Characterization of the TCRß repertoire of peripheral MR1-restricted MAIT cells in psoriasis vulgaris patients.

Psoriasis vulgaris (PV) is an inflammatory skin disease largely driven by aberrant αßT cells. Mucosal-associated invariant T (MAIT) cells, which constitute the largest circulating innate-like αßT cell community in human adults, are characterized by a semi-invariant TCRVα7.2 receptor and MR1-restricted affinity toward microbial metabolites. Limited MAIT TCRα diversity is complemented by a more variable TCRß repertoire, but its footprint in the MAIT repertoire of PV patients has never been tested. Here, we used bulk TCRSeq, MiXCR, VDJTools, and Immunarch pipelines to decipher and compare TCRß clonotypes from flow-sorted, peripheral TCRVα7.2+MR1-5-OP-RU-tet+MAIT cells from 10 PV patients and 10 healthy, matched controls. The resulting TCRß collections were highly private and individually unique, with small public clonotype content and high CDR3ß amino acid length variability in both groups. The age-related increase in the 'hyperexpanded' clonotype compartment was observed in PV, but not in healthy MAIT repertoires. The TCRß repertoires of PV patients were also marked by skewed TRBV/TRBJ pairing, and the emergence of PV-specific, public CDR3ß peptide sequences closely matching the published CDR3ß record from psoriatic skin. Overall, our study provides preliminary insight into the peripheral MAIT TCRß repertoire in psoriasis and warrants further evaluation of its diagnostic and clinical significance.

The miR-20a/miR-92b Profile Is Associated with Circulating γδ T-Cell Perturbations in Mild Psoriasis.

Psoriasis vulgaris (PV) is an autoinflammatory dermatosis of unknown etiology. Current evidence suggests a pathogenic role of γδT cells, but the growing complexity of this population has made the offending subset difficult to pinpoint. The work on γδTCRint and γδTCRhi subsets, which express intermediate and high levels of γδTCR at their surface, respectively, is particularly scarce, leaving their inner workings in PV essentially unresolved. We have shown here that the γδTCRint/γδTCRhi cell composition and their transcriptom are related to the differential miRNA expression by performing a targeted miRNA and mRNA quantification (RT-qPCR) in multiplexed, flow-sorted γδ blood T cells from healthy controls (n = 14) and patients with PV (n = 13). A significant loss of miR-20a in bulk γδT cells (~fourfold decrease, PV vs. controls) largely mirrored increasing Vδ1-Vδ2- and γδintVδ1-Vδ2- cell densities in the bloodstream, culminating in a relative excess of γδintVδ1-Vδ2- cells for PV. Transcripts encoding DNA-binding factors (ZBTB16), cytokine receptors (IL18R1), and cell adhesion molecules (SELPLG) were depleted in the process, closely tracking miR-20a availability in bulk γδ T-cell RNA. Compared to controls, PV was also associated with enhanced miR-92b expression (~13-fold) in bulk γδT cells that lacked association with the γδT cell composition. The miR-29a and let-7c expressions remained unaltered in case-control comparisons. Overall, our data expand the current landscape of the peripheral γδT cell composition, underlining changes in its mRNA/miRNA transcriptional circuits that may inform PV pathogenesis.

