|Year : 2019 | Volume
| Issue : 4 | Page : 549-556
Immune response during influenza virus infection among the population of Assam, Northeast India
Mousumi Dutta1, Prafulla Dutta1, Subhash Medhi2, Biswajyoti Borkakoty1, Dipankar Biswas1
1 Division of Virology, ICMR-Regional Medical Research Centre, N.E.Region, Dibrugarh, Assam, India
2 Department of Bioengineering and Technology, GUIST, Gauhati University, Guwahati, Assam, India
|Date of Submission||29-May-2019|
|Date of Acceptance||16-Apr-2020|
|Date of Web Publication||18-May-2020|
Dr. Biswajyoti Borkakoty
Division of Virology, ICMR-Regional Medical Research Centre, N.E.Region, Dibrugarh-786 001, Assam
Source of Support: None, Conflict of Interest: None
Introduction: The pathogenicity of influenza virus infection is modulated by the cytokine expressions in patients. The present study was aimed to measure some important pro- and anti-inflammatory cytokines in influenza-infected population of Assam, Northeast India. Materials and Methods: Influenza viruses consisting of subtypes influenza A(H1N1)pdm09, H3N2 and influenza-B were detected in patients with symptoms of influenza-like-illness by Real-time reverse transcriptase polymerase chain reaction (RT-PCR) method. Relative messenger ribonucleic acid (mRNA) quantification of four pro-inflammatory cytokines (interleukin [IL]-6, IL-8, interferon-gamma [IFN-γ] and tumour necrosis factor-alpha [TNF-α]) and one anti-inflammatory cytokine (IL-10) were measured in influenza-positive cases and non-influenza controls, by real-time RT-PCR. The plasma concentration of the cytokines was determined using cytometric-bead-array with flow cytometry. Results: Influenza viruses were detected in 14.28% (50/350) of 350 patients screened. The expression of IL-6 was significantly raised in cases compared to controls (P = 0.018). IL-8 and IL-10 were also raised in cases, compared to controls (P = 0.284 and P = 0.018). An increased plasma TNF-α was observed in cases (1.36-fold and P = 0.289). The mRNA expression of IFN-γ was also increased in cases compared to controls (0.87-fold). However, the plasma level of IFN-γ was higher in the non-influenza controls compared to cases. Conclusions: The study revealed a differential cytokine profile during influenza virus infection in the population, which may influence disease severity. An extended study on host immune response may provide better insights for the use of cytokine antagonists in therapeutic treatments among severe cases of influenza virus infection.
Keywords: Cytokine, influenza virus, Northeast India, pathogenicity, real-time polymerase chain reaction
|How to cite this article:|
Dutta M, Dutta P, Medhi S, Borkakoty B, Biswas D. Immune response during influenza virus infection among the population of Assam, Northeast India. Indian J Med Microbiol 2019;37:549-56
|How to cite this URL:|
Dutta M, Dutta P, Medhi S, Borkakoty B, Biswas D. Immune response during influenza virus infection among the population of Assam, Northeast India. Indian J Med Microbiol [serial online] 2019 [cited 2020 Aug 8];37:549-56. Available from: http://www.ijmm.org/text.asp?2019/37/4/549/284512
| ~ Introduction|| |
Influenza virus infection occurs globally in seasonal patterns and results in significant illness. During pandemic years, high frequency of fatal cases was recorded in healthy young and middle aged patients.,, The transfer of this pandemic strain from person to person and its potential virulence factor remains a great concern. Starting from an uncomplicated upper respiratory tract infection, the virus has the potential to cause death by respiratory arrest, involving the severe form of pneumonia. However, the ability of the virus to persist in the human population with increased pathogenicity remains unclear.
In the event of Influenza virus infection, the interplay between the virulence factor of the virus and host resistance can cause disease severity. However, the host resistance can become more hyperactive during influenza A(H1N1)pdm09 virus infection accompanied by excessive inflammatory cytokine response. During viral infections, including influenza virus infection, hyper induction of inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL)-6 and anti-inflammatory cytokine IL-10 has been a hallmark for disease progression and ultimately death of the patient. Higher plasma levels of IL-6, TNF-α and IL-10 are found to be associated with disease severity during influenza A(H1N1)pdm09 infection.,
Population with a different ethnic profile may probably display a different immune response during a particular infection., Assam, being a part of Northeast India with diverse ethnic groups and different topographical conditions, reports influenza virus infections with a seasonal pattern. Thus, an overall scenario of the cytokine profile in this population may be an insight for influenza virus pathogenicity.
