, and endoplasmic reticulum (ER) stress sequentially occur in bronchopulmonary dysplasia (BPD), and all result in DNA damage. When DNA damage becomes irreparable, tumor suppressors increase, followed by apoptosis or senescence. Although cellular senescence contributes to wound healing, its persistence inhibits growth. Therefore, we hypothesized that cellular senescence contributes to BPD progression. Human autopsy lungs were obtained. Sprague-Dawley rat pups exposed to 95% oxygen between Postnatal Day 1 (P1) and P10 were used as the BPD phenotype. N-acetyl-lysyltyrosylcysteine-amide (KYC), tauroursodeoxycholic acid (TUDCA), and Foxo4 dri were administered intraperitoneally to mitigate myeloperoxidase oxidant generation, ER stress, and cellular senescence, respectively. Lungs were examined by histology, transcriptomics, and immunoblotting. Cellular senescence increased in rat and human BPD lungs, as evidenced by increased oxidative DNA damage, tumor suppressors, GL-13 stain, and inflammatory cytokines with decreased cell proliferation and lamin B expression. Cellular senescence-related transcripts in BPD rat lungs were enriched at P10 and P21. Single-cell RNA sequencing showed increased cellular senescence in several cell types, including type 2 alveolar cells. In addition, Foxo4-p53 binding increased in BPD rat lungs. Daily TUDCA or KYC, administered intraperitoneally, effectively decreased cellular senescence, improved alveolar complexity, and partially maintained the numbers of type 2 alveolar cells. Foxo4 dri administered at P4, P6, P8, and P10 led to outcomes similar to TUDCA and KYC. Our data suggest that cellular senescence plays an essential role in BPD after initial inducement by hyperoxia. Reducing myeloperoxidase toxic oxidant production, ER stress, and attenuating cellular senescence are potential therapeutic strategies for halting BPD progression.

A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity.

Although both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted the iTreg pool and established tolerance. In turn, acute depletion of iTreg cells in rescued mice resulted in weight loss and inflammation. Whereas the transcriptional signatures of nTreg and in vivo-derived iTreg cells were closely matched, there was minimal overlap in their T cell receptor (TCR) repertoires. Thus, iTreg cells are an essential nonredundant regulatory subset that supplements nTreg cells, in part by expanding TCR diversity within regulatory responses.

Cystic Fibrosis Plasma Blunts the Immune Response to Bacterial Infection.

Cystic fibrosis (CF) is caused by mutations of the gene encoding the CF transmembrane conductance regulator. It remains unclear whether the abnormal immune response in CF involves extrinsic signals released from the external or internal environment. We sought to characterize the peripheral immune signatures in CF and its association with clinical phenotypes. Healthy peripheral blood mononuclear cells (PBMCs) were cultured with plasma from CF probands (CFPs) or healthy control subjects (HCs) followed by nCounter gene and microRNA (miRNA) profiling. A discovery cohort of 12 CFPs and 12 HCs and a validation cohort of 103 CFPs and 31 HCs (our previous microarray data [GSE71799]) were analyzed to characterize the composition of cultured immune cells and establish a miRNA‒mRNA network. Cell compositions and miRNA profiles were associated with clinical characteristics of the cohorts. Significantly differentially expressed genes and abundance of myeloid cells were downregulated in PMBCs after culture with CF plasma (P < 0.05). Top-ranked miRNAs that increased in response to CF plasma (adjusted P < 0.05) included miR-155 and miR-146a, which target many immune-related genes, such as IL-8. Pseudomonas aeruginosa infection was negatively associated with abundance of monocytes and the presence of those regulatory miRNAs. Extrinsic signals in plasma from patients with CF led to monocyte inactivation and miRNA upregulation in PBMCs. An improved understanding of the immune effects of extrinsic factors in CF holds great promise for integrating immunomodulatory cell therapies into current treatment strategies in CF.

Identification of molecular signatures of cystic fibrosis disease status with plasma-based functional genomics.

