Phosphatidyl Inositol 3 Kinase-Gamma Balances Antiviral and Inflammatory Responses During Influenza A H1N1 Infection: From Murine Model to Genetic Association in Patients

Abstract

Influenza A virus (IAV) infection causes severe pulmonary disease characterized by intense leukocyte infiltration. Phosphoinositide-3 kinases (PI3Ks) are central signaling enzymes, involved in cell growth, survival, and migration. Class IB PI3K or phosphatidyl inositol 3 kinase-gamma (PI3Kγ), mainly expressed by leukocytes, is involved in cell migration during inflammation. Here, we investigated the contribution of PI3Kγ for the inflammatory and antiviral responses to IAV. PI3Kγ knockout (KO) mice were highly susceptible to lethality following infection with influenza A/WSN/33 H1N1. In the early time points of infection, infiltration of neutrophils was higher than WT mice whereas type-I and type-III IFN expression and p38 activation were reduced in PI3Kγ KO mice resulting in higher viral loads when compared with WT mice. Blockade of p38 in WT macrophages infected with IAV reduced levels of interferon-stimulated gene 15 protein to those induced in PI3Kγ KO macrophages, suggesting that p38 is downstream of antiviral responses mediated by PI3Kγ. PI3Kγ KO-derived fibroblasts or macrophages showed reduced type-I IFN transcription and altered pro-inflammatory cytokines suggesting a cell autonomous imbalance between inflammatory and antiviral responses. Seven days after IAV infection, there were reduced infiltration of natural killer cells and CD8+ T lymphocytes, increased concentration of inflammatory cytokines in bronchoalveolar fluid, reduced numbers of resolving macrophages, and IL-10 levels in PI3Kγ KO. This imbalanced environment in PI3Kγ KO-infected mice culminated in enhanced lung neutrophil infiltration, reactive oxygen species release, and lung damage that together with the increased viral loads, contributed to higher mortality in PI3Kγ KO mice compared with WT mice. In humans, we tested the genetic association of disease severity in influenza A/H1N1pdm09-infected patients with three potentially functional PIK3CG single-nucleotide polymorphisms (SNPs), rs1129293, rs17847825, and rs2230460. We observed that SNPs rs17847825 and rs2230460 (A and T alleles, respectively) were significantly associated with protection from severe disease using the recessive model in patients infected with influenza A(H1N1)pdm09. Altogether, our results suggest that PI3Kγ is crucial in balancing antiviral and inflammatory responses to IAV infection.

Investigation of Human IFITM3 Polymorphisms rs34481144A and rs12252C and Risk for Influenza A(H1N1)pdm09 Severity in a Brazilian Cohort

“…Genomic DNA was extracted from clinical respiratory samples by using the PureLink™ Genomic DNA Mini Kit (Invitrogen, USA), as previously described (Garcia et al, 2018 ; Matos et al, 2019 ). After that, the rs34481144A and rs12252C polymorphisms were identified using pre-defined TaqMan TM SNP Genotyping Assays probes (assays IDs: C__26288451_10 and C_175677529_10) (Thermo Fisher Scientific, USA) in a real time PCR assay performed in the Step One Plus equipment (Applied Biosystems, USA) with 5–20 ng DNA, per sample, according to manufacturer's instructions.…”

“…Similarly, host genetic variants may also affect modulation of immune response after viral infection (Falcon et al, 2015 ; Chan et al, 2017 ). Putative associations between genetic polymorphisms in host crucial genes that act against virus infection that are essential for virus-host interplay have been recently explored regarding their association with susceptibility/severity of influenza infections (Zhou et al, 2012 ; Tarnow et al, 2014 ; Cheng et al, 2015 ; Chan et al, 2017 ; Garcia et al, 2018 ; Matos et al, 2019 ).…”

Host DNA released by NETosis in neutrophils exposed to seasonal H1N1 and highly pathogenic H5N1 influenza viruses

“…Depletion of neutrophils in mice infected with a sub-lethal dose of influenza virus resulted in impaired viral clearance and fatal outcome [2,6]. In contrast, an excessive infiltration and activation of neutrophils with the release of reactive oxygen species only resulting in mild pathology [7,8]. Since different influenza virus subtypes vary in their virulence, their direct interaction to neutrophils warrants further investigation.…”

Host DNA released by NETosis in neutrophils exposed to seasonal H1N1 and highly pathogenic H5N1 influenza viruses

“…Depletion of neutrophils in mice infected with asub-lethal dose of in uenza virus resulted in impaired viralclearance and fatal outcome (2,6). In contrast, an excessivein ltration and activation of neutrophils with the release ofreactive oxygen species only resulting in mild pathology (7,8).Since different in uenza virus subtypes vary in their virluence,their direct interaction to neutrophils warrants furtherinvestigation.…”