TNFA and IL10 Polymorphisms and IL-6 and IL-10 Levels Influence Disease Severity in Influenza A(H1N1)pdm09 Virus Infected Patients

Cytokines are key modulators of immune response, and dysregulated production of proinflammatory and anti-inflammatory cytokines contributes to the pathogenesis of influenza A(H1N1)pdm09 virus infection. Cytokine production is impacted by single nucleotide polymorphisms (SNPs) in the genes coding for them. In the present study, SNPs in the IL6, TNFA, IFNG, IL17A, IL10, and TGFB were investigated for their association with disease severity and fatality in influenza A(H1N1)pdm09-affected patients with mild disease (n = 293) and severe disease (n = 86). Among those with severe disease, 41 patients had fatal outcomes. In a subset of the patients, levels of IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17 were assayed in the plasma for their association with severe disease. The frequency of TNFA rs1800629 G/A allele was significantly higher in severe cases and survived severe cases group compared to that of those with mild infection (OR with 95% for mild vs. severe cases 2.95 (1.52–5.73); mild vs. survived severe cases 4.02 (1.84–8.82)). IL10 rs1800896-rs1800872 G-C haplotype was significantly lower (OR with 95% 0.34 (0.12–0.95)), while IL10 rs1800896-rs1800872 G-A haplotype was significantly higher (OR with 95% 12.11 (2.23–76.96)) in fatal cases group compared to that of the mild group. IL-6 and IL-10 levels were significantly higher in fatal cases compared to that of survived severe cases. IL-6 levels had greater discriminatory power than IL-10 to predict progression to fatal outcome in influenza A(H1N1)pdm09 virus-infected patients. To conclude, the present study reports the association of TNFA and IL10 SNPs with severe disease in Influenza A(H1N1)pdm09 virus-infected subjects. Furthermore, IL-6 levels can be a potential biomarker for predicting fatal outcomes in Influenza A(H1N1)pdm09 virus infected subjects.


Introduction
Pandemic H1N1 influenza (pH1N1), a severe acute respiratory illness (SARI) caused by influenza A(H1N1)pdm09, contributed to increased mortality among those infected with different influenza A subtype viruses [1]. In India, Maharashtra, a state in the western part of the country, accounts for 21% of all the influenza A(H1N1)pdm09 cases and 31% of all pH1N1-related deaths [2]. The progression to SARI depends on multiple factors such as age, obesity, the virulence of the virus, genetics of the host, and host immune response [3].
Production of cytokines is affected by the single nucleotide polymorphisms (SNPs) in the genes coding for them. SNPs in the promoter and intron regions are known to affect the stability of the mRNA, transcriptional efficiency, and splicing of the transcribed RNA, and thereby influence cytokine production. Commonly occurring SNPs that are known to influence cytokine levels were reported in the genes coding for TNF-α, IL-6, interferon (IFN)-γ, IL-17A, IL-10, and tumor growth factor (TGF)-β [7][8][9]. SNPs in the cytokine genes were shown to be associated with disease severity in influenza A(H1N1)pdm09 virus infected subjects [10][11][12]. There is a lack of studies investigating the association between cytokine gene polymorphisms and severe influenza A(H1N1)pdm09 infection in India. In the present study, common SNPs in the IL6 (rs1800795), TNFA (rs1800629), IFNG (rs2430561), IL17A (rs2275913), IL10 (rs1800896 and rs1800872), and TGFB (rs1800470 and rs1800471) were investigated for their association with severe disease and fatality in influenza A(H1N1)pdm09 virus infected patients. In a subset of the patients, levels of IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ were assayed in the plasma for their association with severe disease. The effect of SNPs on the plasma level of the cytokines was also studied.

