Endothelial cells are central orchestrators of cytokine amplification during influenza virus infection

Abstract

Cytokine storm during viral infection is a prospective predictor of morbidity and mortality, yet the cellular sources remain undefined. Here, using genetic and chemical tools to probe functions of the S1P(1) receptor, we elucidate cellular and signaling mechanisms that are important in initiating cytokine storm. Whereas S1P(1) receptor is expressed on endothelial cells and lymphocytes within lung tissue, S1P(1) agonism suppresses cytokines and innate immune cell recruitment in wild-type and lymphocyte-deficient mice, identifying endothelial cells as central regulators of cytokine storm. Furthermore, our data reveal immune cell infiltration and cytokine production as distinct events that are both orchestrated by endothelial cells. Moreover, we demonstrate that suppression of early innate immune responses through S1P(1) signaling results in reduced mortality during infection with a human pathogenic strain of influenza virus. Modulation of endothelium with a specific agonist suggests that diseases in which amplification of cytokine storm is a significant pathological component could be chemically tractable.

Graphical abstract

Figure 1

S1P₁ Receptor Agonism Suppresses Early Proinflammatory Cytokine and Chemokine Production and Innate Immune Cell Recruitment during Influenza Virus Infection Mice were infected with 1 × 10⁴ PFU WSN influenza virus, and vehicle (water), AAL-R (0.2 mg/kg) (1 hr postinfection), or CYM-5442 (2 mg/kg) (1,13, 25, and 37 hr postinfection) were administered i.t. to mice. (A) Proinflammatory cytokines and chemokines were measured 48 hr postinfection in BALF by ELISA. (B) Total numbers of innate immune cells were quantified from collagenase-digested lungs by flow cytometry at 48 hr postinfluenza virus infection. (C) Histograms (left) and mean fluorescent intensity (MFI) (right) of CD69 expression on macrophages was quantified on vehicle, AAL-R-, or CYM-5442-treated mice 48 hr postinfluenza virus infection by flow cytometry staining. (D) Histograms (left) and MFI (right) of NK cell CD69 expression quantified as in (C). Data represent average ± SEM from four to mice per group. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005. Results are representative of greater than six independent experiments. See also Figure S1 and Figure S2.

Figure S1

S1P₁ Agonism Suppressed Multiple Proinflammatory Cytokines/Chemokines following Influenza Virus Infection, Related to Figures 1, Figure 5, Figure 6, and Figure 7 (A) Pro-inflammatory cytokines and chemokines were measured in C57BL/6J mice 48 hr post-infection in BALF by ELISA. (B) RAG2^−/− mice were infected and treated with either vehicle or CYM-5442 as done in Figure 1 and pro-inflammatory cytokines and chemokines were measured in BALF by ELISA 48 hr post-influenza infection. (C) Pro-inflammatory cytokines and chemokines were measured in BALF 48 hr post-infection by ELISA in mice treated with vehicle, CYM-5442 or CYM-5442 + rmCCL2. (D) Pro-inflammatory cytokines and chemokines were measured 48 hr post-infection by ELISA in BALF in mice treated with vehicle or CYM-5442 in the presence of either anti-CD11b or isotype control antibody (M7/80) (0.5mg/mouse) 0 and 24 hr post-infection. (E) C57BL/6J or IFN- α/β receptor-deficient mice were infected with 1 × 10⁴ PFU of influenza virus and treated with either vehicle or CYM-5442 and pro-inflammatory cytokines and chemokines were measured 48 hr post-infection by ELISA in BALF fluid. Data represent average ± SEM from 5 mice/group. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005. Results are representative of 2 or more independent experiments.

Figure S2

AAL-R or CYM-5442 Treatment Did Not Alter Viral Titers in the Lung following Influenza Virus Infection, Related to Figure 1 Mice were infected with 1 × 10⁴ PFU WSN influenza virus and either Vehicle (water), AAL-R (0.2mg/kg) (1 hr post-infection) or CYM5442 (2mg/kg) (1,13, 25 and 37 hr-post-infection) were administered i.t. to mice. Viral titers were determined at the indicated times post-infection by plaque assay on MDCK cells. The dotted line indicates the limit of detection of the assay. Data represent average ± SEM from 5 mice/group and represent 2 independent experiments.