Although cystic fibrosis (CF) is attributed to dysfunction of a single gene, the relationships between the abnormal gene product and the development of inflammation and progression of lung disease are not fully understood, which limits our ability to predict an individual patient's clinical course and treatment response. To better understand CF progression, we characterized the molecular signatures of CF disease status with plasma-based functional genomics. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured with plasma samples from CF patients ( n = 103) and unrelated, healthy controls ( n = 31). Gene expression levels were measured with an Affymetrix microarray (GeneChip Human Genome U133 Plus 2.0). Peripheral blood samples from a subset of the CF patients ( n = 40) were immunophenotyped by flow cytometry, and the data were compared with historical data for age-matched healthy controls ( n = 351). Plasma samples from another subset of CF patients ( n = 56) and healthy controls ( n = 16) were analyzed by multiplex enzyme-linked immunosorbent assay (ELISA) for numerous cytokines and chemokines. Principal component analysis and hierarchical clustering of induced transcriptional data revealed disease-specific plasma-induced PBMC profiles. Among 1,094 differentially expressed probe sets, 51 genes were associated with pancreatic sufficient status, and 224 genes were associated with infection with Pseudomonas aeruginosa. The flow cytometry and ELISA data confirmed that various immune modulators are relevant contributors to the CF molecular signature. This study provides strong evidence for distinct molecular signatures among CF patients. An understanding of these molecular signatures may lead to unique molecular markers that will enable more personalized prognoses, individualized treatment plans, and rapid monitoring of treatment response.

Innate immune activity as a predictor of persistent insulin secretion and association with responsiveness to CTLA4-Ig treatment in recent-onset type 1 diabetes.

AIMS/HYPOTHESIS: The study aimed to determine whether discrete subtypes of type 1 diabetes exist, based on immunoregulatory profiles at clinical onset, as this has significant implications for disease treatment and prevention as well as the design and analysis of clinical trials. METHODS: Using a plasma-based transcriptional bioassay and a gene-ontology-based scoring algorithm, we examined local participants from the Children's Hospital of Wisconsin and conducted an ancillary analysis of TrialNet CTLA4-Ig trial (TN-09) participants. RESULTS: The inflammatory/regulatory balance measured during the post-onset period was highly variable. Notably, a significant inverse relationship was identified between baseline innate inflammatory activity and stimulated C-peptide AUC measured at 3, 6, 12, 18 and 24 months post onset among placebo-treated individuals (p ≤ 0.015). Further, duration of persistent insulin secretion was negatively related to baseline inflammation (p ≤ 0.012) and positively associated with baseline abundance of circulating activated regulatory T cells (CD4+/CD45RA-/FOXP3high; p = 0.016). Based on these findings, data from participants treated with CTLA4-Ig were stratified by inflammatory activity at onset; in this way, we identified pathways and transcripts consistent with inhibition of T cell activation and enhanced immunoregulation. Variance among baseline plasma-induced signatures of TN-09 participants was further examined with weighted gene co-expression network analysis and related to clinical metrics. Four age-independent subgroups were identified that differed in terms of baseline innate inflammatory/regulatory bias, rate of C-peptide decline and response to CTLA4-Ig treatment. CONCLUSIONS/INTERPRETATION: These data support the existence of multiple type 1 diabetes subtypes characterised by varying levels of baseline innate inflammation that are associated with the rate of C-peptide decline. DATA AVAILABILITY: Gene expression data files are publicly available through the National Center for Biotechnology Information Gene Expression Omnibus (accession number GSE102234).

Alternatively Activated Macrophages Boost Induced Regulatory T and Th17 Cell Responses during Immunotherapy for Colitis.