Study Subjects
Patients with a history of laboratory confirmed influenza A(H1N1)pdm09 virus infection or ongoing treatment were contacted for enrolment in the study, and blood samples were collected from the consenting study subjects. Patients with influenza A(H1N1)pdm09 virus infection admitted to the collaborating hospitals were also included in the study. All the patients included in the study were affected with influenza A(H1N1)pdm09 virus infection during 2013 to 2018. Written informed consent was obtained from all the participants or their legal guardians before sample collection. The study was reviewed and approved by the institutional ethics committee of ICMR-National Institute of Virology and the collaborating hospitals. Influenza A(H1N1)pdm09 virus infection in the study subjects during the acute phase of the disease was confirmed by a real-time RT-PCR assay. DNA was extracted from the blood samples using a commercial DNA extraction kit (QIAamp blood DNA midi kit, Qiagen, Hilden, Germany) according to the protocol given by the manufacturer. Case history sheets filled by the treating physicians were used to classify the patients into those with mild disease and severe disease. Patients admitted to intensive care unit and requiring ventilator support or the patients with fatal outcomes were classified as those with severe influenza A(H1N1)pdm09 virus disease [10,13]. All the other patients were considered as those with mild disease. All the patients were treated according to the clinical management protocols provided by the directorate general of health services, ministry of health and family welfare, Government of India (https://main.mohfw.gov.in/sites/default/files/2366426352.pdf, accessed on 19 November 2021).

Investigation of Cytokine Levels in the Plasma of Influenza A(H1N1)pdm09 Virus Infected Cases
The cytokine levels were investigated in a subset of samples. The plasma samples collected during the acute phase (within a week of disease onset) of influenza A(H1N1)pdm09 virus infection from fatal (n = 28) and survived cases (n = 28) were examined for the cytokine profile. Plasma samples collected from patients who recovered from mild A(H1N1)pdm09 infection (n = 21) and healthy controls (n = 11) were also assessed for cytokine levels. The level of cytokines IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A were analyzed using BD™ Cytometric Bead Array Human Th1/Th2/Th17 Cytokine Kit (BD Biosciences, San Jose, CA, USA), as per manufacturer's instructions. Briefly, 50 µL plasma was treated with capture beads and phycoerythrin (PE) conjugated detection antibody. The standards provided by the manufacturer were used to prepare the standard curve for each cytokine. Then, the samples, as well as the standards, were acquired and analyzed on a BD FACSCalibur TM flow cytometer (BD Biosciences, San Jose, CA, USA) using BD CellQuestTM Pro software (v5.1). The fcs files were analyzed using flow cytometric analysis program array TM software (v3.0). The detection sensitivity of IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α, and IL-17A were 2.6, 4.9, 2.4, 4.5, 3.8, 3.7, and 18.9 pg/mL, respectively.

Statistical Analysis
For genetic association studies, statistical comparisons were carried out using an online SNP stats program [19] or SPSS version 17 as described earlier [20]. The strength of association was represented in terms of odds ratio (OR) with 95% confidence intervals (CI). The OR with 95% CI was adjusted for age and gender. For cytokine levels and their association with genotypes or disease severity, the Mann-Whitney U test was used to compare two groups while the Kruskal-Wallis test with multiple corrections was used to compare multiple groups. Receiver operating curve analysis was performed to assess the utility of cytokines as biomarkers in predicting fatal outcomes. Graphpad prism software version 7 was used for the analysis.

Demographic Characteristics and Disease Profile of the Study Subjects
A total number of 379 influenza A(H1N1)pdm09 virus-infected patients from Pune and its adjoining districts, Maharashtra, western India were included in the study. Among the patients, 86 were categorized as severe cases based on the requirement of ventilator support and/or fatal outcome. Among the severe cases, 41 patients had fatal outcomes, while the remaining 45 survived. The remaining 293 cases were categorized as having a mild disease. The ratio of males to females is 1.15 for mild cases and 0.97 for severe cases. Severe cases had significantly higher age as compared to mild cases (Table 1). Within the severe cases group, the ratio of male to female was 1.03 among survived severe cases and 1.27 in the fatal cases group. The mean age ± standard deviation (sd) was 51 ± 14.6 in the fatal cases group while it was 50 ± 12.6 in the survived severe cases group and the difference was not statistically significant.

