The economical role of RIOK3 in modulating the Jak1/STAT1 pathway and antiviral immunity against respiratory syncytial virus infection in macrophages: implications for therapeutic potential

Abstract

Respiratory Syncytial Virus (RSV) is a leading cause of lower respiratory tract infections, particularly in vulnerable populations such as infants, the elderly, and immunocompromised individuals. RSV infection can result in mortality rates as high as 20%, attributable not only to viral replication but also to an excessive host immune response. Current therapeutic options are limited, partly due to gaps in understanding the host immune response, especially the role of macrophages and their signaling pathways. This study investigates the role of RIOK3, an unconventional kinase, in modulating the Jak1/STAT1 pathway during RSV infection in macrophages and its impact on viral replication and interferon production. Using both in vitro and in vivo models, including primary bone marrow-derived macrophages (BMM) from control and RIOK3 knockout (KO) mice, we demonstrate that RIOK3 is a critical regulator of the Jak1/STAT1 pathway in macrophages during RSV infection. The absence of RIOK3 enhances viral replication and disrupts the balance of type I interferons. Targeting RIOK3 may represent a promising strategy to enhance antiviral immunity and mitigate RSV-induced inflammation, thus warranting further investigation for therapeutic potential.

Keywords: Jak1/STAT pathway; RIOK3; RSV; macrophage; type I interferon.

Figure 1

RIOK3 enhances JAK1/STAT1 pathway activity by interacting with Jak1 partially depend of its kinase function. (a, b) Sketch maps for kinase-dead RIOK3^K290A plasmid and gRNA inserted CRISPR/Cas9 plasmid construction. (c) Vero cells were transfected with gRNA-inserted plasmid or negative control for 48 h. Cell protein was assayed by WB to detect RIOK3 expression. (d) Vero cells with or without RIOK3-KO were stimulated with human IFN-α (1,000 units/ml) for 0, 15, 30, 60, and 120 min. Protein was collected for WB to analyze expression level of phosphor-Jak1, total Jak1, phospho-STAT1, total STAT1, RIOK3, and β-actin. (e) RIOK3-KO Vero cells were transfected with RIOK3 Over-expression plasmid or Kinase Dead RIOK3^K290A plasmid for 48 h, and then were stimulated with human IFN-α (1,000 units/ml) for 0, 15, 30, 60, and 120 min. Protein was collected for WB to analyze expression level of phosphor-Jak1, total Jak1, phospho-STAT1, total STAT1, RIOK3, and β-actin. (f) BMMs from control or RIOK3^−/− mice were stimulated with murine IFN-α (100 units/ml) for 0, 15, and 30 min. Protein was collected and divided into two parts. One was assayed directly to display the total input volume of RIOK3, Jak1, and β-Actin protein by WB. One was performed immunoprecipitation using Jak1 antibody and both Jak1 and RIOK3 were assayed by WB.

Figure 2

Expression of RIOK3 is dynamically increasing in macrophages during RSV infection. THP1 cells were dynamically infected with RSV for 0, 2, 4, 6, 12, and 24 h at MOI = 5. Cells were harvested to assess mRNA expression of RSV-F (a) and RIOK3 (b) by qRT-PCR analysis, as well as dynamical change of RIOK3 protein level by WB (c).

Figure 3

Lack of RIOK3 is associated with enhanced expression both in RSV-F and type I IFNs during infection. BMM from control or RIOK3^−/− mice were infected with RSV at MOI = 5. At 6, 12, 24, and 48 hpi, RNA was collected for qRT-PCR (a–f), and supernatants were collected for plaque assay (failed) and ELISA (g–k). (g, h) only showed ELISA results of indicated factor at 24 hpi. (l) BMMs from control or RIOK3^−/− mice were stimulated with or without murine IFN-α (100 units/ml) for 6 h and then infected with RSV at MOI = 5. At 12 hpi, cells were collected for RNA extraction and RSV-F mRNA level was assessed by qRT-PCR. Data are expressed as means ± SEM. Significance was determined using Student's t-test; *** p < 0.001, ** p < 0.01, and * p < 0.05 relative to non-infected cells.

Figure 4

Lack of RIOK3 inhibit activation of Jak1/STAT1 pathway. (a) BMMs from control or RIOK3^−/− mice were infected with RSV at MOI = 5. At 0 (un-infection, UI), 2, 4, 6, 12, and 24 hpi, protein was collected for WB. Phospho-STAT1, total STAT1 and β-actin was assayed. (b) BMMs from control or RIOK3^−/− mice were stimulated with murine IFN-α (100 units/ml) for 0, 15, and 30 min. Protein was collected for WB to analyze expression level of phosphor-Jak1, total Jak1, phospho-STAT1, total STAT1, phospho-STAT3, total STAT3, RIOK3, and β-actin. (c–e) BMMs from control or RIOK3^−/− mice were stimulated with murine IFN-α (100 units/ml) for 0, 6, 12, and 24 h. RNA was collected to assess ISGs expression, including Isg15, Oas1, and Mx1. Data are expressed as means ± SEM. Significance was determined using Student's t-test; ** p < 0.01 and * p < 0.05 relative to non-stimulated cells.