Induced regulatory T (iTreg) and Th17 cells promote mucosal homeostasis. We used a T cell transfer model of colitis to compare the capacity of iTreg and Th17 cells to develop in situ following the transfer of naive CD4(+)CD45RB(hi)T cells intoRag1(-/-)C57BL/6 or BALB/c mice, the prototypical Th1/M1- and Th2/M2-prone strains. We found that the frequency and number of Foxp3(+)iTreg cells and Th17 cells were significantly reduced in C57BL/6 mice compared with the BALB/c strain. C57BL/6 mice with colitis were also resistant to natural Treg cell immunotherapy. Pretreatment of C57BL/6Rag1(-/-)mice with IL-4 plus IL-13, or with M2a but not M1 macrophages, dramatically increased the generation of iTreg and Th17 cells. Importantly, M2a transfers, either as a pretreatment or in mice with established colitis, allowed successful immunotherapy with natural Treg cells. M2a macrophages also reduced the generation of pathogenic iTreg cells that lost Foxp3 expression, suggesting that they stabilize the expression of Foxp3. Thus, polarized M2a macrophages drive a directionally concordant expansion of the iTreg-Th17 cell axis and can be exploited as a therapeutic adjuvant in cell-transfer immunotherapy to re-establish mucosal tolerance.

Interleukin-1 antagonism moderates the inflammatory state associated with Type 1 diabetes during clinical trials conducted at disease onset.

It was hypothesized that IL-1 antagonism would preserve ß-cell function in new onset Type 1 diabetes (T1D). However, the Anti-Interleukin-1 in Diabetes Action (AIDA) and TrialNet Canakinumab (TN-14) trials failed to show efficacy of IL-1 receptor antagonist (IL-1Ra) or canakinumab, as measured by stimulated C-peptide response. Additional measures are needed to define immune state changes associated with therapeutic responses. Here, we studied these trial participants with plasma-induced transcriptional analysis. In blinded analyses, 70.2% of AIDA and 68.9% of TN-14 participants were correctly called to their treatment arm. While the transcriptional signatures from the two trials were distinct, both therapies achieved varying immunomodulation consistent with IL-1 inhibition. On average, IL-1 antagonism resulted in modest normalization relative to healthy controls. At endpoint, signatures were quantified using a gene ontology-based inflammatory index, and an inverse relationship was observed between measured inflammation and stimulated C-peptide response in IL-1Ra- and canakinumab-treated patients. Cytokine neutralization studies showed that IL-1α and IL-1ß additively contribute to the T1D inflammatory state. Finally, analyses of baseline signatures were indicative of later therapeutic response. Despite the absence of clinical efficacy by IL-1 antagonist therapy, transcriptional analysis detected immunomodulation and may yield new insight when applied to other clinical trials.

EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice.

Infection and inflammation are invariably associated with activation of the blood coagulation mechanism, secondary to the inflammation-induced expression of the coagulation initiator tissue factor (TF) on innate immune cells. By investigating the role of cell-surface receptors for coagulation factors in mouse endotoxemia, we found that the protein C receptor (ProcR; EPCR) was required for the normal in vivo and in vitro induction of lipopolysaccharide (LPS)-regulated gene expression. In cultured bone marrow-derived myeloid cells and in monocytic RAW264.7 cells, the LPS-induced expression of functionally active TF, assembly of the ternary TF-VIIa-Xa initiation complex of blood coagulation, and the EPCR-dependent activation of protease-activated receptor 2 (PAR2) by the ternary TF-VIIa-Xa complex were required for the normal LPS induction of messenger RNAs encoding the TLR3/4 signaling adaptor protein Pellino-1 and the transcription factor interferon regulatory factor 8. In response to in vivo challenge with LPS, mice lacking EPCR or PAR2 failed to fully initiate an interferon-regulated gene expression program that included the Irf8 target genes Lif, Iigp1, Gbp2, Gbp3, and Gbp6. The inflammation-induced expression of TF and crosstalk with EPCR, PAR2, and TLR4 therefore appear necessary for the normal evolution of interferon-regulated host responses.

Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice.

The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

Increased Expression of Plasma-Induced ABCC1 mRNA in Cystic Fibrosis.