Allele, Genotype, and Haplotype Frequencies in Influenza A(H1N1)pdm09 Virus-Infected Cases
Comparison of allele frequencies among the study groups revealed that the frequency of TNFA rs1800629 A allele was significantly higher in severe cases and survived severe cases group compared to that of those with mild infection (OR with 95% for mild vs. severe cases 2.72 (1.47-4.99); mild vs. survived severe cases 3.49 (1.68-6.99)). Though the frequency of TNFA rs1800629 A allele was higher in fatal cases compared to that of mild cases, it was not statistically significant. The allele frequencies of other SNPs were not different between the influenza A(H1N1)pdm09 infected patient groups (Table 2). Comparison of genotype frequencies of different SNPs among the study groups revealed that the frequency of TNFA rs1800629 G/A allele was significantly higher in severe cases and survived severe cases group compared to that of those with mild infection (OR with 95% for mild vs. severe cases 2.95 (1.52-5.73); mild vs. survived severe cases 4.02 (1.84-8.82)). Though the frequency of TNFA rs1800629 G/A allele was higher in fatal cases compared to mild cases, it was not statistically significant. The genotype frequencies of other SNPs were comparable between the influenza A(H1N1)pdm09-infected patient groups (Table 3).  Comparison of IL10 haplotype frequencies revealed that IL10 rs1800896-rs1800872 G-C haplotype was significantly lower while IL10 rs1800896-rs1800872 G-A haplotype was significantly higher in fatal cases group compared to mild cases (Table 4). Haplotype frequencies of TGFB SNPs were not different between different categories of influenza A (H1N1)pdm09 virus-infected patient groups.

Elevated Plasma IL-6 and IL-10 Level among Fatal Cases during the Acute Phase of Influenza A(H1N1)pdm09 Virus Infection
The proinflammatory cytokine, IL-6 was found to be significantly higher during the acute phase of fatal cases (Mean ± SE, 812 pg/mL ± 451.3, p <0.001) and survived severe cases (Mean ± SE, 112.6 pg/mL ± 53, p < 0.05) compared to that of healthy controls (Mean ± SE, 5.039 pg/mL ± 1.69) and mild recovered cases (Mean ± SE, 6.11 pg/mL ± 3.57). Also, IL-6 level was found significantly higher (p < 0.05) in fatal cases compared to that of survived severe cases ( Figure 1A).The plasma IL-10 level was found significantly higher during the acute phase of fatal cases (Mean ± SE, 36.44 pg/mL ± 12.18, p < 0.001) and survived severe cases (Mean ± SE, 6.567 pg/mL ± 1.43, p < 0.05) compared to that of healthy controls (Mean ± SE, 1.20 pg/mL ± 0.90) and mild recovered cases (Mean ± SE, 0.76 pg/mL ± 0.41). IL-10 level was significantly higher in fatal cases compared to that of survived severe cases (p < 0.05) ( Figure 1B). The other evaluated cytokines such as IL-2, IL-4, IFN-γ, TNF-α, and IL-17A, were observed only in few patients, and the levels were comparable between the study groups. The proinflammatory cytokine, IL-6 was found to be significantly higher during the acute phase of fatal cases (Mean ± SE, 812 pg/ml ± 451.3, p <0.001) and survived severe cases (Mean ± SE, 112.6 pg/ml ± 53, p <0.05) compared to that of healthy controls (Mean ± SE, 5.039 pg/ml ± 1.69) and mild recovered cases (Mean ± SE, 6.11 pg/ml ± 3.57). Also, IL-6 level was found significantly higher (p <0.05) in fatal cases compared to that of survived severe cases ( Figure 1A).The plasma IL-10 level was found significantly higher during the acute phase of fatal cases (Mean ± SE, 36.44 pg/ml ± 12.18, p<0.001) and survived severe cases (Mean ± SE, 6.567 pg/ml ± 1.43, p<0.05) compared to that of healthy controls (Mean ± SE, 1.20 pg/ml ± 0.90) and mild recovered cases (Mean ± SE, 0.76 pg/ml ± 0.41) IL-10 level was significantly higher in fatal cases compared to that of survived severe cases (p <0.05) ( Figure 1B). The other evaluated cytokines such as IL-2, IL-4, IFN-γ TNF-α, and IL-17A, were observed only in few patients, and the levels were comparable between the study groups.

