Genome-Wide DNA Methylation Profiling in CD8 T-Cells and Gamma Delta T-Cells of Asian Indian Patients With Takayasu Arteritis.

Background: Takayasu's Arteritis (TA) is a chronic inflammatory disease that affects aorta and its main branches at their origin. Genetic, pathological and functional studies have shown that CD8 and Gamma delta (γ/δ) T-lymphocytes are involved in inflammatory processes in affected regions of arteries causing vascular damage. The molecular function of these lymphocytes remains unclear and currently no epigenetic studies are available in TA. We primarily performed genome wide methylation analysis in CD8 T cells and γδ T cells of patients with TA and compared with healthy controls. Methods: We recruited 12 subjects in each group namely TA patient and healthy controls. Blood samples were collected after obtaining informed written consent. CD8 T cells and γδ T cells were separated from whole blood. DNA extracted from these cells and were subjected to bisulfite treatment. Finally, bisulfite treated DNA was loaded in Infinium Methylation EPIC array. Bioinformatics analysis was used to identify differential methylation regions which were then mapped to genes. Results: Interleukin (IL)-32 and Lymphotoxin-A were genes significantly hypomethylated in CD8 T-cells. Anti-inflammatory cytokine genes, IL-10, IL-1RN and IL-27 were hypomethylated in γδ T cells of TA patients as compared to healthy controls. Gene enrichment analysis using Gene Ontology (GO) database and Kyoto Encyclopaedia of Genes and Genomes (KEGG) identified that genes involved in T-cell receptor signalling pathways were hypomethylated in CD8 T-cells and hypermethylated in γδ T cells of TA patients. Conclusion: CD8 T-cells might play a major role in immunopathogenesis of inflammation in TA, whereas γδ T cells may play a regulatory role.

High Secretion of Interleukin-6 and Increased MINCLE Receptor Expression Upon Exposure to Mycobacterial Cord Factor Analog Trehalose-6, 6-Dibehenate (TDB) in Patients with Takayasu Arteritis.

INTRODUCTION: Suspicion on the association between Takayasu Arteritis (TA) and Tubcerculosis (TB) has been in vogue for years. Prevalence of TB in TA is reported to be higher. We aimed to study innate immune responses in patients with TA on exposure to Trehalose-6,6-dibehenate (TDB), a synthetic analogue of Trehalose-6,6-Dimycolate (TDM, also known as mycobacterial cord factor) in comparison with healthy controls. MATERIALS AND METHODS: Patients with type V TA, satisfying 1990 ACR criteria, and age and sex matched healthy controls were recruited. PBMCs were cultured with 5µg/ml, 50µg/ml or without any TDB for 48 hours in RPMI medium inside a 5% Co2 incubator. IL-6, TNF-α and IL-17 were measured in cell culture supernatant, which was separated from the cells at the end of the incubation period. Gene expressions of IL-6, IL-8, TNFα, IFN-γ, MINCLE and BCL-10 were quantified in real time PCR using specific primers and SYBR green chemistry. RESULTS: Twenty two TA patients and 21 healthy controls were recruited. Both patients and controls showed response by secreting IL-6 and TNF-α upon stimulation by TDB. Relative induction (TDB stimulated TA sample / unstimulated control) of IL-6 was significantly higher in TA [31.88(0.74-168)] patients as compared to healthy controls [1.931(0.644-8.21); p<0.002], when co-cultured with 50µg/ml TDB. The expression of MINCLE, the TDB receptor was higher in TA samples than healthy controls upon TDB stimulation. CONCLUSION: Stimulation with mycobacterial synthetic analogue led to higher secretion of IL-6 and higher expression of MINCLE in PBMCs of patients with TA as compared to healthy controls.

Study of serial serum myeloid-related protein 8/14 as a sensitive biomarker in Takayasu arteritis: a single centre study.