The ABCC1 gene is structurally and functionally related to the cystic fibrosis transmembrane conductance regulator gene (CFTR). Upregulation of ABCC1 is thought to improve lung function in patients with cystic fibrosis (CF); the mechanism underlying this effect is unknown. We analyzed the ABCC1 promoter single nucleotide polymorphism (SNP rs504348), plasma-induced ABCC1 mRNA expression levels, and ABCC1 methylation status and their correlation with clinical variables among CF subjects with differing CFTR mutations. We assigned 93 CF subjects into disease severity groups and genotyped SNP rs504348. For 23 CF subjects and 7 healthy controls, donor peripheral blood mononuclear cells (PBMCs) stimulated with plasma underwent gene expression analysis via qRT-PCR. ABCC1 promoter methylation was analyzed in the same 23 CF subjects. No significant correlation was observed between rs504348 genotypes and CF disease severity, but pancreatic insufficient CF subjects showed increased colonization with any form of Pseudomonas aeruginosa (OR = 3.125, 95% CI: 1.192-8.190) and mucoid P. aeruginosa (OR = 5.075, 95% CI: 1.307-28.620) compared to the pancreatic sufficient group. A significantly higher expression of ABCC1 mRNA was induced by CF plasma compared to healthy control plasma (p < 0.001). CF subjects with rs504348 (CC/CG) also had higher mRNA expression compared to those with the ancestral GG genotype (p < 0.005). ABCC1 promoter was completely unmethylated; therefore, we did not detect any association between methylation and CF disease severity. In silico predictions suggested that histone modifications are crucial for regulating ABCC1 expression in PBMCs. Our results suggest that ABCC1 expression has a role in CFTR activity thereby increasing our understanding of the molecular underpinnings of the clinical heterogeneity in CF.

Blood-based signatures in type 1 diabetes.

Type 1 diabetes mellitus is one of the most common chronic diseases in childhood. It develops through autoimmune destruction of the pancreatic beta cells and results in lifelong dependence on exogenous insulin. The pathogenesis of type 1 diabetes involves a complex interplay of genetic and environmental factors and has historically been attributed to aberrant adaptive immunity; however, there is increasing evidence for a role of innate inflammation. Over the past decade new methodologies for the analysis of nucleic acid and protein signals have been applied to type 1 diabetes. These studies are providing a new understanding of type 1 diabetes pathogenesis and have the potential to inform the development of new biomarkers for predicting diabetes onset and monitoring therapeutic interventions. In this review we will focus on blood-based signatures in type 1 diabetes, with special attention to both direct transcriptomic analyses of whole blood and immunocyte subsets, as well as plasma/serum-induced transcriptional signatures. Attention will also be given to proteomics, microRNA assays and markers of beta cell death. We will also discuss the results of blood-based profiling in type 1 diabetes within the context of the genetic and environmental factors implicated in the natural history of autoimmune diabetes.

IL-10 produced by induced regulatory T cells (iTregs) controls colitis and pathogenic ex-iTregs during immunotherapy.

"Natural" regulatory T cells (nTregs) that express the transcription factor Foxp3 and produce IL-10 are required for systemic immunological tolerance. "Induced" regulatory T cells (iTregs) are nonredundant and essential for tolerance at mucosal surfaces, yet their mechanisms of suppression and stability are unknown. We investigated the role of iTreg-produced IL-10 and iTreg fate in a treatment model of inflammatory bowel disease. Colitis was induced in Rag1(-/-) mice by the adoptive transfer of naive CD4(+) T cells carrying a nonfunctional Foxp3 allele. At the onset of weight loss, mice were treated with both iTregs and nTregs where one marked subset was selectively IL-10 deficient. Body weight assessment, histological scoring, cytokine analysis, and flow cytometry were used to monitor disease activity. Transcriptional profiling and TCR repertoire analysis were used to track cell fate. When nTregs were present but IL-10 deficient, iTreg-produced IL-10 was necessary and sufficient for the treatment of disease, and vice versa. Invariably, ∼85% of the transferred iTregs lost Foxp3 expression (ex-iTregs) but retained a portion of the iTreg transcriptome, which failed to limit their pathogenic potential upon retransfer. TCR repertoire analysis revealed no clonal relationships between iTregs and ex-iTregs, either within mice or between mice treated with the same cells. These data identify a dynamic IL-10-dependent functional reciprocity between regulatory T cell subsets that maintains mucosal tolerance. The niche supporting stable iTregs is limited and readily saturated, which promotes a large population of ex-iTregs with pathogenic potential during immunotherapy.