Expression of Oxidative Stress and Inflammation-Related Genes in Nasal Mucosa and Nasal Polyps from Patients with Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is a prevalent, multifaceted inflammatory condition affecting the nasal cavity and the paranasal sinuses, frequently accompanied by formation of nasal polyps (CRSwNP). This apparently uniform clinical entity is preceded by heterogeneous changes in cellular and molecular patterns, suggesting the presence of multiple CRS endotypes and a diverse etiology. Alterations of the upper airway innate defense mechanisms, including antimicrobial and antioxidant capacity, have been implicated in CRSwNP etiology. The aim of this study was to investigate mRNA expression patterns of antioxidative enzymes, including superoxide dismutase (SOD) and peroxiredoxin-2 (PRDX2), and innate immune system defense players, namely the bactericidal/permeability-increasing fold-containing family A, member 1 (BPIFA1) and PACAP family members, particularly adenylate-cyclase-activating polypeptide receptor 1 (ADCYAP1) in nasal mucosa and nasal polyps from CRSwNP patients. Additional stratification based on age, sex, allergic comorbidity, and disease severity was applied. The results showed that ADCYAP1, BPIFA1, and PRDX2 transcripts are differentially expressed in nasal mucosa and scale with radiologically assessed disease severity in CRSwNP patients. Sinonasal transcriptome is not associated with age, sex, and smoking in CRSwNP. Surgical and postoperative corticosteroid (CS) therapy improves endoscopic appearance of the mucosa, but variably reverses target gene expression patterns in the nasal cavity of CRSwNP patients. Transcriptional cross-correlations analysis revealed an increased level of connectedness among differentially expressed genes under inflammatory conditions and restoration of basic network following CS treatment. Although results of the present study imply a possible engagement of ADCYAP1 and BPIFA1 as biomarkers for CRSwNP, a more profound study taking into account disease severity and CRSwNP endotypes prior to the treatment would provide additional information on their sensitivity.

Differential Skewing of Circulating MR1-Restricted and γδ T Cells in Human Psoriasis Vulgaris.

Psoriasis vulgaris (PV) is a chronic, recurrent inflammatory dermatosis mediated by aberrantly activated immune cells. The role of the innate-like T cells, particularly gammadelta T (γδT) cells and MR1-restricted T lymphocytes, is incompletely explored, mainly through animal models, or by use of surrogate lineage markers, respectively. Here, we used case-control settings, multiparameter flow cytometry, 5-OP-RU-loaded MR1-tetramers, Luminex technology and targeted qRT-PCR to dissect the cellular and transcriptional landscape of γδ and MR1-restricted blood T cells in untreated PV cases (n=21, 22 matched controls). High interpersonal differences in cell composition were observed, fueling transcriptional variability at healthy baseline. A minor subset of canonical CD4+CD8+MR1-tet+TCRVα7.2+ and CD4+CD8-MR1-tet+TCRVα7.2+ T cells was the most significantly underrepresented community in male PV individuals, whereas Vδ2+ γδ T cells expressing high levels of TCR and Vδ1-δ2- γδ T cells expressing intermediate levels of TCR were selectively enriched in affected males, partly reflecting disease severity. Our findings highlight a formerly unappreciated skewing of human circulating MAIT and γδ cytomes during PV, and reveal their compositional changes in relation to sex, CMV exposure, serum cytokine content, BMI, and inflammatory burden. Complementing numerical alterations, we finally show that flow-sorted, MAIT and γδ populations exhibit divergent transcriptional changes in mild type I psoriasis, consisting of differential bulk expression for signatures of cytotoxicity/type-1 immunity (EOMES, RUNX3, IL18R), type-3 immunity (RORC, CCR6), and T cell innateness (ZBTB16).

HLA-A, -B, -C, -DRB1, -DQA1, and -DQB1 allele and haplotype frequencies defined by next generation sequencing in a population of East Croatia blood donors.

Next-generation sequencing (NGS) is increasingly used in transplantation settings, but also as a method of choice for in-depth analysis of population-specific HLA genetic architecture and its linkage to various diseases. With respect to complex ethnic admixture characteristic for East Croatian population, we aimed to investigate class-I (HLA-A, -B, -C) and class-II (HLA-DRB1, -DQA1, -DQB1) HLA diversity at the highest, 4-field resolution level in 120 healthy, unrelated, blood donor volunteers. Genomic DNA was extracted and HLA genotypes of class I and DQA1 genes were defined in full-length, -DQB1 from intron 1 to 3' UTR, and -DRB1 from intron 1 to intron 4 (Illumina MiSeq platform, Omixon Twin algorithms, IMGT/HLA release 3.30.0_5). Linkage disequilibrium statistics, Hardy-Weinberg departures, and haplotype frequencies were inferred by exact tests and iterative Expectation-Maximization algorithm using PyPop 0.7.0 and Arlequin v3.5.2.2 software. Our data provide first description of 4-field allele and haplotype frequencies in Croatian population, revealing 192 class-I and class-II alleles and extended haplotypic combinations not apparent from the existing 2-field HLA reports from Croatia. This established reference database complements current knowledge of HLA diversity and should prove useful in future population studies, transplantation settings, and disease-associated HLA screening.