The aim of the study was to explore utility of serial serum myeloid-related protein 8/14 (MRP8/14) as a biomarker of clinical disease activity and angiographic progression in Takayasu arteritis (TA). Serum MRP8/14 levels were assayed by commercial ELISA for 85 TA patients and 24 healthy controls at baseline, and for 56 and 21 TA patients during follow-up visits R1 and R2, respectively. Disease was categorised as active, indeterminate and stable according to Indian Takayasu Arteritis score (ITAS 2010), ITAS-A(CRP) and angiography. Patients were divided into responders and non-responders/relapsers based on treatment response. Non-parametric tests were used for inter-group comparisons at baseline and during follow-up time points. Generalised Estimating Equation was used to study association between changes in serial MRP8/14 levels and disease activity. At baseline, median MRP8/14 levels were higher in patients with TA than healthy controls [7353 (4524 to11283) vs 4896 (3194 to 8474.5) ng/ml, p = 0.011]. Patients with active disease had higher levels [8552 (5463-12488)] than stable disease [5292.5 (3140.5-7310)], p = 0.002, and healthy controls [4896 (3194-8474.5)], p = 0.001. Changes in serial MRP8/14 level were associated with changes in disease activity, independent of steroid dose, p = 0.000. At R1, MRP 8/14 levels were lower than baseline in responders (n = 38) [9146.0 (6296.8-13693.8) vs 6501 (4314.8-8304.5), p = 0.004], but did not change in non-responders/relapsers (n = 14) [6693.5(4210.8-10516.3) vs 7755.0(5342-10741.0), p = 0.42]. Similar trend was observed at R2. MRP8/14 levels increased during follow-up in 66% and 26.3% of angiographic progressors and non-progressors, respectively. MRP8/14 in TA may act as a novel biomarker with prognostic implications.

Soluble-HLA-E: A follow up biomarker in Takayasu arteritis, independent of HLA-E genotype.

AIM: Disease activity assessment in Takayasu arteritis (TA) is challenging. Human leukocyte antigen E (HLA-E) is shed from endothelium into serum as a soluble molecule (sHLA-E) in response to inflammation. We aimed to study: (i) utility of sHLA-E as a biomarker of disease activity; and (ii) association of HLA-E polymorphism rs1264457 with clinical disease in Asian-Indian TA patients. MATERIALS AND METHODS: In phase-1, sHLA-E levels were estimated in sera of 50 consecutive TA patients at baseline visit and 27 healthy controls. Serial estimations were performed in 27 of them. In phase-2, DNA of 150 TA patients and 264 healthy controls were genotyped for rs1264457 polymorphism. RESULTS: At baseline visit, disease was classified as active, stable and grumbling in 23, 18 and nine patients, respectively. sHLA-E levels were higher in active TA (43; interquartile range [IQR]: 25.3-64.6) pg/mL) than stable disease (12.9; IQR: 7.6-21.6 pg/mL) (P = 0.001). At first follow-up visit, sHLA-E levels were numerically higher in active disease than stable disease (P = 0.06) but this trend was blunted at second follow-up. sHLA-E levels increased in 54% versus 25% of patients with persistently active/relapsing and persistent stable course, respectively. rs1264457 polymorphism was not associated with susceptibility to TA and did not affect sHLA-E levels. CONCLUSION: sHLA-E level is useful as a biomarker of disease activity and course in TA patients. rs1264457 polymorphism is neither associated with susceptibility nor did it influence sHLA-E levels in TA.

Addition of second-line steroid sparing immunosuppressants like mycophenolate mofetil improves outcome of Immunoglobulin G4-related disease (IgG4-RD): a series from a tertiary care teaching hospital in South India.

IgG4-related disease (IgG4-RD) is a systemic fibro-inflammatory disease. This disease may be associated with elevated serum and tissue IgG4 levels. Early treatment prevents fibrosis and organ damage. We retrospectively studied the clinicopathologic correlation and outcome of treatment in IgG4-RD. This single-center retrospective study was done using electronic records of patients subjected to assay of serum IgG4 levels in our laboratory by nephelometry. There were 473 patients with suspected IgG4-RD. Of them, 41 patients fulfilled comprehensive diagnostic criteria for IgG4-RD and 432 had diseases other than IgG4-RD. Clinical and histopathological data including tissue IgG4/IgG ratio, other relevant laboratory findings as well as management data of 41 patients with IgG4-RD were analyzed. There were 29 males and 12 females with mean age of 44.1 ± 2.19 years. Thirteen patients had definite, 19 had probable and 9 had possible IgG4-RD. Male predominance, multiple organ involvement and IgG4 responder Index were significantly higher in definite IgG4-RD as compared to probable and possible IgG4-RD. Serum IgG4 level was elevated in 37 patients (90.2%). Glucocorticoids were used in 35 patients (85.4%) and second-line immunosuppressive agent in 23 patients (65.7%). Of the 21 patients on follow-up, 19 (90.7%) had clinical improvement at the first follow-up visit. Nine (90%) out of the ten patients who were assessed by IgG4 responder index, also had shown improved score with treatment. Patients with IgG4-RD in our series showed favorable responses to treatment with glucocorticoids and addition of steroid sparing immunosuppressive agents (mainly mycophenolate mofetil) helped successful tapering of steroids, while maintaining the improvement.

