Detection of Novel Biomarkers in Pediatric Autoimmune Hepatitis by Proteomic Profiling.

Autoimmune hepatitis (AIH) is characterized by immune-mediated hepatocyte injury resulting in the destruction of liver cells, causing inflammation, liver failure, and fibrosis. Pediatric (AIH) is an autoimmune inflammatory disease that usually requires immunosuppression for an extended period. Frequent relapses after treatment discontinuation demonstrate that current therapies do not control intrahepatic immune processes. This study describes targeted proteomic profiling data in patients with AIH and controls. A total of 92 inflammatory and 92 cardiometabolic plasma markers were assessed for (i) pediatric AIH versus controls, (ii) AIH type 1 versus type 2, (iii) AIH and AIH-autoimmune sclerosing cholangitis overlapping syndrome and (iv) correlations with circulating vitamin D levels in AIH. A total of 16 proteins showed a nominally significant differential abundance in pediatric patients with AIH compared to controls. No clustering of AIH subphenotypes based on all protein data was observed, and no significant correlation of vitamin D levels was observed for the identified proteins. The proteins that showed variable expression include CA1, CA3, GAS6, FCGR2A, 4E-BP1 and CCL19, which may serve as potential biomarkers for patients with AIH. CX3CL1, CXCL10, CCL23, CSF1 and CCL19 showed homology to one another and may be coexpressed in AIH. CXCL10 seems to be the central intermediary link for the listed proteins. These proteins were involved in relevant mechanistic pathways for liver diseases and immune processes in AIH pathogenesis. This is the first report on the proteomic profile of pediatric AIH. The identified markers could potentially lead to new diagnostic and therapeutic tools. Nevertheless, considering the complex pathogenesis of AIH, more extensive studies are warranted to replicate and validate the present study's findings.

Enhanced Innate and Acquired Immune Responses in Systemic Sclerosis Primary Peripheral Blood Mononuclear Cells (PBMCs).

Chronic immune activation in systemic sclerosis is supported by the production of a plethora of cytokines with proven regulatory activities of the immune responses. This study aimed to explore PBMCs' cytokine profiles in SSc patients versus controls, as well as to investigate the balance between pro- and anti-inflammatory cytokines in association with disease duration. PBMCs were isolated from 18 SSc patients and 17 controls and further subjected to in vitro stimulation with lipopolysaccharide and heat-killed Candida albicans. Cytokine production was measured after 24 h and 7 days, respectively, using ELISA kits for interleukin (IL)-1ß, IL-1 receptor antagonist (IL-1Ra), IL-6, tumor necrosis factor (TNF), IL-10, IL-17, and interferon-gamma (IFN-gamma). IL-1 ß, IL-6, and TNF levels were increased in SSc patients compared with healthy volunteers irrespective of the stimulus used. IL-1Ra and Il-17 concentrations were not statistically different between groups, even though a trend toward higher levels in patients compared with their matched controls was also observed. Most cytokines demonstrated a stable course with disease progression, except for IL-10 levels, which declined over time. In conclusion, the results of this pilot study reveal that in patients with SSc a persistently enhanced immune response is established and maintained regardless of stimulus or disease duration.

Immune Profiling of Patients with Systemic Sclerosis through Targeted Proteomic Analysis.

High-throughput proteomic analysis could offer new insights into the pathogenesis of systemic sclerosis (SSc) and reveal non-invasive biomarkers for diagnosis and severity. This study aimed to assess the protein signature of patients with SSc compared to that of healthy volunteers, decipher various disease endotypes using circulating proteins, and determine the diagnostic performance of significantly expressed plasma analytes. We performed targeted proteomic profiling in a cohort of fifteen patients with SSc and eighteen controls using the Olink® (Olink Bioscience, Uppsala, Sweden)Target 96 Inflammation Panels. Seventeen upregulated proteins involved in angiogenesis, innate immunity, and co-stimulatory pathways discriminated between patients with SSc and healthy controls (HCs) and further classified them into two clusters, a low-inflammatory and a high-inflammatory endotype. Younger age, shorter disease duration, and lack of reflux esophagitis characterized patients in the low-inflammatory endotype. TNF, CXCL9, TNFRSF9, and CXCL10 positively correlated with disease progression, while the four-protein panel comprising TNF, CXCL9, CXCL10, and CX3CL1 showed high diagnostic performance. Collectively, this study identified a distinct inflammatory signature in patients with SSc that reflects a persistent T helper type 1 (Th 1) immune response irrespective of disease duration, while the multi-protein panel might improve early diagnosis in SSc.

