TNF, IL6, and IL1B Polymorphisms Are Associated with Severe Influenza A (H1N1) Virus Infection in the Mexican Population

Abstract

Background: Hypercytokinemia is the main immunopathological mechanism contributing to a more severe clinical course in influenza A (H1N1) virus infections. Most patients infected with the influenza A (H1N1) pdm09 virus had increased systemic levels of pro-inflammatory cytokines; including interleukin IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α). We propose that single-nucleotide polymorphisms (SNPs) in the promoter regions of pro-inflammatory genes are associated with the severity of influenza A (H1N1) pdm09 virus infection.

Methods: 145 patients with influenza A (H1N1) (pA/H1N1), 133 patients with influenza-like illness (ILI), and 360 asymptomatic healthy contacts (AHCs) were included. Eleven SNPs were genotyped in six genes (TNF, LT, IL1B, IL6, CCL1, and IL8) using real-time PCR; the ancestral genotype was used for comparison. Genotypes were correlated with 27 clinical severity variables. Ten cytokines (GM-CSF, TNF-α, IL-2, IL-1β, IL-6, IL-8, IFN-γ, IL-10, IL-5, and IL-4) were measured on a Luminex 100.

Results: The IL6 rs1818879 (GA) heterozygous genotype was associated with severe influenza A (H1N1) virus infection (odds ratio [OR] = 5.94, 95% confidence interval [CI] 3.05-11.56), and two IL1B SNPs, rs16944 AG and rs3136558 TC, were associated with a decreased risk of infection (OR = 0.52 and OR = 0.51, respectively). Genetic susceptibility was determined (pA/H1N1 vs. AHC): the LTA rs909253 TC heterozygous genotype conferred greater risk (OR = 1.9), and a similar association was observed with the IL1B rs3136558 CC genotype (OR = 1.89). Additionally, severely ill patients were compared with moderately ill patients. The TNF-238 GA genotype was associated with an increased risk of disease severity (OR = 16.06, p = 0.007). Compared with ILIs, patients with severe pA/H1N1 infections exhibited increased serum IL-5 (p <0.001) and IL-6 (p = 0.007) levels.

Conclusions: The TNF gene was associated with disease severity, whereas IL1B and IL6 SNPs were associated with influenza A (H1N1) virus infection.

Fig 1. Flowchart for the collection and classification of patient samples in the study.

The blood samples were collected from suspected influenza inpatients at INER from 2009–2012. Abbreviations: ILI: influenza-like illness; RT-PCR: real-time PCR.

Fig 2. Haplotype for TNF gene at Chr 6 in ILI patients.

Linkage disequilibrium between the four single nucleotide polymorphisms identified at chromosome 6 in the TNF gene (generated for the ILI patients in Haploview 4.2).

Fig 3. Concentrations of IL-6, IL-5, and GM-SCF in p AH1N1, ILI patients and AHC at admission.

Box and whisker plots of cytokine levels in sera from the AHC, ILI, and pA/H1N1 groups. Boxes represents the 25–75 quartile, the whiskers represent the range of values, the middle line inside the box is the median, and the outlier values are represented as circles outside the boxes. The p-values (** and * representing <0.001 and <0.05, respectively) were calculated by one-way ANOVA and are presented above the graphs.