Serum 25-hydoxyvitamin D concentrations in relation to Hashimoto's thyroiditis: a systematic review, meta-analysis and meta-regression of observational studies.

PURPOSE: Available evidence on the relation between vitamin D status and Hashimoto's thyroiditis (HT) remains inconsistent. We conducted a meta-analysis of serum 25-hydoxyvitamin [25(OH)D] concentrations in HT, and examined how the strength of this relationship varies as a function of several moderating factors. METHODS: Twenty-six observational, case-control studies, published before Feb 20, 2018, were located using Google Scholar, PubMed, Web of Science, SCOPUS, LILACS and SCIELO. Study quality was assessed and random-effects models were used, along with univariate mixed-effect meta-regression, for all analyses. RESULTS: The 25 studies (2695 cases, 2263 controls) confirmed lower serum 25(OH)D concentrations in HT compared to healthy controls, with Cohen's d - 0.62 (95% CI - 0.89, - 0.34; P = 1.5 × 10-5) and substantial heterogeneity between studies. HT showed an odds ratio (OR) of 3.21 (1.94-5.3; P = 5.7 × 10-6) for 25(OH)D deficiency (cut-off 20 ng/mL) against healthy controls. A corrected Cohen's d of - 0.43 [(- 0.76, - 0.09), P = 0.013] was obtained by trim-and-fill adjustment for publication bias. The association was consistent across Asian and European studies, pediatric and adult population, high- and moderate-quality studies. Near-equatorial latitudes (< 35° N/S, P = 3.4 × 10-4) and moderate-income economy (gross national income (GNI) 1000 < US$ < 12,000, P = 0.012) were associated with more discrepant 25(OH)D concentrations between the groups. Higher latitude (P = 0.0047), and higher mean body mass index (P = 0.006, 10 studies) were associated with smaller Cohen's d by univariate meta-regression, with evidence of nonlinear moderation by GNI (P = 3.5 × 10-6), and mean serum thyrotropin in affected individuals (P = 0.017, 21 studies). CONCLUSION: The present work shows a significant association between circulating 25(OH)D and HT, partly resolves mixed findings by identifying the empirical moderators contributing to overall heterogeneity, and highlights HT patient groups and the conditions under which the association is strongest.

Quality control project of NGS HLA genotyping for the 17th International HLA and Immunogenetics Workshop.

The 17th International HLA and Immunogenetics Workshop (IHIW) organizers conducted a Pilot Study (PS) in which 13 laboratories (15 groups) participated to assess the performance of the various sequencing library preparation protocols, NGS platforms and software in use prior to the workshop. The organizers sent 50 cell lines to each of the 15 groups, scored the 15 independently generated sets of NGS HLA genotyping data, and generated "consensus" HLA genotypes for each of the 50 cell lines. Proficiency Testing (PT) was subsequently organized using four sets of 24 cell lines, selected from 48 of 50 PS cell lines, to validate the quality of NGS HLA typing data from the 34 participating IHIW laboratories. Completion of the PT program with a minimum score of 95% concordance at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci satisfied the requirements to submit NGS HLA typing data for the 17th IHIW projects. Together, these PS and PT efforts constituted the 17th IHIW Quality Control project. Overall PT concordance rates for HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 were 98.1%, 97.0% and 98.1%, 99.0%, 98.6%, 98.8%, 97.6%, 96.0%, 99.1%, 90.0% and 91.7%, respectively. Across all loci, the majority of the discordance was due to allele dropout. The high cost of NGS HLA genotyping per experiment likely prevented the retyping of initially failed HLA loci. Despite the high HLA genotype concordance rates of the software, there remains room for improvement in the assembly of more accurate consensus DNA sequences by NGS HLA genotyping software.

Expression of TIGIT and FCRL3 is Altered in T Cells from Patients with Distinct Patterns of Chronic Autoimmune Thyroiditis.

