Influence of genetic variants and mRNA expression of interleukin IL17A gene in asthma susceptibility.

Asthma is a chronic respiratory disease characterized by coughing, wheezing, shortness of breath, chest tightness, overproduction of mucus, and expiratory airflow limitation, which affects >300 million people worldwide. It is triggered by the dynamic interplay of genetic factors and environmental exposure. Th17 cells are an emerging subset of CD4+ T cells, which secrete IL-17A. This proinflammatory cytokine has recently been associated with asthma, autoimmune diseases, and inflammatory disorders. The present case-control study was focused on identifying the involvement of the IL-17A gene in asthma pathogenesis among 150 clinically diagnosed asthma patients and 150 healthy controls (HCs) of South Indian origin. To carry out the study, we aimed to screen the genetic variants of rs2275913G/A and rs8193036C/T and also estimated the serum cytokine levels of the IL-17A cytokine of recruited subjects. Further, we evaluated mRNA expression in selected subjects to correlate with the genetic variants. The results revealed that the mean IL-17A serum levels (161.6 ± 380.1 pg/ml vs. 86.75 ± 90.01 pg/ml) and IgE levels (257.7 ± 133.3 pg/ml vs. 311.2 ± 160.5 pg/ml) in asthma patients were significantly high as compared to healthy controls (p < 0.05). The ROC curves were constructed to compare the cytokine levels of asthma patients and HC, and the area under the curve (AUC) for IL-17A cytokine was 0.64, indicating that the test was satisfactory and significant (95 % CI: 0.575-0.709; p < 0.001). Genotyping of rs2275913G/A polymorphism indicated a 1.6-fold risk (95 % CI-1.02-2.56; p = 0.04) for asthma patients compared to healthy controls, whereas no significant association was observed for rs8193036C/T polymorphism with asthma susceptibility. Under genetic models, GA and AA models showed a protective effect against the disease for rs2275913G/A. In contrast, no statistically significant result was observed among the models of rs8193036C/T when adjusted with age and sex. The mRNA expression levels of the gene were statistically high in patients compared to the HCs, with a 1.8-fold change (p < 0.0001). We conclude that the results indicate IL-17A rs2275913G/A is likely to contribute to protection against the disease, while IL-17A rs8193036C/T shows no association with the disease. However, no correlation was identified in serum cytokine levels concerning genotypes. This comprehensive information in the present study might contribute to developing novel therapeutic strategies for treating inflammatory diseases like asthma. Further studies are warranted to understand the diverse functions of IL-17A concerning its longitudinal stability and its response to clinical interventions with large sample sizes in various ethnicities.

Interaction of TLR4 and TLR8 in the Innate Immune Response against Mycobacterium Tuberculosis.

The interaction and crosstalk of Toll-like receptors (TLRs) is an established pathway in which the innate immune system recognises and fights pathogens. In a single nucleotide polymorphisms (SNP) analysis of an Indian cohort, we found evidence for both TLR4-399T and TRL8-1A conveying increased susceptibility towards tuberculosis (TB) in an interdependent manner, even though there is no established TLR4 ligand present in Mycobacterium tuberculosis (Mtb), which is the causative pathogen of TB. Docking studies revealed that TLR4 and TLR8 can build a heterodimer, allowing interaction with TLR8 ligands. The conformational change of TLR4-399T might impair this interaction. With immunoprecipitation and mass spectrometry, we precipitated TLR4 with TLR8-targeted antibodies, indicating heterodimerisation. Confocal microscopy confirmed a high co-localisation frequency of TLR4 and TLR8 that further increased upon TLR8 stimulation. The heterodimerisation of TLR4 and TLR8 led to an induction of IL12p40, NF-κB, and IRF3. TLR4-399T in interaction with TLR8 induced an increased NF-κB response as compared to TLR4-399C, which was potentially caused by an alteration of subsequent immunological pathways involving type I IFNs. In summary, we present evidence that the heterodimerisation of TLR4 and TLR8 at the endosome is involved in Mtb recognition via TLR8 ligands, such as microbial RNA, which induces a Th1 response. These findings may lead to novel targets for therapeutic interventions and vaccine development regarding TB.

Toll-like receptor 2 polymorphisms and their effect on the immune response to ESAT-6, Pam3CSK4 TLR2 agonist in pulmonary tuberculosis patients and household contacts.