Therefore, the present study was aimed to measure the cytokine profile with respect to messenger ribonucleic acid (mRNA) expression and plasma concentration of the cytokines during influenza virus infection in the population of Assam, Northeast India.
| ~ Materials and Methods|| |
Patients and samples
A prospective case-control study was carried out in influenza-like-illness (ILI) patients attending out-patients department of primary health centres as well as hospitalized cases in Dibrugarh district of Assam, Northeast India. The study was performed at ICMR-Regional Medical Research Centre, Dibrugarh, Assam (India) from January 2016 to December 2017. Clinical specimens of nasopharyngeal/throat swabs and blood samples were collected from patients >12 years of age with symptoms of ILI and also from healthy controls. Informed consents were obtained from all the patients before sample collection. The study was approved by the Institutional Human Ethics Committee of ICMR-Regional Medical Research Centre, Dibrugarh, Assam, India.
The samples were collected from patients presented with ILI within 3-7 days of illness. Nasopharyngeal/throat swabs were collected in viral transport medium from the patients and transported to the laboratory in cold condition (+4°C). Three ml whole blood samples were collected in ethylenediaminetetraacetic acid vials and plasma was separated immediately by centrifugation at 2000 rpm for 10 min at 4°C before storage at −80°C. Peripheral blood mononuclear cells (PBMC) were isolated from whole-blood samples using an equal amount of phosphate buffer saline and lymphoprep (Alere Technologies AS, Oslo, Norway).
Molecular detection of influenza viruses
Nasopharyngeal/throat swabs were processed and viral ribonucleic acid (RNA) was extracted from 140 μl of the clinical specimens using QIAamp Viral RNA mini kit (Qiagen, GmbH, Hilden, Germany) according to the manufacturer's instruction with proper biosafety measures. The extracted RNA was subjected to the detection of influenza A (influenza A(H1N1)pdm09 and H3N2) and influenza B viruses by real-time reverse transcriptase-polymerase chain reaction based on TaqMan probe assay. Patients who tested positive for influenza viruses were considered as cases and those tested negative were referred to as non-influenza control groups.
Cytokine messenger ribonucleic acid expression profile
Total RNA was extracted from PBMC using RNeasy Mini Kit (Qiagen, GmbH, Hilden, Germany) according to the manufacturer's instruction for measuring the cytokines IL-6, IL-8, IL-10, TNF-α and IFN-γ. The extracted RNA was eluted in 30 μl nuclease-free water, and stored in aliquots at −80°C until use. To avoid contamination with genomic deoxyribonucleic acid (DNA), the extracted RNA was treated with DNA-Free DNase (Promega, Madison, USA) according to the manufacturer's instruction. The quality of extracted RNA was analysed by measuring optical density at 260/280 nm with EPOCH Microplate spectrophotometer (BioTek). The RNA was than reverse transcribed to cDNA using random primers, MMLV reverse transcriptase (Promega, Madison, USA). The real-time PCR reaction was performed in duplicate using the SYBR Green PCR Master Mix (Qiagen). The PCR condition was 95°C for 2 min, followed by 40 cycles of 95°C for 5 s and 60°C for 10 s. The sense and antisense primers used in real-time PCR for measuring the cytokines are listed in [Table 1]. The expression of a housekeeping gene (GAPDH gene) was used with every sample in duplicate for normalisation. Relative quantification of mRNA level was performed using the comparative delta-delta cycle threshold (CT) method for measuring the relative expression level of the respective gene.
|Table 1: Primers used in real.time reverse transcriptase polymerase chain reaction assay|
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Estimation of cytokine levels in plasma samples
The plasma levels of five cytokines (IL-6, IL-8, IL-10, TNF-α and IFN-γ) were measured using cytometric bead array flex sets from BD Biosciences (CA) as per the manufacturer's instructions. Briefly, 50 μl of a mixture of bead population with distinct fluorescence intensities, coated with specific antibodies for capturing different cytokines and chemokines, was incubated with 50 μl of plasma for 1 h at room temperature. These cytokine-captured beads were then mixed with 50 μl of phycoerythrin-conjugated detection antibodies to form sandwich complexes. After incubation for 2 h at room temperature in the dark, followed by a washing step, the samples were subjected to flow cytometry using FACS AriaII Flow cytometry analyser using BD FACS Diva v7 software (BD Biosciences, CA) for acquiring data. Data analysis was performed using FCAP Array v3 software (BD Biosciences, CA), and the levels of cytokines were expressed as picogram per millilitre (pg/ml).