BACKGROUND: Co-inhibitory receptors (IR), such as TIGIT and FCRL3, provide a checkpoint against highly destructive immune responses. Co-expression of TIGIT and FCRL3, in particular, has been linked to the HELIOS+ subset of regulatory CD4+FOXP3+T-cells. Of these, CD4+FOXP3-exon(E)2+ cells have higher expression of IR and exhibit strongest suppressive properties. Nevertheless, how the expression of TIGIT, FCRL3, HELIOS, and FOXP3E2 is regulated in chronic autoimmune thyroiditis (AT), is not known. METHODS: Thirty patients with AT [encompassing spontaneously euthyroid (euAT), hypothyroid-untreated and L-thyroxine-treated cases)] and 10 healthy controls (HC) were recruited. FCRL3, TIGIT, HELIOS and FOXP3E2 mRNA expression levels in peripheral blood (PB) T cells were measured via quantitative real-time PCR and compared to clinicopathological factors. RESULTS: The TIGIT and FCRL3 expression levels from T cells of AT cases were inversely related to the thyroid volume, and were significantly increased in hypothyroid patients (on+off L-thyroxine), but not euAT cases. The FCRL3 expression in PB T cells positively correlated with thyroid-peroxidase autoantibody levels; by contrast, T cells from aged AT patients and combined samples (AT+HC) accumulated more TIGIT mRNA. The patients with higher TIGIT mRNA levels had a greater prevalence of hypothyroidism, showing higher peak thyrotropin levels at diagnosis or at follow-up. CONCLUSIONS: Multiple IR, namely FCRL3 and TIGIT, but not the transcription factors HELIOS and FOXP3E2, showed increased mRNA levels in PB T cells from end-stage, long-standing and/or more aggressive AT, in proportion to disease severity. A relation with major clinical subphenotypes was observed, thereby identifying IR as potentially important players in AT.

miR-29a-3p/T-bet Regulatory Circuit Is Altered in T Cells of Patients With Hashimoto's Thyroiditis.

OBJECTIVE: Hashimoto's thyroiditis (HT) is a common autoimmune thyroid disorder that frequently evolves from asymptomatic, T-cell mediated chronic inflammation toward overt hypothyroidism. Previously, we have demonstrated a role for T-bet, a T helper 1/CD8+ T cell transcription factor (TF), and FoxP3, a regulatory T cell TF, in disease progression and severity, but the basis behind their altered mRNA expression remains unknown. In this study, we aimed to leverage the role for microRNAs, representing negative transcriptional regulators, across the spectrum of HT clinical presentations using the same, well-characterized RNA sample cohort. METHOD: Ten hypothyroid, untreated patients (hypoHT), 10 hypothyroid cases rendered euthyroid by l-thyroxine therapy (substHT), 11 spontaneously euthyroid HT subjects (euHT), and 10 healthy controls (ctrl) were probed for three candidate immunoregulatory miRNA (miR-9-5p, miR-29a-3p, and miR-210-3p) using quantitative real-time PCR measurements. Data were normalized to U6snRNA and fold difference in expression calculated by the efficiency corrected 2-ΔΔCt model. RESULTS: Compared to healthy controls, peripheral blood (PB) T cells of HT patients exhibited significantly diminished miR-29a-3p expression levels [median expression levels (IQR), HT vs CTRL, 0.62 (0.44-1.01) vs 1.373 (0.63-2.7), P = 0.046], and a similar, but not significant decline in miR-210-3p abundance [HT vs CTRL, 0.64 (0.39-1.31) vs 1.2 (0.5-2.56), P = 0.24, Wilcoxon test]. A significant inverse correlation was observed between the two differentially expressed transcripts, T-bet mRNA and miR-29a-3p. Moreover, altered miR-29a-3p/T-bet expression in T cells of untreated HT patients was related to low serum FT4, high serum thyrotropin, and decreased thyroid volumes. Of note, miR-210-3p expression was positively correlated to HIF1α, and inversely to FoxP3 mRNA levels, but no evidence of differential expression for any of these miRNA-mRNA pairs was observed. Finally, miR-9-5p expression levels were no different in HT vs control comparisons, or related to clinicopathological features. CONCLUSION: T cell miR-29a-3p is downregulated in HT patients and associated with clinical and biochemical parameters of progressive thyroid injury, plausibly subsequent to altered control of T-bet expression in PB T cells. As such miR-29a-3p/T-bet axis should be further explored as a biomarker or as a plausible target for therapeutic interventions in HT.

