Mapping the innate signaling cascade essential for cytokine storm during influenza virus infection

Abstract

During pathogenic influenza virus infection, robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced tissue destruction all contribute to morbidity and mortality. Earlier we reported that modulation of sphingosine-1-phosphate-1 receptor (S1P1R) signaling provided a chemically tractable approach for the effective blunting of cytokine storm, leading to the improvement of clinical and survival outcomes. Here, we show that S1P1R agonist treatment suppresses global cytokine amplification. Importantly, S1P1R agonist treatment was able to blunt cytokine/chemokine production and innate immune cell recruitment in the lung independently of endosomal and cytosolic innate sensing pathways. S1P1R signaling suppression of cytokine amplification was independent of multiple innate signaling adaptor pathways for myeloid differentiation primary response gene 88 (MyD88) and IFN-β promoter stimulator-1 signaling, indicating a common pathway inhibition of cytokine storm. We identify the MyD88 adaptor molecule as responsible for the majority of cytokine amplification observed following influenza virus challenge.

Keywords: pathology; pulmonary.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

S1P1R agonism blunts innate cellular and cytokine responses independently of TLR3 or -7 signaling. TLR3−/− (A) or TLR7−/− (B) mice were infected with 1 × 104 PFU WSN influenza virus and either vehicle (water) or CYM5442 (2 mg/kg) were administered intratracheally to mice. Proinflammatory cytokines and chemokines were measured 48 h postinfection in BALF by ELISA. Total numbers of activate macrophages/monocytes and NK cells (C and E) were quantified from collagenase-digested lungs at 48 h postinfluenza virus infection. (D and F) Total numbers of neutrophils isolated from collagenase-digested lungs. *P < 0.05, **P < 0.01, ***P < 0.005. Results are representative of two to three independent experiments and five mice per group.

Fig. 2.

S1P1R agonist inhibition of influenza virus induced cytokine amplification is independent of endosomal and cytosolic innate sensing pathways. The 3d (A) or IPS-1−/− (B) mice were infected with 1 × 104 PFU WSN influenza virus and either vehicle (water) or CYM5442 (2 mg/kg) were administered intratracheally to mice. Proinflammatory cytokines and chemokines were measured 48 h postinfection in BALF by ELISA. (C) Total numbers of activated macrophages/monocytes, NK cells, and neutrophils were quantified from collagenase-digested lungs at 48 h postinfluenza virus infection in 3d mice. Percentage (D) and total numbers (E) of CD69 expressing macrophages in the lung of vehicle or CYM-5442 treated IPS-1+/+ or IPS-1−/− mice 48 h postinfluenza virus infection. (F) Total numbers of neutrophils in the lungs of IPS-1+/+ or IPS-1−/− mice 48 h postinfluenza virus infection. *P < 0.05, **P < 0.01, ***P < 0.005. Results are representative of two to three independent experiments and five mice per group.

Fig. 3.

IL-1R signaling is required for cytokine amplification following influenza virus infection. (A) IL-1R−/− or IL-1R+/+ control mice were infected with 1 × 104 PFU WSN influenza virus and proinflammatory cytokines and chemokines were measured 48 h postinfection in BALF by ELISA. (B) Bone marrow chimeras between IL-1R+/+ and IL-1R−/− mice by injection of either IL-1R+/+ bone marrow cells into lethally irradiated IL-1R−/− mice or vice versa. IL-1R+/+ bone marrow cells injected into irradiated IL-1R+/+ mice and IL-1R−/− bone marrow cells into irradiated IL-1R−/− mice as controls. Chimeric mice were infected with 1 × 104 PFU WSN influenza virus and proinflammatory cytokines and chemokines were measured 48 h postinfection in BALF by ELISA. (C) IL-1R−/− or IL-1R+/+ control mice were infected with 1 × 104 PFU WSN influenza virus and either vehicle (water) or CYM5442 (2 mg/kg) were administered intratracheally to mice. Proinflammatory cytokines and chemokines were measured 48 h postinfection in BALF by ELISA. *P < 0.05, **P < 0.01, ***P < 0.005. Results are representative of two to three independent experiments and five mice per group.

Fig. 4.

MyD88/TRIF signaling is required for cytokine amplification following influenza virus infection. Wild-type or MyD88−/−/TRIF−/− double-knockout mice were infected with 1 × 104 PFU WSN influenza virus and either vehicle (water) or CYM5442 (2 mg/kg) were administered intratracheally to mice. (A) Proinflammatory cytokines and chemokines were measured 48 h postinfection in BALF by ELISA. Percentage (B) and total numbers (C) of activated macrophages/monocytes or (D) NK cells were quantified from collagenase-digested lungs at 48 h postinfluenza virus infection in MyD88−/−/TRIF−/− mice. *P < 0.05, **P < 0.01, ***P < 0.005. Results are representative of two independent experiments and five mice per group.

Fig. 5.

MyD88 is the predominant signaling pathway required for cytokine amplification following influenza virus infection. Wild-type, MyD88−/− mice (A and B) were infected with 1 × 104 PFU WSN influenza virus and either treated with vehicle or CYM-5442 (B) and proinflammatory cytokines and chemokines were measured 48 h postinfection in BALF by ELISA. (C) Total numbers of macrophages/monocytes and neutrophils were quantified from collagenase-digested lungs at 48 h postinfluenza virus infection in MyD88−/− mice. (D) Percentage of CD69 expressing macrophages in the lung of MyD88+/+ or MyD88−/− (vehicle or CYM-5442 treated) mice 48 h postinfluenza virus infection. *P < 0.05, **P < 0.01, ***P < 0.005. Results are representative of two independent experiments and five mice per group.