The clinical characteristics of the patients were analysed using IBM SPSS Statistics 20, trial version (IBM, New York, USA). The results of mRNA and plasma cytokine levels were calculated in Microsoft Excel 2007. Comparison of delta CT values and fold change between influenza-infected cases and non-influenza control groups were calculated in GraphPad prism, trial version (GraphPad software, CA) as previously studied. The P value was calculated using a two-tailed Fisher's exact test, and P ≤ 0.05 were considered statistically significant. Descriptive statistics included medians and interquartile ranges for non-normal distribution, performed by column statistics.
| ~ Results|| |
A total of 350 patients of >12 years of age with symptoms of ILI were recruited from January 2016 to December 2017. The Clinical and demographic details of patients are shown in [Table 2]. There was a significant difference in age between influenza-infected cases and non-influenza controls (P = 0.004). The male-to-female ratio was found to be almost equal among cases and controls (P = 0.638). However, the clinical characteristics such as cough, sore throat, breathlessness, vomiting and oxygen requirement were higher in cases, as compared to non-influenza controls. Most of the patients included in this study were outpatients, with 15 hospitalised patients, of which 6 were found to be positive for influenza viruses.
Detection of influenza viruses in influenza-like-illness patients
Influenza viruses were detected in 14.28% (50/350) of the ILI patients. Thus, 50 samples were found positive and 300 samples were found negative for influenza viruses. Among the detected influenza viruses, 70% (35/50) were influenza A viruses (influenza A(H1N1)pdm09 62.85%, 22/35 and H3N2 37.14%, 13/35) and 30% (15/50) were influenza-B viruses. The 50 influenza-positive samples were considered as cases and 50 (of 300 influenza-negative samples) samples that tested negative for influenza viruses were considered as non-influenza controls. In this study, influenza virus detection was higher in females, with 58% (29/50) than that of males with 42% (21/50). Influenza virus infects almost all age groups in a population. The distribution of influenza virus infection among the different age groups in the study population is shown in [Figure 1]. The incidence of influenza virus infection was found all around the year. However, the highest positivity of the influenza virus was detected in the month of June and July, followed by February, March and April in all the years.
Cytokine messenger ribonucleic acid expression in influenza-infected cases and non-influenza controls
The mRNA expression of all the five cytokines was measured in 50 representative numbers of samples that are positive for influenza viruses and 50 ILI patients negative for the virus that serves as non-influenza controls. The relative mRNA expression revealed a significantly increased level of IL-6 (P = 0.02) and IL-8 (P = 0.02) in influenza-infected cases as compared to non-influenza controls. The expression of IL-10 (P = 0.27), TNF-α (P = 0.36) and IFN-γ (P = 0.25) was also higher in cases as compared to non-influenza controls. The comparative expression of fold change among influenza-infected cases and non-influenza controls is shown in [Table 3]. During the study, three influenza subtypes were detected among influenza-infected cases, including influenza A(H1N1)pdm09, H3N2 and influenza-B. The average mRNA fold change observed in cases infected by different subtypes of influenza virus, when compared with non-influenza controls as the standard, is shown in [Figure 2].
|Table 3: Expression profile of targeted cytokines among influenza infected cases and non-influenza controls|
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|Figure 2: Scattered plot representation of comparative expression level (fold change) of targeted cytokines among cases infected with different influenza subtypes. Fold change is calculated with non-influenza control group as standard. Straight line indicates mean of each group|
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Plasma cytokines expression in influenza-infected cases and non-influenza controls
A total of 50 plasma samples from influenza-infected cases and 50 plasma samples from the non-influenza control group were tested to determine the overall differences in cytokine profile induced by the virus. Among the five cytokines studied, two cytokines, IL-6 (P = 0.01) and IL-10 (P = 0.01) showed a significantly higher level in influenza-infected cases compared to the non-influenza control group. Whereas, no significant differences were observed in IL-8 (P = 0.28) and TNF-α (P = 0.28) concentration, although it was higher in cases than those of non-influenza controls. On the other hand, the plasma level of IFN-γ was found to be higher in non-influenza controls (P = 0.10) as compared to cases. [Figure 3] represents the plasma cytokine concentrations of targeted cytokine in influenza-infected cases and non-influenza controls. Notably, all the cytokine responses showed an increase in the cytokine concentration except IFN-γ, in the influenza-infected cases when compared to non-influenza controls. The plasma cytokine levels were also determined in cases infected with different influenza subtypes influenza A(H1N1)pdm09, H3N2 and influenza-B. [Figure 4] shows the difference in plasma cytokine expression among the cases infected by three different influenza subtypes.
|Figure 3: Plasma cytokine concentrations of targeted cytokines (interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-alpha and interferon-gamma) in influenza infected cases and non-influenza controls. Two-tailed P < 0.05 (*in figure) were considered to be statistically significant|
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|Figure 4: Plasma cytokine levels in cases infected with influenza A(H1N1)pdm09, H3N2 and influenza-B subtypes|
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| ~ Discussion|| |
In this study, we presented some pro and anti-inflammatory cytokine levels following influenza virus infection in patients from Assam, Northeast India, along with non-influenza control group.