Anti-C1q Antibody is Associated with Renal and Cutaneous Manifestations in Asian Indian Patients with Systemic Lupus Erythematosus.

INTRODUCTION: C1q play an important role in clearance of immune complexes and apoptotic cell debris. Impaired clearance leads to exposure of C1 native antigen and development of anti-C1q antibody formation. Anti-C1q antibody is well studied in Systemic Lupus Erythematosus (SLE). Significance of anti-C1q Ab in Indian SLE patients and their clinical manifestations is not clear. AIM: The aim of this study was to investigate associations between anti-C1q antibody and clinical as well as serological markers of SLE. MATERIALS AND METHODS: Retrospective study of SLE patients fulfilling either American College of Rheumatology (ACR) 1990 or Systemic Lupus International Collaborating Clinics (SLICC) 2012 classification criteria were recruited from inpatients and outpatients services of the Clinical immunology and Rheumatology Department, Christian Medical College at Vellore, India between March 2013 and January 2015. Anti-C1q antibody was assayed by ELISA (Demeditec Diagnostics GmbH, Germany). Logistic regression analysis was performed to find the association of anti-C1q antibodies with serological and clinical parameters in SLE including Lupus Nephritis (LN). RESULTS: Sixty nine patients (54.76%) out of 126 SLE patients had LN. Anti-C1q levels were higher in patients with LN as compared to those without (p<0.05). Anti-C1q antibody was also significantly associated with positive C1q immunofluorescence staining in renal biopsy specimens (p<0.05). Overall, renal Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) {OR 1.35 (1.08-1.69)}, low C4 {OR 3.11 (1.04-9.26)} and mucocutaneous manifestation {OR 4.72 (1.38-16.05)} were independently associated with anti-C1q levels in serum. CONCLUSION: Renal SLEDAI, low C4 and mucocutaneous manifestations were independently associated with raised anti C1q antibody in SLE patients.

Interleukin-17F and interleukin-6 gene polymorphisms in Asian Indian patients with Takayasu arteritis.

OBJECTIVES: To assess genetic association between single nucleotide polymorphisms (SNPs) in genes encoding T-helper cytokines and Takayasu Arteritis (TA) susceptibility in Asian Indian population. METHODS: In Phase-1, the genomic DNA of 120 TA patients and 119 healthy controls were genotyped for SNPs rs1800795 (interleukin (IL)-6), rs763780 (IL-17F), rs1800871, rs1800872, rs1800896 (IL-10) and rs1800468, rs1800469, rs1800470 (transforming growth factor-ß). Allele frequencies between cases and controls were compared using chi-squared test and also reassessed empirically (pe) by 10,000 permutations. In Phase-2, additional 98 TA patients and 101 controls were genotyped for replicating the significant associations noted in Phase-1 of the study. RESULTS: All 8 SNPs in Phase 1 were in Hardy-Weinberg proportions. The G allele at rs763780 (IL-17F) was significantly associated with TA (p=0.014). We also found that rs1800795 (IL-6) was associated with tuberculosis (p=0.001) under a dominant model. In Phase-2 replication part of the study, the rs763780 showed a trend towards association with TA (p=0.08), and the magnitude and direction of the odds ratio (OR) also were consistent with results of Phase-1. In the combined analysis, protective association of the G allele of rs763780 with TA was again significant [OR (95% CI)=0.44 (0.25-0.77); p=0.0029]. The G allele was also significantly associated (p<0.05) with underlying tuberculosis (TB) and occurrence of syncope in TA. CONCLUSION: G allele of rs763780 in IL-17F gene was protectively associated against susceptibility to TA. GG genotypes of rs1800795 in IL-6 was also associated with occurrence of tuberculosis in our patients with TA.