Inhibiting Protein Kinase D Promotes Airway Epithelial Barrier Integrity in Mouse Models of Influenza A Virus Infection

Abstract

RationaleProtein kinase D (PKD) is a serine/threonine kinase family that is involved in a wide array of signaling pathways. Although PKD has been implicated in immune responses, relatively little is known about the function of PKD in the lung or during viral infections.ObjectivesWe investigated the hypothesis that PKD is involved in multiple aspects of host response to viral infection.MethodsThe selective PKD inhibitor CRT0010166 was administered to C57BL/6 mice prior to and during challenge with either inhaled double-stranded RNA or Influenza A Virus. PKD signaling pathways were investigated in human bronchial epithelial cells treated with CRT0010166, double-stranded RNA, and/or infected with Influenza A Virus.MeasurementsTotal protein and albumin accumulation in the bronchoalveolar fluid was used to asses inside/out leak. Clearance of inhaled FITC-dextran out of the airspace was used to assess outside/in leak. Cytokines and neutrophils in bronchoalveolar lavage were assayed with ELISAs and cytospins respectively. Viral RNA level was assessed with RT-PCR and protein level assessed by ELISA.Main ResultsPKD inhibition prevented airway barrier dysfunction and pro-inflammatory cytokine release. Epithelial cells express PKD3, and PKD3 siRNA knock-down inhibited polyI:C induced cytokine production. Lung epithelial-specific deletion of PKD3 (CC10-Cre x PKD3-floxed mice) partially attenuated polyI:C-induced barrier disruption in vivo. Mechanistically, we found that PKD promoted cytokine mRNA transcription, not secretion, likely through activating the transcription factor Sp1. Finally, prophylactic CRT treatment of mice promoted barrier integrity during influenza virus infection and reduced viral burden.ConclusionsInhibiting PKD promotes barrier integrity, limit pathogenic cytokine levels, and restrict Influenza A Virus infection. Therefore, PKD is an attractive target for novel antiviral therapeutics.

Protein kinase D3 promotes neutrophil migration during viral infection

“…To determine the role of PKD3 in this process, we next measured epithelial barrier function in WT and PKD3 −/− mice. In contrast to the barrier protection and reduced inflammation observed with CRT‐treated mice, 18 PKD3 −/− mice were not protected from poly(I:C)‐mediated inside‐out barrier disruption as seen by total protein and serum albumin leak into the airspace (Figure 2a). PKD3 −/− mice were also not protected from outside‐in barrier disruption as seen by loss of inhaled 4‐kDa FITC–dextran from the airspace and accumulation in the serum (Figure 2b).…”

“…To determine whether PKD3 has a neutrophil‐intrinsic role in promoting neutrophil recruitment to the lung following dsRNA challenge, we generated a new line of mice by breeding PKD3‐floxed mice 18 with mice expressing Cre recombinase driven by the Ly6G promoter. Resulting dual transgenic PKD3‐floxed × Ly6G‐Cre mice were challenged with inhaled poly(I:C) and compared with single transgenic controls.…”

“…We and others have found that PKD3 is highly expressed in the lung tissue and immune cells 4,18,22 . We therefore hypothesized that PKD3 may play an underappreciated role in immune cell accumulation in the lung.…”

“…PKD has also been implicated in promoting production of neutrophil chemoattractants in the lung 16 and facilitating neutrophil activation and migration 17 . We recently reported that PKD3 is highly expressed in airway epithelial cells, where it contributes to chemokine production, neutrophil accumulation and barrier dysfunction in response to polyinosinic:polycytidylic acid [poly(I:C)], a synthetic double‐stranded RNA (dsRNA), and viral infection 18 …”

Bioinformatics insights into the genes and pathways on severe COVID-19 pathology in patients with comorbidities

“…Specific inhibition of these factors significantly alters the functions of specialized inflammatory cells suggesting that these factors may help in early diagnosis and could be predictors of clinical symptoms ( Guo et al, 2022a ). IDO1 is also an important immunomodulatory gene in T lymphocyte activation and in modulating immune tolerance ( Veazey et al, 2020 ; Makaremi et al, 2022 ). In preclinical models, pharmacological inhibitors of IDO1 have therapeutic utility in various diseases, including cancer, HIV and influenza ( Yang et al, 2020b ; Chang et al, 2020 ; Chi et al, 2020 ).…”