The pathogenesis of influenza virus infection is known to be associated with differential cytokine production.,, The cytokines such as ILs, IFN-γ and TNF-α plays an important role in the regulation of immune response and inflammation towards infection.
Our study recorded the cytokine profiles of 50 influenza-infected cases and equal number of non-influenza controls. The influenza positivity in both out-patient and hospitalised cases did not show any significant difference in our study. While previous studies observed significant difference of influenza virus association in community-based surveillance and hospitalised cases. Conventionally, community-based surveillance of ILI cases represents a broader knowledge of population-level influenza virus activity, while hospital based surveillance relies only with the individuals seeking personal medical care and illness severity. Previous studies found that influenza virus positivity was significantly higher in individuals >50 years of age than that of middle age group of 11–50 years. Similarly, in our study population influenza viruses were detected in patients >40 years of age and non-influenza control group was mostly <30 years. Influenza positivity was detected in both males and females (mean age = 40.68), with a higher frequency in females. The clinical features, primarily suggestive of ILI has been recorded in both cases and non-influenza controls, among which cough, breathlessness, vomiting and oxygen requirement, were found to be significantly higher in cases compared to non-influenza controls.
During the study, the mRNA expression and cytokine production of IL-6, IL-8 and IL-10 were considerably higher in influenza-infected cases as compared to non-influenza controls. IL-6, a cytokine of innate immunity, has been an important mediator of fever and acute-phase reaction during influenza virus infection,, Previous studies reported increased IL-6 expression in critically ill patients infected with influenza A(H1N1)pdm09.,, Likewise, the mRNA expression and plasma cytokine of IL-6 in our study population were significantly higher in influenza-infected cases as compared to non-influenza controls. Thus, increased level of IL-6 may act as an inflammatory mediator for influenza virus infection.
IL-8, a chemokine of innate immunity, plays an important role in acute lung injury through neutrophil-mediated inflammatory response., Several studies reported high levels of IL-8 in severe influenza A(H1N1)pdm09 infected cases., The plasma level of IL-8 in our study population was higher in cases as compared to non-influenza controls, along with a higher fold change in mRNA expression. Patients with influenza virus infection in our study were found to have a higher rate of breathlessness (12%) and oxygen requirement (12%) compared to non-influenza controls (1.66% and 1%, respectively). Nevertheless, an increase in IL-8 could probably be a marker for influenza severity.
IL-10, an anti-inflammatory cytokine, is found to be associated with severe influenza A(H1N1)pdm09 and seasonal H3N2 virus infection., IL-10, downregulates the expression of type 1 helper cells, can block the master switch nuclear factor kappa B, MHC-Class II antigens and co-stimulatory molecules in macrophages. In the present study, mRNA expression and plasma levels of IL-10 were significantly higher in influenza-infected cases as compared to non-influenza controls. Thus, higher secretion of IL-10 may be a counter-response to increasing levels of pro-inflammatory cytokines.
TNF-α, a pro-inflammatory cytokine of innate immunity, plays an important role in recruiting various host cells that include monocytes and lymphocytes to the site of infection. It has been known that influenza virus infection induces TNF-α expression in humans., It induces lung tissue destruction during influenza infection, thus creating systemic symptoms., In our study population, plasma levels of TNF-α were apparently higher in influenza-infected cases as compared to non-influenza controls, with higher fold change in mRNA expression. IFN-γ is a cytokine of innate and adaptive immunity, which plays an important role in the activation of macrophage and inhibition of Th17 pathway and control of the intracellular pathogen. Previous study shows high systemic levels of IFN-γ levels in influenza A(H1N1)pdm09 infected hospitalised cases. Similarly, the present study shows an increased mRNA fold change in cases as compared to non-influenza controls. Whereas, the plasma concentration of IFN-γ in non-influenza controls were higher compared to influenza-infected cases. The changes in gene expression and protein levels of IFN-γ may be due to post-translational downregulation during the process of infection.
| ~ Conclusions|| |
Increased level of IL-6, IL-8 and IL-10 in influenza-infected cases indicates that these may be a hallmark for disease outcome and severity as studied earlier. Further, in-depth studies that can target differential immune response in an extended population during influenza virus infection for therapeutic strategies and modulating disease severity remains awaited.
The authors are grateful to the Department of Science and Technology, Government of India, Ministry of Science and Technology for financial assistance received under the grant SR/WOS-A/LS-212/2016(G). The authors acknowledge the entire members of virology division and Regional Viral Research Diagnostic Laboratory, RMRC, Dibrugarh, for their assistance and laboratory facilities during the study period. Authors also acknowledge the institutional support of Regional Medical Research Centre, Dibrugarh for implementation of the project.
Financial support and sponsorship
The study was supported by Department of Science and Technology, Ministry of Science and Technology, Government of India.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]