Figure 2

S1P₁ Receptor Agonism Suppresses Early Proinflammatory Cytokine and Chemokine Production and Recruitment of Activated Innate Immune Cells during Human Pathogenic H1N1:2009 Swine Influenza Virus Infection (A–D) Mice were infected with 1 × 10⁵ PFU A/Wisconsin/WSLH34939/09 influenza virus, and either vehicle (water), CYM-5442 (2 mg/kg) (1, 13, 25, and 37 hr postinfection), or RP-002 (2 mg/kg on 1 and 25 hr postinfection) were administered i.t. to mice. Proinflammatory cytokines and chemokines were measured 48 hr postinfection in BALF by ELISA in either CYM-5442- (A) or RP-002-treated mice (C). Total numbers of innate immune cells were quantified from collagenase-digested lungs by flow cytometry at 48 hr postinfluenza virus infection in mice treated with either CYM-5442 (B) or RP-002 (D). Data represent average ± SEM from four to five mice per group. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005. Results are representative of two independent experiments. See also Figure S3.

Figure S3

RP-002 Treatment Does Not Alter Viral Titers in the Lung following Human Pathogenic Influenza Virus Infection, Related to Figure 2 and Figure 3 Mice were infected with 1 × 10⁵ PFU A/Wisconsin/WSLH34939/09 influenza virus and either Vehicle (water) or RP-002 (2mg/kg on 1 and 25 hr post-infection) were administered i.t. to mice. Viral titers were determined at the indicated times post-infection by plaque assay on MDCK cells. Data represent average ± SEM from 5 mice/group and represent 2 independent experiments.

Figure 3

Oral Administration of an S1P₁ Agonist Suppresses Early Innate Cytokine and Chemokine Production and Significantly Improves Survival to Lethal Infection with H1N1 2009 Swine Influenza Virus Mice were infected with 2 × 10⁵ PFU A/Wisconsin/WSLH34939/09 and treated by gavage with either vehicle (water) or RP-002 (6 mg/kg on 1 and 25 hr postinfection for cytokine and cell recruitment assays at 48 hr, with a third dose administered at 49 hr for the 16 day survival experiment). (A) Levels of proinflammatory cytokines and chemokines were analyzed in the BALF 48 hr postinfection by ELISA. (B) Total numbers of activated (CD69⁺) macrophages/monocytes and NK cells were detected in collagenase-digested lungs 48 hr postinfection by flow cytometry. (C) Mice were monitored daily for survival for 16 days postinfection. For (A) and (B), data represent the average ± SEM of four to five mice per group, whereas (C) had ten mice per group. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005. Data are representative of three independent experiments. See also Figure S3.

Figure 4

S1P₁ Receptor Is Expressed on Pulmonary Endothelium and Lymphocytes, but Not on Pulmonary Epithelial Cells (A–C) Flow cytometry histograms showing eGFP fluorescence from heterozygous S1P₁-eGFP on lung endothelial and epithelial cells. (A) CD4 and CD8 T cells and B cells. (B) Macrophages, neutrophils, dendritic cells, and NK cells (C) prior to influenza virus infection. (D) Relative expression of S1P₁-eGFP on lung cell populations before or 48 hr after influenza virus infection. The numbers in the upper-right corner of each plot represent the relative eGFP expression as calculated by dividing the MFI of eGFP on S1P₁-eGFP transgenic mice over eGFP MFI of C57BL/6J littermate controls. Data represent average ± SD from two to five mice per group. Results are representative of three independent experiments. See also Figure S4.

Figure S4

S1P₁ Receptor Is Expressed on Pulmonary Lymphocytes and Endothelial Cells, Related to Figure 4 (A) Western blot of FACS purified lung cell populations from uninfected and 48 hr post-influenza virus infected S1P1-eGFP mice. (B) S1P₁ receptor expression is not altered after CYM-5442 treatment. Flow cytometry histograms showing eGFP fluorescence on lung endothelial, epithelial cells, CD4, CD8 T cells and B cells in S1P₁-eGFP mice infected with influenza virus and treated with or without CYM-5442.

Figure 5

S1P₁ Receptor Agonism Suppresses Early Proinflammatory Immune Responses Independently of Lymphocytes Rag2^−/− mice were infected and treated with either vehicle or CYM-5442, as in Figure 1. (A) Proinflammatory cytokines and chemokines were measured in BALF by ELISA 48 hr postinfluenza infection. (B) Total numbers of innate immune cell recruitment were quantified from collagenase-digested lungs by flow cytometry at 48 hr postinfluenza virus infection. Data represent average ± SEM from five mice per group. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005. Results are representative of two independent experiments. See also Figure S1 and Figure S5.

Figure S5

S1P₁ Agonism Suppresses Innate Immune Cell Activation following Influenza Infection in Lymphocyte-Deficient Mice, Related to Figure 5 Mean fluorescence intensity of CD69 expression was quantified on vehicle or CYM-5442 treated macrophages/monocytes or NK cells in Rag2^−/− mice 48 hr post-influenza virus infection by flow cytometry staining. Data represent average ± SEM representing 5 mice/group. ^∗p < 0.05; and ^∗∗∗p < 0.0005. Results are representative of two independent experiments.