IL-6 and IL-10 Levels Among Influenza A(H1N1)pdm09 Virus-Infected Subjects with Different Genotypes of IL-6 and IL-10
When IL-6 and IL-10 levels were compared between levels among influenza A(H1N1)pdm09 virus-infected subjects with different genotypes of IL-6 and IL-10, the cytokine levels were not significantly different between subjects with different IL6 or IL10 genotypes. However, irrespective of the IL6 and IL10 genotypes, the cytokine levels were elevated in fatal cases compared to that of survived severe cases (Figure 2A-C).

IL-6 and IL-10 Levels among Influenza A(H1N1)pdm09 Virus-Infected Subjects with Different Genotypes of IL-6 and IL-10
When IL-6 and IL-10 levels were compared between levels among influenza A(H1N1) pdm09 virus-infected subjects with different genotypes of IL-6 and IL-10, the cytokine levels were not significantly different between subjects with different IL6 or IL10 genotypes. However, irrespective of the IL6 and IL10 genotypes, the cytokine levels were elevated in fatal cases compared to that of survived severe cases (Figure 2A-C).

209
The utility of IL-6 and IL-10 levels in predicting fatal outcomes in influenza 210 A(H1N1)pdm09 virus infected subjects with severe disease was investigated using re-211 ceiver operating curve analysis. The analysis revealed that the area under curve (AUC) 212 value for IL-6 in discriminating survived severe cases and fatal cases was 0.81 (p <0.0001) 213 while it was 0.73 (p <0.01)for IL-10 ( Figure 3A,B).

217
Cytokines are the key mediators of inflammation and were associated with the dis-218 ease severity in influenza A(H1N1)pdm09 virus infection [21][22][23]. In the present study, 219 the SNPs in the IL6, TNFA, IFNG, IL17A, IL10, and TGFB genes were investigated for their 220 association with disease severity and fatality in influenza A(H1N1)pdm09 virus-infected 221

Utility of IL-6 and IL-10 Levels as A Marker for Predicting Fatality in Influenza A(H1N1)pdm09 Virus-Infected Subjects
The utility of IL-6 and IL-10 levels in predicting fatal outcomes in influenza A(H1N1)pdm09 virus infected subjects with severe disease was investigated using receiver operating curve analysis. The analysis revealed that the area under curve (AUC) value for IL-6 in discriminating survived severe cases and fatal cases was 0.81 (p < 0.0001) while it was 0.73 (p < 0.01)for IL-10 ( Figure 3A,B). The utility of IL-6 and IL-10 levels in predicting fatal outcomes in influenza 210 A(H1N1)pdm09 virus infected subjects with severe disease was investigated using re-211 ceiver operating curve analysis. The analysis revealed that the area under curve (AUC) 212 value for IL-6 in discriminating survived severe cases and fatal cases was 0.81 (p <0.0001) 213 while it was 0.73 (p <0.01)for IL-10 ( Figure 3A,B).

217
Cytokines are the key mediators of inflammation and were associated with the dis-218 ease severity in influenza A(H1N1)pdm09 virus infection [21][22][23]. In the present study, 219 the SNPs in the IL6, TNFA, IFNG, IL17A, IL10, and TGFB genes were investigated for their 220 association with disease severity and fatality in influenza A(H1N1)pdm09 virus-infected 221

