Commensal bacteria calibrate the activation threshold of innate antiviral immunity

Abstract

Signals from commensal bacteria can influence immune cell development and susceptibility to infectious or inflammatory diseases. However, the mechanisms by which commensal bacteria regulate protective immunity after exposure to systemic pathogens remain poorly understood. Here, we demonstrate that antibiotic-treated (ABX) mice exhibit impaired innate and adaptive antiviral immune responses and substantially delayed viral clearance after exposure to systemic LCMV or mucosal influenza virus. Furthermore, ABX mice exhibited severe bronchiole epithelial degeneration and increased host mortality after influenza virus infection. Genome-wide transcriptional profiling of macrophages isolated from ABX mice revealed decreased expression of genes associated with antiviral immunity. Moreover, macrophages from ABX mice exhibited defective responses to type I and type II IFNs and impaired capacity to limit viral replication. Collectively, these data indicate that commensal-derived signals provide tonic immune stimulation that establishes the activation threshold of the innate immune system required for optimal antiviral immunity.

Figure 1. Systemic LCMV T1b Infection Results in Delayed Viral Clearance and an Impaired LCMV-Specific CD8⁺ T Cell Response in ABX Mice

(A) Viral titer in the serum of CNV or ABX C57BL/6 mice after LCMV T1b infection (L.o.D., limit of detection). (B and C) LCMV-specific (B) DbGP33 and (C) DbGP276 tetramer⁺ CD8⁺ T cells per 10⁶ peripheral blood mononuclear cells (PBMC) at d7 and d14 p.i. (D) Serial dilution of LCMV-specific IgG antibody titers in serum of CNV or ABX mice at d23 p.i. Naive serum from CNV mice used for baseline. (E) Expression of inhibitory receptors PD-1, 2B4, CD160, LAG-3 on DbGP33 tetramer⁺ CD8⁺ T cells isolated from the spleen of CNV (black line) or ABX (red line) mice. Shaded histograms represent CD44^lo CD8⁺ T cells. Numbers in italics represent mean fluorescence intensity (MFI). (F and G) Splenocytes from d31 infected mice were incubated with GP33 peptide for 5 hr in the presence of BFA and assessed for production of IFN-γ, TNF-α, MIP-1α, CD107a, and IL-2. FACS plots gated on live, CD8α⁺ cells. (G) Proportion of GP33 peptide responsive CD8⁺ T cells producing multiple effector molecules. Data representative of three independent experiments with n = 5 mice per group. Data shown are the mean ± SEM. Serum viral titer statistics determined by two-part t test for each time point. *p < 0.05, **p < 0.01. See also Figure S1.

Figure 2. Alterations in Commensal Bacterial Communities Exacerbate Lung Pathology and Mortality to Influenza Virus

CNV or ABX C57BL/6 mice were infected i.n. with influenza virus PR8-GP33. (A and B) Time course of weight loss (A) and blood oxygen (B) saturation after infection (representative exp. n = 5: † signifies mice below 70% initial weight were sacrificed). (C) Influenza virus genome copies in the lung at d12 p.i. assessed by RT-PCR and displayed as TCID₅₀/gram of lung tissue. (D) Survival curve after PR8-GP33 infection; CNV n = 27, ABX n = 25. (E–J) H&E-stained lung section of CNV (E and F) or ABX (G–J) mice at d12 p.i. Black box and arrows highlight (E and F) epithelial hyperplasia, (G and H) epithelial cell necrosis, (I) cellular debris and exudate in lumen, and (J) loss of bronchiole epithelium (scale bar represents 50 µm in E, G, I, and J; 20 µm in F and H). (K) Disease score of bronchiole epithelial degeneration at d12 p.i. Data representative of five independent experiments with n = 5–6 mice per group. Survival statistics determined by log rank test. Viral titer statistics determined by two-part t test. *p < 0.05, **p < 0.01, and ***p < 0.001. Data shown are mean ± SEM. See also Figure S2.

Figure 3. ABX Mice Have a Diminished Influenza Virus-Specific Adaptive Immune Response

(A and B) Total number of influenza virus-specific DbGP33 tetramer⁺ CD8⁺ T cells isolated from the (A) BAL or (B) lung parenchyma at d7 p.i. (C) DbNP366 tetramer⁺ CD8⁺ T cells isolated from the lung parenchyma at d7 p.i. (D) PR8-specific IgM and IgG titers in the serum at d12 p.i. (E) Phenotypic profile of DbGP33 tetramer⁺ CD8⁺ T cells isolated from the lung of CNV (solid line) or ABX (dotted line) mice at d7, d10, and d12 p.i. Gray shaded histograms are CD44^lo CD8⁺ T cells isolated from the lung. Numbers in italics represent MFI. Data representative of three independent experiments with n = 4–5 mice per group. *p < 0.05 and **p < 0.01. Data shown are mean ± SEM. See also Figure S3.