Monocytes in type 1 diabetes families exhibit high cytolytic activity and subset abundances that correlate with clinical progression.

Monocytes are immune regulators implicated in the pathogenesis of type 1 diabetes (T1D), an autoimmune disease that targets insulin-producing pancreatic ß cells. We determined that monocytes of recent onset (RO) T1D patients and their healthy siblings express proinflammatory/cytolytic transcriptomes and hypersecrete cytokines in response to lipopolysaccharide exposure compared to unrelated healthy controls (uHCs). Flow cytometry measured elevated circulating abundances of intermediate monocytes and >2-fold more CD14+CD16+HLADR+KLRD1+PRF1+ NK-like monocytes among patients with ROT1D compared to uHC. The intermediate to nonclassical monocyte ratio among ROT1D patients correlated with the decline in functional ß cell mass during the first 24 months after onset. Among sibling nonprogressors, temporal decreases were measured in the intermediate to nonclassical monocyte ratio and NK-like monocyte abundances; these changes coincided with increases in activated regulatory T cells. In contrast, these monocyte populations exhibited stability among T1D progressors. This study associates heightened monocyte proinflammatory/cytolytic activity with T1D susceptibility and progression and offers insight to the age-dependent decline in T1D susceptibility.

Mast cell activation and neutrophil recruitment promotes early and robust inflammation in the meninges in EAE.

The meninges are often considered inert tissues that house the CSF and provide protection for the brain and spinal cord. Yet emerging data demonstrates that they are also active sites of immune responses. Furthermore, the blood-CSF barrier surrounding meningeal blood vessels, together with the blood-brain barrier (BBB), is postulated to serve as a gateway for the pathological infiltration of immune cells into the CNS in multiple sclerosis (MS). Our previous studies using mast cell-deficient (Kit(W/Wv)) mice demonstrated that mast cells resident in the dura mater and pia mater exacerbate experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, by facilitating CNS inflammatory cell influx. Here we examined the underlying mechanisms that mediate these effects. We demonstrate that there are dramatic alterations in immune associated gene expression in the meninges in pre-clinical disease, including those associated with mast cell and neutrophil function. Meningeal mast cells are activated within 24 h of disease induction, but do not directly compromise CNS vascular integrity. Rather, through production of TNF, mast cells elicit an early influx of neutrophils, cells known to alter vascular permeability, into the meninges. These data add to the growing evidence that inflammation in the meninges precedes CNS immune cell infiltration and establish that mast cells are among the earliest participants in these disease-initiating events. We hypothesize that mast cell-dependent neutrophil recruitment and activation in the meninges promotes early breakdown of the local BBB and CSF-blood barrier allowing initial immune cell access to the CNS.

Requirements for growth and IL-10 expression of highly purified human T regulatory cells.

Human regulatory T cells (T(R)) cells have potential for the treatment of a variety of immune mediated diseases but the anergic phenotype of these cells makes them difficult to expand in vitro. We have examined the requirements for growth and cytokine expression from highly purified human T(R) cells, and correlated these findings with the signal transduction events of these cells. We demonstrate that these cells do not proliferate or secrete IL-10 even in the presence of high doses of IL-2. Stimulation with a superagonistic anti-CD28 antibody (clone 9.3) and IL-2 partially reversed the proliferative defect, and this correlated with reversal of the defective calcium mobilization in these cells. Dendritic cells were effective at promoting T(R) cell proliferation, and under these conditions the proliferative capacity of T(R) cells was comparable to conventional CD4 lymphocytes. Blocking TGF-ß activity abrogated IL-10 expression from these cells, while addition of TGF-ß resulted in IL-10 production. These data demonstrate that highly purified populations of T(R) cells are anergic even in the presence of high doses of IL-2. Furthermore, antigen presenting cells provide proper co-stimulation to overcome the anergic phenotype of T(R) cells, and under these conditions they are highly sensitive to IL-2. In addition, these data demonstrate for the first time that TGF-ß is critical to enable human T(R) cells to express IL-10.

