Inflammatory response of mast cells during influenza A virus infection is mediated by active infection and RIG-I signaling

Abstract

Influenza A virus (IAV) is a major respiratory pathogen of both humans and animals. The lung is protected from pathogens by alveolar epithelial cells, tissue-resident alveolar macrophages, dendritic cells, and mast cells. The role of alveolar epithelial cells, endothelial cells, and alveolar macrophages during IAV infection has been studied previously. In this study, we address the role of mast cells during IAV infection. Respiratory infection with A/WSN/33 causes significant disease and immunopathology in C57BL/6 mice but not in B6.Cg-Kit(W-sh) mice, which lack mast cells. During in vitro coculture, A/WSN/33 caused mast cells to release histamine, secrete cytokines and chemokines, and produce leukotrienes. Moreover, when mast cells were infected with IAV, the virus did not replicate within mast cells. Importantly, human H1N1, H3N2, and influenza B virus isolates also could activate mast cells in vitro. Mast cell production of cytokines and chemokines occurs in a RIG-I/MAVS-dependent mechanism; in contrast, histamine production occurred through a RIG-I/MAVS-independent mechanism. Our data highlight that, following IAV infection, the response of mast cells is controlled by multiple receptors. In conclusion, we identified a unique inflammatory cascade activated during IAV infection that could potentially be targeted to limit morbidity following IAV infection.

Figure 1. B6.Cg-Kit^W-sh is less sensitive to A/WSN/33

Age-matched C57BL/6 (B6) or B6.Cg-Kit^W-sh (W^sh) mice were infected nasally with 1500 PFU of A/WSN/33 (WSN). (A) Body weights were measured daily. Each graph is normalized to each mouse’s starting body weight. (B) Damage to the lung was assessed by measuring the total protein levels in the BALF at the indicated time points. (C) Total leukocyte recruitment to the BALF was measured day 7 post infection. (D) Lung viral titers were determined at indicated time points post-infection by plaque assay on MDCK cells. Dotted line represents the limit of detection of the plaque assay (LOD = 2 PFU/mL). N.D. = none detected. (E) Formalin fixed lungs were paraffin embedded, then sectioned and stained with H&E for analysis by microscopy. Pictures of representative lung sections were taken using 10x magnification objectives from naïve and A/WSN/33 infected (7d or 10d) C57BL/6 and W^sh mice. Data are representative of 2–4 independent experiments consisting of 4–8 mice per group. Statistically significant differences were determined using a Mann-Whitney U-test (*p<0.05).

Figure 2. B6.Cg-Kit^W-sh mice reconstituted with BMCMC were susceptible to WSN

(A) Age-matched C57BL/6 (B6), B6.Cg-Kit^W-sh (W^sh), or B6.Cg-Kit^W-sh mice reconstituted with 3×10⁶ BMCMC 10 weeks prior were infected nasally with 1500 PFU of A/WSN/33 (WSN). Body weights were measured daily. († = mouse had to be euthanized). The graph is normalized to each mouse’s starting body weight. (B) Age-matched C57BL/6 mice and B6.Cg-Kit^W-sh mice, reconstituted with either 1×10⁶ or 5×10⁶ BMCMC, were infected nasally 10 weeks later with 1500 PFU of A/WSN/33 (WSN) and total lymphocyte, neutrophil, and macrophage counts in the BALF was measured 7 days after infection. Data are representative of two independent experiments consisting of 4–8 mice per group. Statistically significant differences were determined using a Mann-Whitney U-test (A) or one-way ANOVA (B) (*p<0.05).

Figure 3. B6.Cg-Kit^W-sh mice had less detection of cytokines in the lung during A/WSN/33 infection

Age-matchedC57BL/6 (B6) or B6.Cg-Kit^W-sh (W^sh) mice were infected nasally with 1500 PFU of A/WSN/33 (WSN). Cytokine and chemokine levels in the BALF were assessed 7 days after infection using Milliplex^™ multiplex assays. Statistically significant differences were determined using a Mann-Whitney U-test (*p<0.05). Data are representative of two independent experiments consisting of 4–6 mice per group.

Figure 4. In vitro BMCMC activation with A/WSN/33 results in mast cell activation

BMCMC were derived by culturing total bone marrow with IL-3 for 5 weeks, supplementing with stem cell factor for the last 2 weeks. 2.5×10⁵ FcRε1⁺ and CD117⁺ mast cells were treated with media, A/WSN/33 (WSN), or a positive control of calcium ionophore (Ca²⁺) at 40nM or LPS at 5μg/mL. (A) Histamine levels were measured by an EIA at the indicate time points. As a control histamine levels from unstimulated BMCMC were assayed after 6 hours of culture. Cytokine/chemokine (B) and LTB₄ (C) secretion were measured 6 hours after stimulation by either Milliplex^™ multiplex analysis or an EIA, respectively. Each virus was added at an MOI of 1. Data are representative of 2–4 independent experiments. N.D. = none detected.

