key: cord-0876963-4fym7j09 authors: Çalık Başaran, Nursel; Tan, Çağman; Özışık, Lale; Özbek, Begüm; İnkaya, Ahmet Çağkan; Alp, Şehnaz; Ersoy, Ebru Ortaç; Ayvaz, Deniz Çağdaş; Tezcan, İlhan title: Evaluation of the peripheral blood T and B cell subsets and IRF‐7 variants in adult patients with severe influenza virus infection date: 2022-01-20 journal: Health Sci Rep DOI: 10.1002/hsr2.492 sha: f99dd23a80ef3c96a9b93814433e8cad1ae97cf6 doc_id: 876963 cord_uid: 4fym7j09 BACKGROUND AND AIMS: Influenza virus is one of the leading infections causing death among human being. Despite known risks, primary immune deficiency due to Interferon Regulatory Factor‐7 (IRF7) gene defect was reported as a possible cause of the risk factors for complicated influenza. We aimed to investigate the changes in peripheral T and B cell subsets in adult patients with severe seasonal influenza virus infection and the investigation of variants of IRF7 gene. METHODS: In this study, 32 patients, hospitalized due to influenza infection‐related acute respiratory failure were included. RESULTS: The median age of the patients was 76 years (26‐96), and 13/32 (40.6%) were in the intensive care unit. Central memory Th, effector memory Th, TEMRA Th, cytotoxic T lymphocytes (CTL), central memory CTL of the patients were found to be increased, naive CTL were decreased. There was a significant increase in the percentage of effector memory Th, and a decrease in the percentage of naive CTL in patients ≥65 years‐old compared to patients <65 years old (P = .039, and P = .017, respectively). IRF7 gene analysis revealed two different nucleotide changes in three patients; c.535 A > G; p.Lys179Glu ( K179E ) and c584A > T; p.His195Leu ( H195L ), located in the fourth exon of the IRF7 gene. DISCUSSION: The increases in central and effector memory Th, central memory CTL and decrease of naive CTLs may be secondary to the virus infection. K179E (rs1061502) and H195L (rs139709725) variants were not reported to be related with susceptibility to an infection yet. It is conceivable to investigate for novel variants in other genes related to antiviral immunity. Influenza virus infection will continue to be a problem today and in the future, as in the past, because a persistent and effective prevention method could not be developed due to the frequently changing nature of the virus. Seasonal influenza infection affects 3 to 5 million individuals, and responsible for up to 650 000 deaths annually worldwide. 1 Clinical presentation ranges from mild to severe and even to death in patients with high risk. It is well defined in the literature that especially young children, elderly people, and patients with chronic diseases are susceptible to infection and severe complications. 2 However, healthy adults may also experience severe influenza infections, complications, and mostly developed complication is pneumonia and related respiratory failure causing death. 3 Influenza virus activates all components of the immune system. Ciancanelli et al defined a loss of function mutation in the interferon regulator factor 7 (IRF7) gene in a child and his family members, all experienced only severe influenza infection reminding susceptibility to influenza virus in 2015. 9 They reported compound heterozygous mutations -p.Phe410Val(F410V) and p.Gln421X(Q421X)-and showed that in fibroblast type I IFN, in plasmacytoid dendritic cells (pDC) type I and III IFN generation were affected and resulted in increase of influenza A virus replication. They also showed that IFN generation diminished in airway epithelial cells causing faster and increased viral shedding. 9 The defined IRF7 mutation was accepted as the first single monogenic disease among primary immunodeficiencies, which makes the patient susceptible to influenza virus and has taken its place in the classification of primary immunodeficiencies. 10 After antigen presentation of antigen presenting cell (APC) to the CD4 and CD8+ T lymphocytes via major histocompatibility complex (MHC) I and II in the regional lymph nodes, mature T lymphocytes gain effector cell function and migrate to the infection site, and show antiviral activity. Mature CD4+ T lymphocytes help differentiation of antigen-specific cytotoxic CD8+ T lymphocytes, B cell activation, and production of virus-specific antibodies, activate alveolar macrophages via cytokines. Specific CD8+ lymphocyte response against virus peaks 7 days after the onset of infection, differentiates into cytotoxic effector cells in order to eliminate virus. After the initial proliferation of antigen specific CD8+ T lymphocytes to control the infection, a pool of effector antigen-specific memory T cells is generated. Then effector T lymphocytes start to die and memory T cells proliferate and migrate to the secondary lymph organs in order to fight against a second encounter with the virus, and long-term immunity. Naive T cells express CD8 + CD45RA + CCR7+, and with maturation CCR7 Demographic parameters and clinical characteristics (co-morbid diseases, presence of fever, cough, sputum, dyspnea, and other symptoms, type of respiratory support), and influenza test results were recorded. Presence of infiltration on chest X-ray or computerized chest tomography, and laboratory test results (white blood cell count, lymphocyte, neutrophil, platelet counts, hemoglobin, hematocrit, transaminases, blood-urea-nitrogen, serum creatinine, erythrocyte sedimentation rate, c-reactive protein, calcitonin) were also recorded. The respiratory failure (type 1 or type 2) was defined as the presence of hypoxemia or hypercarbia in the arterial blood gases tests or the requirement of respiratory support during the hospitalization period of the patients with influenza infection. 