Figure 6

S1P₁ Receptor Agonism Actively Suppresses Recruitment of Innate Immune Cells through Downregulation of Chemokine Expression on Lung Endothelial Cells (A) Relative mRNA expression of chemokines from purified lung endothelial cell populations at 36 hr postinfluenza virus infection. (B) Protein expression of CCL2 and CXCL10 measured by ELISA on FACS-purified vascular and lymphatic lung endothelial cells 48 hr postinfluenza virus infection. (C) LysM-GFP mice were infected with 1 × 10⁴ PFU of influenza virus. At 2 days later, bone marrow cells were harvested from infected LysM-GFP mice and transferred into C57BL/6J mice that were either left uninfected or infected with 1 × 10⁴ PFU of influenza virus 2 hr prior. Mice receiving bone marrow cells from LysM-GFP-positive mice were treated with either vehicle or CYM-5442 (2 mg/kg 1, 13, 25, and 37 hr postinfection), and at 48 hr postinfection, total lung cells were harvested from lung homogenates and the total numbers of GFP-expressing granulocytes (CD11b⁺, Ly6G⁺) and macrophages/monocytes (CD11b⁺F480⁺) were quantified. (D) Total number of LysM-GFP-positive granulocytes and macrophages/monocytes per lung of uninfected or infected mice treated with either vehicle or CYM-5442. (E) Exogenous intratracheal administration of chemokine restores recruitment of innate immune cells. However, it does not resurrect cytokine/chemokine production after CYM-5442 treatment. Mice were infected with 1 × 10⁴ PFU WSN influenza virus, and either vehicle (water) or CYM-5442 (2 mg/kg 1, 13, 25, and 37 hr postinfection) were administered to mice i.t. Following administration of CYM-5442, recombinant mouse CCL2 was administered (50 μg) i.t. directly into the lungs. The bar graph shows the total numbers of macrophages/monocytes, neutrophils, and NK cells in the lung 48 hr postinfluenza virus infection. (F) Proinflammatory cytokines and chemokines were measured in BALF 48 hr postinfection by ELISA in mice treated with vehicle, CYM-5442, or CYM-5442 + rmCCL2, as in (E). Data represent average ± SEM from five mice/group. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005. Results are representative of two independent experiments. See also Figure S1 and Figure S6.

Figure S6

S1P₁ Agonism Does Not Alter Levels of Adhesion Molecule Expression on Pulmonary Endothelial Cells, Related to Figure 6 Expression of cellular adhesion molecules on pulmonary endothelial cells 48 hr post influenza virus infection in C57BL/6J mice treated with either vehicle or CYM-5442. Data represent average ± SEM from 5 mice/group and represents 2 independent experiments.

Figure S7

Inhibition of Inflammatory Macrophage/Monocyte Recruitment Does Not Alter Cytokine/Chemokine Responses following Influenza Virus Infection, Related to Figure 7 C57BL/6J or CCR2 receptor deficient mice were infected with 1 × 10⁴ PFU of influenza virus and treated with either vehicle or CYM-5442 (2mg/kg 1, 13, 25, and 36 hr post infection. (A and B) (A) Total numbers of innate immune cells were quantified from lung digests by flow cytometry at 48 hr post-influenza virus infection and (B) pro-inflammatory cytokines and chemokines were measured 48 hr post-infection by ELISA in BALF fluid. Data represent average ± SEM from 5 mice/group. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005. Results are representative of two independent experiments.

Figure 7

Proinflammatory Cytokine Responses Are Independent of Innate Immune Cell Recruitment and Dependent on Type I Interferon Signaling (A) Total numbers of innate immune cells were quantified from lung digests by flow cytometry at 48 hr postinfluenza virus infection in mice treated 1 hr postinfection with vehicle or CYM-5442 in the presence of either anti-CD11b or isotype control antibody (M7/80) (0.5 mg/mouse) 0 and 24 hr postinfection. (B) Proinflammatory cytokines and chemokines were measured 48 hr postinfection by ELISA in BALF in mice treated as in (A). (C) C57BL/6J or IFN αβ receptor-deficient mice were infected with 1 × 10⁴ PFU of influenza virus and treated with either vehicle or CYM-5442 (2 mg/kg 1, 13, 25, and 36 hr postinfection. Total numbers of innate immune cells were quantified from lung digests by flow cytometry at 48 hr postinfluenza virus infection, and (D) proinflammatory cytokines and chemokines were measured 48 hr postinfection by ELISA in BALF fluid. Data represent average ± SEM from five mice per group. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005. Results are representative of two independent experiments. See also Figure S1 and Figure S7.