Toll-like receptors (TLRs) play a pivotal role in organizing the effective immune response through inducing the pro-inflammatory cytokines for control of tuberculosis infection and TLR polymorphisms are associated with altered cytokine levels have been described. Therefore, the main aim of the present study was to confirm whether TLR2 (C2029T, G2258A) polymorphisms effects the cytokine production in PTB patients and Household contacts (HHC), healthy controls (HC). The polymorphisms were performed by amplification refractory mutation system-polymerase chain reaction (ARMS) & Restriction Fragment Length Polymorphism (RFLP) in 336 subjects. Cytokine levels were estimated in Pam3CSK4, antigen ESAT-6 stimulated culture supernatants by Enzyme-Linked Immunosorbent Assay. Under the over-dominant model GA genotype of G2258A SNP and CT genotype of the co-dominant model in C2029T SNP showed a susceptible effect in patients, whereas in HHC, CT genotype showed a protective effect. A significant decreased TNF-α, IL-12 and increased IL-1ß levels were observed after Pam3CSK4, antigen ESAT-6stimulation; our results showed the following associations: TLR2 G2258A SNP of GA with decreased TNF-α; TLR2 C2029T SNP of CT, TT with decreased IL-12 and increased IL-1ß levels. Regression analysis demonstrated that age, BCG, gender and T allele were significantly associated with TB. Pre-mRNA secondary structure of the A, T alleles are more stable than G, C alleles. Altogether, we suggest that cytokine levels, 2029T allele, TLR2 polymorphisms were considered as predictive markers for identification of high-risk individuals in TB.

Role of IFN-γ and IL-6 cytokines and their association in determining susceptibility to chronic idiopathic urticaria.

Chronic Idiopathic Urticaria (CIU) is a common skin disorder, which may occur spontaneously. The aim of the present study was to assess the serum levels of interferon (IFN)-γ and interleukin (IL)-6 and to examine the association of IFN-γ+874 T/A and IL-6-174 G/C cytokine gene polymorphisms. To accomplish this, ELISA-based cytokine serum levels of IFN-γ (n=30) and IL-6 (n=30) in CIU patients (n=100) and Healthy Controls (HC) (n=200) were performed. Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was performed to verify the positional significance. A significant (p<0.0001) increase in the serum cytokine levels of IFN-γ and IL-6 was recorded in CIU patients compared to HC. The AT and TT genotypes of IFN-γ and GG genotype of IL-6 were found to be significantly associated with CIU. In conclusion, our findings show a significant increase in the cytokine levels of IFN-γ and IL-6, highlighting their regulatory role in the development of disease. In addition to this, association studies have revealed that TT genotype of IFN-γ +874 T/A and GG genotype of IL-6-174 G/C were susceptible towards the CIU.

Association of interleukin-10 gene promoter polymorphism in allergic patients.

AIM: Allergic diseases are increasing alarmingly worldwide affecting >30% of the population, including India. Allergy is the result of interaction of the epitopes on the protein with the immunoglobulin E (IgE). T helper cell-2 cytokines promote allergen-specific IgE antibody and induce eosinophil-dominated inflammatory tissue responses. Interleukin-10 (IL-10), an antiinflammatory cytokine, plays a major role in the development of the allergy. The cytokine gene polymorphism of -592C→A (rs1800872) and -1082G→A (rs1800896) of IL-10 may influence the expression of the protein. Hence, the current study was aimed to evaluate the persistent association between these variants in the susceptibility of the disease. METHODS: The allelic and genotype frequencies corresponding to IL-10 (-592C→A; -1082G→A) were determined in 94 allergic patients and 100 controls. Genomic typing was performed with polymerase chain reaction with sequence-specific primers. RESULT: The genotype AA at -592 position (p<0.000; odds ratio [OR] 9.92; 95% confidence interval [CI]=5.06-19.42) and GG at IL-10-1082 position (p<0.04; OR=2.47; 95% CI=1.003-4.96) was associated significantly in patients compared with controls. A considerable frequency of A-A haplotype in the patients and C-A, C-G haplotypes in controls was observed. A highly noteworthy difference was found in diplotype frequencies of A/A-A/A and A/A-G/A in patients and A/C-G/G and A/C-G/A in the controls. CONCLUSION: Our results indicate that haplotype and diplotype frequencies of the IL-10 locus may confer susceptibility to allergic patients.