Discussion
Cytokines are the key mediators of inflammation and were associated with the disease severity in influenza A(H1N1)pdm09 virus infection [21][22][23]. In the present study, the SNPs in the IL6, TNFA, IFNG, IL17A, IL10, and TGFB genes were investigated for their association with disease severity and fatality in influenza A(H1N1)pdm09 virus-infected subjects. The results revealed that TNFArs1800629 A allele and G/A genotype were associated with severe disease but not fatal outcomes. Our earlier study [15] with a smaller sample size also reported the association of TNFArs1800629 A allele and G/A genotype with severe disease, and this was also confirmed in the present study. Contradicting our study, a study of a Mexican population showed the association of TNFArs1800629G allele with severe disease [24]. TNFA rs1800629 is located in the promoter region, and this SNP, in combination with other SNPs, might influence TNF-α production. TNFA rs361525 was shown to be associated with severe disease in Mexican population [10]. Thus, the haplotype structure might vary between populations and influence genetic associations of TNFA SNPs with the disease in different populations. The lack of association of TNFArs1800629 with fatality suggests the role of additional factors including other genetic factors and comorbidities in progression to a fatal outcome and needs investigation.
The results also suggested thatIL10 rs1800896-rs1800872 G-C haplotype was associated with protection to fatal outcome, while IL10 rs1800896-rs1800872 G-A haplotype was associated with fatal outcomes in influenza A(H1N1)pdm09 virus infected-subjects. Similar to our study, IL10 rs1800896-rs1800872 G-A haplotype was reported to be associated with protection against severe disease in a Mexican population [24]. IL10 rs1800896-rs1800872 G-A haplotype might be associated with dysregulated production of IL-10 while IL10 rs1800896-rs1800872 G-C haplotype might be associated with optimal IL-10 production. Thus, these IL10 haplotypes might be associated with contrasting outcomes. The present study also reported the lack of association between SNPs in the IL6, IFNG, IL17A, and TGFB genes and disease severity of Influenza A(H1N1)pdm09 virus infection.
The present study demonstrated an elevated level of IL-6, indicative of raised inflammatory responses during the acute phase of influenza A(H1N1)pdm09 virus infection. Previous studies also indicated a higher level of IL-6 in severe influenza A(H1N1)pdm09 disease [21,22,25]. Besides, our study also revealed an increased level of plasma IL-10 during the acute phase of the infection and is in line with the previous findings [23,26]. Together, our study reported that there is a surge in plasma IL-6 and IL-10 level during the acute phase of influenza A(H1N1)pdm09 virus infection and also, this cytokine surge is more pronounced in fatal cases, as compared to that of severe survived cases, indicating their critical role in the pathogenesis of severe influenza A(H1N1)pdm09 disease. In some instances, IL-6 may get altered to resemble IL-10, a potent inhibitor of dendritic cells and macrophages [27,28], and thereby suppressing their microbicidal activities and production of proinflammatory cytokines. Thus, the immunosuppressive effect of IL-10 in synergy with IL-6, probably supports viral persistence or secondary infection, resulting in the more severe clinical outcome in influenza A(H1N1)pdm09 virus [23]. The utility of IL-6 and IL-10 in discriminating fatal and nonfatal cases was also investigated. The results revealed that IL-6 has greater discriminatory power than IL-10 and can be utilized as a biomarker to predict fatal outcomes in influenza A(H1N1)pdm09 virus-infected subjects and undertake better treatment strategies to prevent the same. Since IL-6 is nonspecifically upregulated in all type of infections [29], it cannot be used as a biomarker of influenza A(H1N1)pdm09 virus infection but can be used as a biomarker to predict fatal outcomes in subjects with confirmed influenza A(H1N1)pdm09 virus infection.
The study also revealed that investigated IL6 and IL10 SNPs were not associated with plasma IL-6 and IL-10 levels. An earlier study from the Mexican population also reported no association between plasma cytokine levels and cytokine gene SNPs [24]. However, a study using respiratory syncytial virus stimulated macrophages showed the association between cytokine levels and cytokine SNPs [8]. It is plausible that association between plasma cytokine levels and cytokine SNPs are not usually observed since plasma cytokines represent total cytokines produced by different cell types under specific stimulation as well as nonspecific production of cytokines.
It was shown that age, obesity, underlying other chronic diseases, secondary bacterial coinfections, and early administration of oseltamivir influence disease severity in influenza A(H1N1)pdm09 virus-infected subjects [30]. In the present study, details of the existing comorbidities in the study subjects were not available and are a limitation.

Conclusions
The present study suggests that TNFA and IL10 SNPs and IL-6 and IL-10 levels are associated with disease severity in influenza A(H1N1)pdm09 virus-infected subjects. Further, IL-6 levels might have utility as a biomarker for predicting fatal outcomes in influenza A(H1N1)pdm09 virus-infected subjects.  Informed Consent Statement: Written informed consent was obtained from all the participants or their legal guardians before sample collection.

Data Availability Statement:
The data presented in this study are available in the article.