GWAS-identified hyperuricemia-associated IGF1R variant rs6598541 has a limited role in urate mediated inflammation in human mononuclear cells.

Gout is a common autoinflammatory joint diseases characterized by deposition of monosodium urate (MSU) crystals which trigger an innate immune response mediated by inflammatory cytokines. IGF1R is one of the loci associated with both urate levels and gout susceptibility in GWAS to date, and IGF-1-IGF-1R signaling is implicated in urate control. We investigate the role of IGF-1/IGF1R signaling in the context of gouty inflammation. Also, we test the gout and urate-associated IGF1R rs6598541 polymorphism for association with the inflammatory capacity of mononuclear cells. For this, freshly isolated human peripheral blood mononuclear cells (PBMCs) were exposed to recombinant IGF-1 or anti-IGF1R neutralizing antibody in the presence or absence of solubilized urate, stimulated with LPS/MSU crystals. Also, the association of rs6598541 with IGF1R and protein expression and with ex vivo cytokine production levels after stimulation with gout specific stimuli was tested. Urate exposure was not associated with IGF1R expression in vitro or in vivo. Modulation of IGF1R did not alter urate-induced inflammation. Developing urate-induced trained immunity in vitro was not influenced in cells challenged with IGF-1 recombinant protein. Moreover, the IGF1R rs6598541 SNP was not associated with cytokine production. Our results indicate that urate-induced inflammatory priming is not regulated by IGF-1/IGF1R signaling in vitro. IGF1R rs6598541 status was not asociated with IGF1R expression or cytokine production in primary human PBMCs. This study suggests that the role of IGF1R in gout is tissue-specific and may be more relevant in the control of urate levels rather than in inflammatory signaling in gout.

Epigenetic, transcriptional, and functional characterization of myeloid cells in familial Mediterranean fever.

Familial Mediterranean fever (FMF) is a periodic fever syndrome caused by variation in MEFV. FMF is known for IL-1ß dysregulation, but the innate immune landscape of this disease has not been comprehensively described. Therefore, we studied circulating inflammatory proteins, and the function of monocytes and (albeit less extensively) neutrophils in treated FMF patients in remission. We found that monocyte IL-1ß and IL-6 production was enhanced upon stimulation, in concordance with alterations in the plasma inflammatory proteome. We did not observe changes in neutrophil functional assays. Subtle differences in chromatin accessibility and transcriptomics in our small patient cohort further argued for monocyte dysregulation. Together, these observations suggest that the MEFV-mutation-mediated primary immune dysregulation in monocytes leads to chronic inflammation that is subsequently associated with counterregulatory epigenetic/transcriptional changes reminiscent of tolerance. These data increase our understanding of the innate immune changes in FMF, aiding future management of chronic inflammation in these patients.

Regulation of SOCS3-STAT3 in urate-induced cytokine production in human myeloid cells.

OBJECTIVE: Hyperuricaemia is necessary for gout. High urate concentrations have been linked to inflammation in mononuclear cells. Here, we explore the role of the suppressor of cytokine signaling 3 (SOCS3) in urate-induced inflammation. METHODS: Peripheral blood mononuclear cells (PBMCs) from gout patients, hyperuricemic and normouricemic individuals were cultured for 24h with varying concentrations of soluble urate, followed by 24h restimulation with lipopolysaccharides (LPS)±monosodium urate (MSU) crystals. Transcriptomic profiling was performed using RNA-Sequencing. DNA methylation was assessed using Illumina Infinium® MethylationEPIC BeadChip system (EPIC array). Phosphorylation of signal transducer and activator of transcription 3 (STAT3) was determined by flow cytometry. Cytokine responses were also assessed in PBMCs from patients with JAK2 V617F tyrosine kinase mutation. RESULTS: PBMCs pre-treated with urate produced more interleukin-1beta (IL-1ß) and interleukin-6 (IL-6) and less interleukin-1 receptor anatagonist (IL-1Ra) after LPS simulation. In vitro, urate treatment enhanced SOCS3 expression in control monocytes but no DNA methylation changes were observed at the SOCS3 gene. A dose-dependent reduction in phosphorylated STAT3 concomitant with a decrease in IL-1Ra was observed with increasing concentrations of urate. PBMCs with constitutively activated STAT3 (JAK2 V617F mutation) could not be primed by urate. CONCLUSION: In vitro, urate exposure increased SOCS3 expression, while urate priming, and subsequent stimulation resulted in decreased STAT3 phosphorylation and IL-1Ra production. There was no evidence that DNA methylation constitutes a regulatory mechanism of SOCS3. Elevated SOCS3 and reduced pSTAT3 could play a role in urate-induced hyperinflammation since urate priming had no effect in PBMCs from patients with constitutively activated STAT3.