Association of increased eomesodermin, BCL6, and granzyme B expression with major clinical manifestations of Hashimoto's thyroiditis - an observational study.

PURPOSE: Studies of cytotoxic T cells and their respective lineage master regulators have been limited in Hashimoto's thyroiditis (HT). It is unclear whether their transcriptomes are changed in HT patients and how these changes are associated with the thyroid damage, major clinical manifestations, and disease progression. METHODS: We explored the gene expression patterns of selected transcription factors [eomesodermin (EOMES), BACH2, BCL6, TCF1] and cytolytic molecules [granzyme B (GZMB)] in peripheral blood (PB) T cells of 10 healthy controls and 30 HT patients of various subtypes (hypothyroid, untreated HT; L-thyroxine (T4)-treated HT, and spontaneously euthyroid HT) using real-time quantitative PCR. RESULTS: EOMES (Mann-Whitney P = 0.044), GZMB (P = 0.028), and BCL6 mRNA (P = 0.001) were overrepresented in PB T cells from HT and showed levels varying by age, thyroid volume and disease severity. BCL6 transcripts were predominantly enriched in severely affected, hypothyroid cases, both on and off LT4. Increased EOMES RNA expression was associated with advancing age, lower thyroid volumes and higher peak adjusted TSH levels over the course of the disease. The body mass-adjusted, steady-state maintenance dose of LT4 increased with GZMB and BCL6 levels in PB T cells of hypothyroid cases, mostly postmenopausal women having long-standing, non-goitrous and atrophic disease form. CONCLUSIONS: Our exploratory results suggest a role for GZMB, EOMES, and BCL6 in the context of HT, thyroid injury, and aggressive/advanced disease forms. Functions enriched within differentially expressed transcripts could be an important new target in understanding the pathogenesis of HT.

Altered expression of CTLA-4, CD28, VDR, and CD45 mRNA in T cells of patients with Hashimoto's thyroiditis - a pilot study.

INTRODUCTION: CD28/T-cell receptor (TCR)/cytotoxic T-lymphocyte antigen 4 (CTLA4) complex controls T-cell tolerance and autoimmunity in Hashimoto's thyroiditis (HT). In addition, CD45 protein tyrosine phosphatase (PTPase) and vitamin D receptor (VDR) cooperatively interact with the TCR complex to affect autoimmune processes central to the pathogenesis of HT. Nevertheless, their role in HT aetiology has been less well established. In this study, we aimed to explore mRNA expression levels of CTLA4, CD28, CD45, and VDR in T-cells, across different outcomes of HT. MATERIAL AND METHODS: The study included 45 HT patients and 13 euthyroid, healthy controls. T-lymphocytes were isolated from peripheral blood mononuclear cells, total mRNA was extracted from T-cells, and gene expression was studied by reverse transcription-polymerase chain reaction (RT-PCR) and ImageQuant method relative to glyceraldehyde-3-phosphate dehydrogenase RT-PCR products. RESULTS: Nominally higher expression levels of VDR, CTLA4, CD28, and CD45RAB mRNA were found in unsorted T-lymphocytes of healthy controls when compared to the HT patients. No difference was observed between hypothyroid/untreated, spontaneously euthyroid and LT4-treated HT patients. VDR mRNA expression was linked to both T3 levels and CTLA4 gene expression, whilst CD45RB mRNA expression coincided with CTLA4 and CD28 transcript levels. Conversely, older age and lower T3 levels were associated with increased abundance of CD45R0 isoform in HT patients. CONCLUSIONS: The results suggest a cross talk between endocrine and immune functions in HT pathology: an altered peripheral T cell mRNA profile with reduced VDR, CTLA4, CD28, and CD45RAB transcript levels is accompanied by age-related shift from naive to memory/late-differentiated T cell CD45R mRNA signature and associated with thyroid hormone status in the HT patients.