An autoinflammatory disease due to homozygous deletion of the IL1RN locus.

We describe a patient with an autoinflammatory disease in which the main clinical features are pustular rash, marked osteopenia, lytic bone lesions, respiratory insufficiency, and thrombosis. Genetic studies revealed a 175-kb homozygous deletion at chromosome 2q13, which encompasses several interleukin-1 family members, including the gene encoding the interleukin-1-receptor antagonist (IL1RN). Mononuclear cells, obtained from the patient and cultured, produced large amounts of inflammatory cytokines, with increasing amounts secreted after stimulation with lipopolysaccharide. A similar increase was not observed in peripheral-blood mononuclear cells from a patient with neonatal-onset multisystem inflammatory disorder (NOMID). Treatment with anakinra completely resolved the symptoms and lesions.

Blockade of interleukin-23 signaling results in targeted protection of the colon and allows for separation of graft-versus-host and graft-versus-leukemia responses.

Allogeneic stem cell transplantation is the most potent form of effective adoptive immunotherapy. The graft-versus-leukemia (GVL) effect mediated by the allogeneic graft, however, is typically coexpressed with graft-versus-host disease (GVHD), which is the major complication of allogeneic stem cell transplantation. In this study, we used genetic and antibody-based strategies to examine the effect that blockade of interleukin 23 (IL-23) signaling had on GVH and GVL reactivity in murine transplantation recipients. These studies demonstrate that the selective protection of the colon that occurs as a consequence of inhibition of IL-23 signaling reduces GVHD without loss of the GVL effect. The separation of GVH and GVL reactivity was noted in both acute and chronic hematologic malignancy models, indicating that this approach was not restricted by the kinetic profile of the underlying leukemia. Furthermore, a potent GVL response could be mounted in the colon under conditions where tumor cells migrated to this site, indicating that this organ did not serve as a sanctuary site for subsequent systemic relapse in GVHD-protected animals. These studies demonstrate that blockade of IL-23 signaling is an effective strategy for separating GVH and GVL responses and identify IL-23 as a therapeutic target for the regulation of alloresponses in humans.

Characterizing T cell responses to enzymatically modified beta cell neo-epitopes.

Introduction: Previous studies verify the formation of enzymatically post-translationally modified (PTM) self-peptides and their preferred recognition by T cells in subjects with type 1 diabetes (T1D). However, questions remain about the relative prevalence of T cells that recognize PTM self-peptides derived from different antigens, their functional phenotypes, and whether their presence correlates with a specific disease endotype. Methods: To address this question, we identified a cohort of subjects with T1D who had diverse levels of residual beta cell function. Using previously developed HLA class II tetramer reagents, we enumerated T cells that recognize PTM GAD epitopes in the context of DRB1*04:01 or PTM IA2 epitopes in the context of DQB1*03:02 (DQ8). Results: Consistent with prior studies, we observed higher overall frequencies and a greater proportion of memory T cells in subjects with T1D than in HLA matched controls. There were significantly higher numbers of GAD specific T cells than IA2 specific T cells in subjects with T1D. T cells specific for both groups of epitopes could be expanded from the peripheral blood of subjects with established T1D and at-risk subjects. Expanded neo-epitope specific T cells primarily produced interferon gamma in both groups, but a greater proportion of T cells were interferon gamma positive in subjects with T1D, including some poly-functional cells that also produced IL-4. Based on direct surface phenotyping, neo-epitope specific T cells exhibited diverse combinations of chemokine receptors. However, the largest proportion had markers associated with a Th1-like phenotype. Notably, DQ8 restricted responses to PTM IA2 were over-represented in subjects with lower residual beta cell function. Neo-epitope specific T cells were present in at-risk subjects, and those with multiple autoantibodies have higher interferon gamma to IL-4 ratios than those with single autoantibodies, suggesting a shift in polarization during progression. Discussion: These results reinforce the relevance of PTM neo-epitopes in human disease and suggest that distinct responses to neo-antigens promote a more rapid decline in beta cell function.

