RGS10 Reduces Lethal Influenza Infection and Associated Lung Inflammation in Mice

Abstract

Seasonal influenza epidemics represent a significant global health threat. The exacerbated immune response triggered by respiratory influenza virus infection causes severe pulmonary damage and contributes to substantial morbidity and mortality. Regulator of G-protein signaling 10 (RGS10) belongs to the RGS protein family that act as GTPase activating proteins for heterotrimeric G proteins to terminate signaling pathways downstream of G protein-coupled receptors. While RGS10 is highly expressed in immune cells, in particular monocytes and macrophages, where it has strong anti-inflammatory effects, its physiological role in the respiratory immune system has not been explored yet. Here, we show that Rgs10 negatively modulates lung immune and inflammatory responses associated with severe influenza H1N1 virus respiratory infection in a mouse model. In response to influenza A virus challenge, mice lacking RGS10 experience enhanced weight loss and lung viral titers, higher mortality and significantly faster disease onset. Deficiency of Rgs10 upregulates the levels of several proinflammatory cytokines and chemokines and increases myeloid leukocyte accumulation in the infected lung, markedly neutrophils, monocytes, and inflammatory monocytes, which is associated with more pronounced lung damage. Consistent with this, influenza-infected Rgs10-deficent lungs contain more neutrophil extracellular traps and exhibit higher neutrophil elastase activities than wild-type lungs. Overall, these findings propose a novel, in vivo role for RGS10 in the respiratory immune system controlling myeloid leukocyte infiltration, viral clearance and associated clinical symptoms following lethal influenza challenge. RGS10 also holds promise as a new, potential therapeutic target for respiratory infections.

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“…To address whether NE release inside Mtb-infected airways is affected by Duox1, NE concentrations were assessed by ELISA in the BAL fluid, sera, and lung lysates of Mtb-infected WT and Duox1 KO mice as above (Figures 5D-F). In addition, NE enzymatic activity was measured as previously described (28) and the results show no effect of Duox1 (Figures 5G, H). Additionally, tissue distribution of MPO inside the lung tissue was evaluated by immunohistochemistry (Figure 6).…”

“…Following a washing step, the cells were resuspended in 100 µl of 1X PBS containing 1% bovine serum albumin and blocked using TruStain FcX ™ anti-mouse (CD16/32) antibody (Cat#: 101320; BioLegend, San Diego, CA) on ice and protected from light for 10 minutes. Multicolor cell surface staining was performed separately using two different antibody cocktails for the characterization of lymphoid (CD45, CD3, CD4, CD8, and NK1.1) and myeloid cells (CD11b, CD11c, F4/80, Ly6G, Ly6C, and CD115) as previously described (20,28,29). The cells were subsequently stained with surface antibodies (Table 1) at a 1:100 dilution at the same time to detect the number of cells from each of the myeloid and lymphoid cell subsets following the protocol as described previously (20).…”

“…Enzymatic activity of NE was evaluated using the NE activity assay kit (Cat#600610; Cayman Chemical; Ann Arbor, MI) as previously described (28). Neutrophil elastase at 18 mU/ml was used to make a standard curve ranging from 0.156 mU/ml to 10 mU/ml.…”

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“…RGS10 encodes an important anti-inflammatory protein and has previously been implicated in vitro in regulating macrophage activity, specifically limiting activation of the NF-ĸB pathway, reducing expression of tumour necrosis factor (TNF), and regulating macrophage M1 to M2 repolarisation 99 . In murine models challenged with a lethal dose of influenza A virus, loss of RGS10 resulted in increased cytokine and chemokine activity, and a more pronounced recruitment of neutrophils and monocytes to the site of infection 100 .…”

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“…According to the respective manufacturer’s instructions, measurement of cytokine levels, including interleukin-6 (IL-6), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-9 (MMP-9), was completed using commercial ELISA kits. MPO-DNA complexes were detected in the mouse serum using MPO (1:200), the secondary mouse anti-DNA-POD (1:500) and TMB substrate as previously described [ 22 ].…”