The Expression of T Cell FOXP3 and T-Bet Is Upregulated in Severe but Not Euthyroid Hashimoto's Thyroiditis.

Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disorder characterized by progressive thyroid failure. Th1 and Treg subset of CD4(+) cells have been implicated in the pathogenesis; however, less is known about their respective roles across the spectrum of HT clinical presentations. To shed more light on CD4(+) subsets role in HT, we investigated the mRNA expression levels of several Th1/Treg-associated transcription factors (T-bet/ETS1, HIF1α/BLIMP1/FOXP3) in peripheral blood T cells of 10 hypothyroid, untreated HT patients, 10 hypothyroid patients undergoing hormone replacement therapy, 12 euthyroid HT subjects, and 11 healthy controls by the qRT-PCR. Compared to euthyroid HT patients and controls, both hypothyroid (2.34-fold difference versus controls, P < 0.01) and thyroxine-supplemented patients (2.5-fold, P < 0.001) showed an increased FOXP3 mRNA expression in T cells. Similarly, mRNA expression levels of T-bet were upregulated in severely affected but not in euthyroid HT subjects (2.37-fold and 3.2-fold, hypothyroid and thyroxine-supplemented HT patients versus controls, resp., P < 0.01). By contrast, no differences in mRNA expression levels of ETS1, BLIMP1, and HIF1α were observed across the study groups. In summary, severe but not euthyroid HT was associated with robust upregulation of T-bet and FOXP3 mRNA in peripheral T cells, independent of the thyroid hormone status but proportional to disease activity.

Pilot study of variants of the IL-23R and STAT3 genes reveals no association with Hashimoto's thyroiditis in the Croatian population.

Interleukin-23 receptor (IL-23R) and signal transducer and activator of transcription 3 (STAT3) polymorphisms are common risk factors for a number of T helper (Th) 17-mediated autoimmune diseases. However, the importance of genetic variations in Th17 pathways to thyroid autoimmunity, and particularly Hashimoto's thyroiditis (HT), is not fully understood. In this study, we genotyped three single nucleotide polymorphisms (SNPs) within the IL-23R (rs11209026/p.Arg381Gln, rs7530511) and STAT3 (rs744166) genes in 217 Croatian patients with HT and 161 healthy controls using fluorescence resonance energy transfer technology and melting curve analysis of polymerase chain reaction products. None of the tested SNPs or IL-23R haplotypes was associated with HT susceptibility or disease severity. These results suggest that the studied IL-23R/STAT3 polymorphisms affecting Th17 signaling efficiency are not major determinants of HT risk in the Croatian population. Further work is necessary to determine if these loci contribute modestly or conditionally to the risk of HT.

Lack of association of vitamin D receptor gene 3'-haplotypes with psoriasis in Croatian patients.

Cis-acting regulatory variants in biologically relevant pathways and target tissues are a common source of phenotypic variations and individual disease susceptibility. In the skin, vitamin D receptor (VDR) is a master regulator of epidermal barrier function, inflammation, stem cell proliferation and microbial defense; therefore, we tested whether VDR 3'-regulatory haplotypes, a portion of which affect VDR transcriptional efficiency, allelic symmetry and mRNA turnover, were associated with psoriasis vulgaris. For this purpose, three VDR tag polymorphisms that capture most of the variability of the VDR 3'-regulatory element (rs1544410, rs7975232 and rs731236) were genotyped by the polymerase chain reaction restriction fragment length polymorphism method in 180 Caucasian patients with chronic plaque psoriasis and 366 ethnically matched, healthy controls of the Croatian origin. We found no evidence of association for any of the selected polymorphisms. Similarly, none of the 3'-VDR restriction haplotypes were associated with the risk for development of psoriasis in Croatian patients. These results show that neither VDR 3'-restriction polymorphisms nor common 3'-regulatory haplotypes contribute to psoriasis risk in the Croatian population.