12 The study was car- For the detection of nucleotide changes in IRF7 gene and analysis of T and B lymphocyte subgroups, 3 mL peripheral blood was drawn to a tube with EDTA. Genomic DNA extraction, and lymphocyte subgroup analyzes with flow cytometry were performed on the same day. Isolated DNA samples were frozen and stored at À20 C for IRF7 gene analysis. The median age was 76 years , and 21/32 (65%) were female, 13/32 (40.6%) were in ICU. Non-invasive mechanical ventilation (NIMV) applied to 13/32 (40.6%) of the patients and invasive mechanical ventilation (IMV) was required in 5/32 (15.6%) of the patients. Median length of hospitalization was 11.5 (4-56) days, and 3/32 (9.3%) were died in the hospital. The most prevalent underlying diseases were Chronic Obstructive Pulmonary Disease (COPD) which was present in 13/32 (40.6%), and chronic heart diseases were the second most prevalent one (12/32, 37.5%) in our patient population. The demographic and clinical characteristics of patients were given on Median WBC count was 7000/uL (3200-26 000), and lymphocyte count was 988/uL (92-3078). B lymphocyte subsets were analyzed in 30 patients, and T lymphocyte subsets were analyzed in 16 patients. The median T and B lymphocyte subsets of the patients, and comparison with normal population were given in Table 3 Note: All values are given as median (minimum-maximum), and "n" presented as cells/micL. P values <0.05, which was considered statistically significant, was shown as bold. In this study, 32 adult patients hospitalized due to severe influenza virus infection with respiratory failure were evaluated in terms of T and B lymphocyte subgroups and mutation screening was performed for IRF7 gene. To the best of our knowledge, this is the first study conducted on T and B lymphocyte subsets and IRF7 gene analysis in adults with severe influenza infection. Central memory Th, effector memory Th, TEMRA Th, CTLs, central memory CTLs were found to be increased, naive CTLs were decreased. The increase in the effector memory Th, and a decrease in the naive CTL percentages were significant in elderly patients compared with younger patients with severe influenza infection. Age was one of the most important risk factors for severe influenza infection, complications, and mortality. 2 In our study group, the median age of the patients was 67 years, and 19/32 patients were aged 65 and over. In a study, the average age of 221 patients hospitalized due to severe acute respiratory infection caused by influenza was reported to be 74.1 years old and 75.6% of patients were over 65 years old. 15 Whereas, in another study, the mean age of 88 patients with H1N1 influenza-related severe respiratory infection was reported as 48 ± 15 years. 16 The difference in the most affected age groups of the patients can be explained by distinct influenza strains, for example, H1N1 was associated with more severe infections in younger individuals, or difference in the study designs. 16 At least one chronic medical disease was present in 90.7% of our patients. COPD, and chronic heart diseases were the leading comorbidities which was compatible with the literature. 2, 15 Lymphopenia was present in 62.5% of our patients. When com- and Graves' disease and Graves' ophthalmopathy, however, our patients had none of them. 22, 23 To the best of our knowledge, there is no relationship between the other variant rs139709725 / H195L and a disease in the literature yet. These variants located in the fourth exon of the IRF7 gene are located in the CAD domain structure of the gene, however, there is no information in the literature that these variants make a significant change in the protein structure. tion and a significant decrease in IFN production. 24 Also recently, it has been reported that mutations in IRF9, RIG-1, and TLR3 genes cause susceptibility to severe influenza virus infection. [25] [26] In the presence of an IRF9 homozygous "dysfunction" mutation, the formation of the ISGF3 complex cannot be achieved and therefore cannot respond appropriately to the type I IFN response due to virus infection. 25 Although there are multiple risk factors for complication of influenza infection, a genetic predisposition other than IRF7 in these patients may also cause susceptibility to severe influenza virus infections. Genetic variants in IRF7 or IRF9, RIG-1, and TLR3 affecting the immune response may cause predisposition to influenza virus in addition to the traditional risk factors. The limitations of our study were the small sample size, and the cross-sectional design of the study. There is a lack of knowledge on the distribution of the lymphocyte subsets before or after the virus infections, so we cannot conclude the causality of these changes on complication of influenza infection. In conclusion, the effector memory Th and CTLs were increased and naive T cells were decreased in adults with severe influenza infection compared to healthy controls. We could not detect a previously identified variant of IRF7, but we cannot disregard other genetic defects for severe influenza infections in our patients. Genetic analysis especially in young patients with severe influenza infection without a known chronic disease will contribute to the literature. None declared. Research Unit, project number THD-2017-14742. Authors declare no conflict of interest. Nursel Çalık Başaran, corresponding author of the manuscript, confirm that I had full access to all of the data in the study and take complete responsibility for the integrity of the data and the accuracy of the data analysis. 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