Probiotic normalization of systemic inflammation in siblings of type 1 diabetes patients: an open-label pilot study.

The incidence of type 1 diabetes (T1D) has increased, coinciding with lifestyle changes that have likely altered the gut microbiota. Dysbiosis, gut barrier dysfunction, and elevated systemic inflammation consistent with microbial antigen exposure, have been associated with T1D susceptibility and progression. A 6-week, single-arm, open-label pilot trial was conducted to investigate whether daily multi-strain probiotic supplementation could reduce this familial inflammation in 25 unaffected siblings of T1D patients. Probiotic supplementation was well-tolerated as reflected by high participant adherence and no adverse events. Community alpha and beta diversity were not altered between the pre- and post-supplement stool samplings. However, LEfSe analyses identified post-supplement enrichment of the family Lachnospiraceae, producers of the anti-inflammatory short chain fatty acid butyrate. Systemic inflammation was measured by plasma-induced transcription and quantified with a gene ontology-based composite inflammatory index (I.I.com). Post-supplement I.I.com was significantly reduced and pathway analysis predicted inhibition of numerous inflammatory mediators and activation of IL10RA. Subjects with the greatest post-supplement reduction in I.I.com exhibited significantly lower CD4+ CD45RO+ (memory):CD4+ CD45RA+ (naïve) T-cell ratios after supplementation. Post-supplement IL-12p40, IL-13, IL-15, IL-18, CCL2, and CCL24 plasma levels were significantly reduced, while post-supplement butyrate levels trended 1.4-fold higher. Probiotic supplementation may modify T1D susceptibility and progression and warrants further study.

Lactiplantibacillus plantarum 299v supplementation modulates ß-cell ER stress and antioxidative defense pathways and prevents type 1 diabetes in gluten-free BioBreeding rats.

The increasing incidence of Type 1 diabetes has coincided with the emergence of the low-fiber, high-gluten Western diet and other environmental factors linked to dysbiosis. Since Lactiplantibacillus plantarum 299 v (Lp299v) supplementation improves gut barrier function and reduces systemic inflammation, we studied its effects in spontaneously diabetic DRlyp/lyp rats provided a normal cereal diet (ND) or a gluten-free hydrolyzed casein diet (HCD). All rats provided ND developed diabetes (62.5±7.7 days); combining ND with Lp299v did not improve survival. Diabetes was delayed by HCD (72.2±9.4 days, p = .01) and further delayed by HCD+Lp299v (84.9±14.3 days, p < .001). HCD+Lp299v pups exhibited increased plasma propionate and butyrate levels, which correlated with enriched fecal Bifidobacteriaceae and Clostridiales taxa. Islet transcriptomic and histologic analyses at 40-days of age revealed that rats fed HCD expressed an autophagy profile, while those provided HCD+Lp299v expressed ER-associated protein degradation (ERAD) and antioxidative defense pathways, including Nrf2. Exposing insulinoma cells to propionate and butyrate promoted the antioxidative defense response but did not recapitulate the HCD+Lp299v islet ERAD transcriptomic profile. Here, both diet and microbiota influenced diabetes susceptibility. Moreover, Lp299v supplement modulated antioxidative defense and ER stress responses in ß-cells, potentially offering a new therapeutic direction to thwart diabetes progression and preserve insulin secretion.