Hyperuricemia remodels the serum proteome toward a higher inflammatory state.

Gout is an autoinflammatory disease triggered by a complex innate immune response to MSU crystals and inflammatory triggers. While hyperuricemia is an obligatory risk factor for the development of gout, the majority of individuals with hyperuricemia never develop gout but have an increased risk of developing cardiometabolic disorders. Current management of gout aims at MSU crystal dissolution by lowering serum urate. We apply a targeted proteomic analysis, using Olink inflammation panel, to a large group of individuals with gout, asymptomatic hyperuricemia, and normouricemic controls, and we show a urate-driven inflammatory signature. We add in vivo evidence of persistent immune activation linked to urate exposure and describe immune pathways involved in the pathogenesis of gout. Our results support a pro-inflammatory effect of asymptomatic hyperuricemia and pave the way for new research into targetable mechanisms in gout and cardiometabolic complications of asymptomatic hyperuricemia.

Trained immunity and inflammation in rheumatic diseases.

BACKGROUND: Rheumatic diseases include a variety of autoimmune and autoinflammatory conditions that are characterised by musculoskeletal involvement and systemic disease. Both innate and adaptive immunity can contribute to the complex inflammatory processes that take part in the pathogenesis of these debilitating disorders. FINDINGS: Over the past decade, studies have led to a paradigm-shift around the concept of immune memory, generating the knowledge that cells of the innate immune system can develop a de facto memory mediated by epigenetic reprograming and metabolic changes (trained immunity). Here we provide an overview of current data that describe features of trained immunity in rheumatic diseases. We link evidence on inflammatory mediators and cytokine production, immunometabolism and epigenetic regulation of immunological programs, and outline the fact that trained immunity could play mechanistic roles in rheumatic diseases such as gout, rheumatoid arthritis, systemic lupus erythematosus or systemic sclerosis. CONCLUSION: This review describes recent findings in several important rheumatic disorders and emphasizes changes in the functional program of innate immune cells that are reminiscent of a trained immune phenotype. Further assessment of trained immunity in rheumatic disease can provide targetable mechanisms that could potentially alter the disease symptomatology and evolution.

Longitudinal study of neuropathy, microangiopathy, and autophagy in sural nerve: Implications for diabetic neuropathy.

OBJECTIVES: The progression and pathophysiology of neuropathy in impaired glucose tolerance (IGT) and type 2 diabetes (T2DM) is poorly understood, especially in relation to autophagy. This study was designed to assess whether the presence of autophagy-related structures was associated with sural nerve fiber pathology, and to investigate if endoneurial capillary pathology could predict the development of T2DM and neuropathy. PATIENTS AND METHODS: Sural nerve physiology and ultrastructural morphology were studied at baseline and 11 years later in subjects with normal glucose tolerance (NGT), IGT, and T2DM. RESULTS: Subjects with T2DM had significantly lower sural nerve amplitude compared to subjects with NGT and IGT at baseline. Myelinated and unmyelinated fiber, endoneurial capillary morphology, and the presence and distribution of autophagy structures were comparable between groups at baseline, except for a smaller myelinated axon diameter in subjects with T2DM and IGT compared to NGT. The baseline values of the subjects with NGT and IGT who converted to T2DM 11 years later demonstrated healthy smaller endoneurial capillary and higher g-ratio versus subjects who remained NGT. At follow-up, T2DM showed a reduction in nerve conduction, amplitude, myelinated fiber density, unmyelinated axon diameter, and autophagy structures in myelinated axons. Endothelial cell area and total diffusion barrier was increased versus baseline. CONCLUSIONS: We conclude that small healthy endoneurial capillary may presage the development of T2DM and neuropathy. Autophagy occurs in human sural nerves and can be affected by T2DM. Further studies are warranted to understand the role of autophagy in diabetic neuropathy.