Figure 5. Human influenza virus isolates can activate mast cells

BMCMC were derived by culturing total bone marrow with IL-3 for 5 weeks, supplementing with stem cell factor for the last 2 weeks. 2.5×10⁵ FcRε1⁺ and CD117⁺ mast cells were treated with media or 50μL of each virus for 6 hours at which time supernatants were collected and analyzed for cytokine and chemokine analysis with Milliplex^™ multiplex analysis (A–D). Expression of IL-6 is shown for each virus: Mouse adapted strains of IAV H1N1: A/WSN/33 and A/PR/8/34 (EID50/ml log10=8.8); Human derived isolates of IAV H1N1 which are either seasonal or pandemic viruses: A/Solomon Island/03/2006 (EID₅₀/ml log₁₀=7.2), A/FtMonmouth/1/47 (EID₅₀/ml log₁₀=7.4), A/CA/04/2009 (EID₅₀/ml log₁₀=6.9), A/CA/07/2009 (EID₅₀/ml log₁₀=8.4), A/Netherlands/2290/2009 (EID₅₀/ml log₁₀=7.4), and A/WI/629-D02452/2009 (EID₅₀/ml log₁₀=7.2); Human derived isolates of IAV H3N2: A/Brisbane/10/2007 (EID₅₀/ml log₁₀=8.4), A/Sydney/5/97 (EID₅₀/ml log₁₀=7.9), and A/Hong Kong/8/68 (EID₅₀/ml log₁₀=8.4); and Human derived isolates of influenza B virus B/FL/4/2006 (EID₅₀/ml log₁₀=8.4). Similar results were observed with CCL2 and CCL4 (data not shown). Data are representative of two independent experiments. All IAV isolates, except A/PR/8/34, induced an IL-6 response which was statistically significantly different than the medium control, as determined by a one-way ANOVA (p<0.05).

Figure 6. Mast cells are infected by A/WSN/33

BMCMC were derived by culturing total bone marrow with IL-3 for 5 weeks, supplementing with stem cell factor for the last 2 weeks. 2.5×10⁵ FcRε1⁺ and CD117⁺ mast cells were treated with medium, A/WSN/33, or A/PR/8/34. Each virus was added at an MOI of 1. BMCMC were harvested after 5 hours. BMCMC were fixed, permeabilized, and stained for intracellular NS-1 using mAb NS1-1A7 for 30 minutes. BMCMC were then stained with PE-labeled anti-mouse IgG F(ab′) fragments. Representative histograms are shown for 2 independent experiments. Shaded histogram = PE-labeled anti-mouse IgG F(ab′) fragments alone; Black histogram = media treated BMCMC; Blue histogram = A/PR/8/34 infected BMCMC; Red histogram = A/WSN/33 infected BMCMC.

Figure 7. Mast cell activation by A/WSN/33 is dependent on RIG-I

BMCMC were generated by culturing total bone marrow from either C57BL/6 or RIG-I^−/− mice with IL-3 for 5 weeks, supplementing with stem cell factor for the last 2 weeks. 2.5×10⁵ FcRε1⁺ and CD117⁺ mast cells were treated with media or A/WSN/33 (WSN). Virus was added at an MOI of 1. Six hours after inoculation IL-6 (A), LTB4 (B), and histamine (C) levels were measured by Milliplex^™ multiplex analysis or EIA assays. Similar data was observed for CCL2 and CCL4 expression (data not shown). Statistically significant differences were determined using a one-way ANOVA (***p<0.001; *p<0.05; NS=not significant). Data are representative of 2–4 independent experiments. White bars = Media treated; Black bars = A/WSN/33 treated.

Figure 8. Mast cell activation is partially dependent on STING and STAT6

BMCMC were generated by culturing total bone marrow from either C57BL/6 or CARD9^−/− mice (A), C57BL/6 or STING^−/− mice (B), or C57BL/6 or STAT6^−/− mice (C) with IL-3 for 5 weeks, supplementing with stem cell factor for the last 2 weeks. 2.5×10⁵ FcRε1⁺ and CD117⁺ mast cells were treated with media or A/WSN/33 (WSN). Virus was added at an MOI of 1. Six hours after inoculation IL-6 levels were measured by Milliplex^™ multiplex analysis. Similar data was observed for CCL2 and CCL4 expression (data not shown). Statistically significant differences were determined using a one-way ANOVA (***p<0.001; **p<0.01; NS=not significant). Data are representative of 2–4 independent experiments. White bars = Media treated; Black bars = A/WSN/33 treated.