Figure 4. Innate Antiviral Immune Response Is Diminished in ABX Mice after Influenza Virus or LCMV Infection

(A) Total numbers of alveolar macrophages, inflammatory monocytes, neutrophils and plasmacytoid dendritic cells isolated from BAL at d3 p.i. (B) Total numbers of dendritic cells (DCs) isolated from the lung parenchyma and mediastinal lymph node (medLN). (C) Expression of CD86 and CD40 on naive or d3 p.i. DCs isolated from the lung and medLN of CNV or ABX mice. (D) Fold induction of antiviral defense gene expression in the lung at d3 post-influenza virus infection relative to lung of naive CNV mice as assessed by RT-PCR. (E) Fold induction of antiviral defense genes in the spleen 12 hr after LCMV (T1b) infection relative to spleen of naive CNV mice as assessed by RT-PCR. (F) IFN-β levels in the serum at 12 hr post LCMV infection as detected by ELISA. (G) Expression of MHC-I and CD86 on peritoneal macrophages from CNV or ABX mice 24 hr post LCMV infection. Data representative of two or more independent experiments with n = 3–5 mice per group. *p < 0.05, **p < 0.01. Data shown are mean ± SEM. See also Figure S4.

Figure 5. Naive Macrophages from ABX Mice Have an Attenuated Antiviral Defense Gene Profile

RNA was extracted from sort-purified peritoneal macrophages isolated from naive CNV or ABX mice. Extracted RNA was hybridized to an Affymetrix GeneChip microarray to assess gene expression. (A) Frequency and total number of elevated genes in CNV macrophages compared to macrophages isolated from ABX mice. (B) Highly enriched biological pathways and functions found within the subset of elevated genes from CNV macrophages as assessed by Ingenunity pathways analysis. Red bars indicate the percent of genes in a pathway upregulated in macrophages isolated from CNV mice. Yellow line indicates p value calculated by Fisher’s exact test. (C) Heat map of key antiviral defense genes in macrophages isolated from CNV or ABX mice. Red, high expression; blue, low expression. See also Figures S5 and S6.

Figure 6. Macrophages from Naive ABX Mice Have a Diminished Ability to Respond to IFN Stimulation and Viral Infection In Vitro

(A–D) Peritoneal Macrophages isolated from CNV or ABX mice were stimulated with IFN-γ or IFN-β in vitro. Histograms of STAT1 phosphorylation in macrophages after (A) IFN-γ stimulation (0.4, 4, 20, 200ng/mL) or (C) IFN-β stimulation (10³ units/mL for 5, 10, and 15 min). MFI of pSTAT1 in macrophages after (B) IFN-γ or (D) IFN-β stimulation. (E and F) Peritoneal macrophages sorted from naive CNV or ABX mice were infected in vitro with (E) influenza virus (X31-GP33, MOI of5)or(F) LCMV (cl-13 strain, MOI of 0.2). (E) Induction of antiviral defense genes in macrophages at 6 and 24 hr p.i. as assessed by RT-PCR. (F) LCMV viral titers in supernatant at 24–96 hr p.i. Data representative of two or more independent experiments with n = 3–5 mice per group. *p < 0.05, **p < 0.01, and ***p < 0.001. Data shown are mean ± SEM. See also Figure S7.

Figure 7. In Vivo Antiviral Macrophage Response Is Impaired in ABX Mice after LCMV or Influenza Virus Infection

(A and B) CNV or ABX mice were inoculated with LCMV (T1b) or PBS i.v. and (A) splenocytes at 6 hr p.i. or (B) PECs at 12 hr p.i. were immediately fixed to preserve the in vivo STAT1 phosphorylation status of macrophages. (C) CNV or ABX mice were infected with influenza virus (PR8-GP33). At d3 p.i., alveolar macrophages were sorted from the BAL and in vivo induction of antiviral defense genes was assessed by RT-PCR. Gene expression displayed as fold induction over naive alveolar macrophages from CNV mice. Data representative of two independent experiments with n = 3–5 mice per group. (D–F) CNV or ABX mice were infected with influenza virus (PR8-GP33). Mice received 30 µg of poly I:C (ABX+pIC group) or PBS (CNV & ABX group) i.n. at d −1 and 100 µg of poly I:C or PBS i.p. at d3. Weight loss (D) and blood oxygen (E) saturation after infection (representative exp. n = 4–6: † signifies mice below 70% initial weight were sacrificed). Weight loss statistics determined by two-way ANOVA. (F) Survival curve after influenza virus infection. Survival curve is a combination of two independent experiments. CNV n = 10, ABX n = 8, ABX+pIC n = 12. Survival statistics determined by log rank test. *p < 0.05, **p < 0.01, and ***p < 0.001. Data shown are mean ± SEM.