The sst1 resistance locus regulates evasion of type I interferon signaling by Chlamydia pneumoniae as a disease tolerance mechanism

Abstract

The sst1, "supersusceptibility to tuberculosis," locus has previously been shown to be a genetic determinant of host resistance to infection with the intracellular pathogen, Mycobacterium tuberculosis. Chlamydia pneumoniae is an obligate intracellular bacterium associated with community acquired pneumonia, and chronic infection with C. pneumoniae has been linked to asthma and atherosclerosis. C. pneumoniae is a highly adapted pathogen that can productively infect macrophages and inhibit host cell apoptosis. Here we examined the role of sst1 in regulating the host response to infection with C. pneumoniae. Although mice carrying the sst1 susceptible (sst1(S) ) locus were not impaired in their ability to clear the acute infection, they were dramatically less tolerant of the induced immune response, displaying higher clinical scores, more severe lung inflammation, exaggerated macrophage and neutrophil influx, and the development of fibrosis compared to wild type mice. This correlated with increased activated caspase-3 in the lungs of infected sst1(S) mice. Infection of sst1(S) macrophages with C. pneumoniae resulted in a shift in the secreted cytokine profile towards enhanced production of interferon-β and interleukin-10, and induced apoptotic cell death, which was dependent on secretion of interferon-β. Intriguingly macrophages from the sst1(S) mice failed to support normal chlamydial growth, resulting in arrested development and failure of the organism to complete its infectious cycle. We conclude that the sst1 locus regulates a shared macrophage-mediated innate defense mechanism against diverse intracellular bacterial pathogens. Its susceptibility allele leads to upregulation of type I interferon pathway, which, in the context of C. pneumoniae, results in decreased tolerance, but not resistance, to the infection. Further dissection of the relationship between type I interferons and host tolerance during infection with intracellular pathogens may provide identification of biomarkers and novel therapeutic targets.

Figure 1. C. pneumoniae infected B6.C3H-sst1 mice develop diffuse lung inflammation compared to B6 mice.

C57BL/6 (B6) or B6.C3H-sst1 congenic mice were infected with C. pneumoniae as described in the Methods. At day 3 and day 6 post infection, mice were euthanized and lungs removed for histologic analysis. A–D: routine H & E staining for day 3 (A–B) and day 6 (C–D). Black arrow heads indicate areas of patchy inflammatory infiltrate. E–F: CAE staining for detection of neutrophils, indicated by red arrow heads. G–H: immunohistochemistry for the macrophage marker CD68, shown by brown staining. Graphs at the bottom represent quantified data from (I) neutrophil images E–F and (J) macrophage images G–H. Original magnification: A–D, 40×; E–F, 400×; G–H, 100×. Shown above are images from one of four infected mice from each genetic background. The result is a representative of two independent experiments. ***, p≤0.001.

Figure 2. Cytokine production from the lung homogenates of C. pneumoniae infected mice.

C57BL/6 (B6) or B6.C3H-sst1 congenic mice were infected with C. pneumoniae (Cp) as described in the Methods. At day 3 and day 6 post infection, mice were euthanized and lung homogenates prepared for detection of a panel of inflammatory cytokines and chemokines. Shown above are a subset of the cytokines assayed: (A) IL-6, (B) IL-1β, (C) IL-10, (D) IFN-β, (E) IFN-γ and (F) MCP-1. Each data point represents one mouse, and the horizontal bar represents the mean. Significance: *, p<0.05; **, p≤0.01; ***, p≤0.001 infected C57BL/6 vs. B6.C3H-sst1 mice. The result is representative of two independent experiments.

Figure 3. In vitro cytokine production from C. pneumoniae infected BMDM.

BMDMs were prepared from C57BL/6 (B6) or B6.C3H-sst1 congenic mice, as described in the Methods. Cells were infected with C. pneumoniae at an MOI of 3∶1, and supernatant harvested at 24 hpi for assay of the indicated cytokines. (A–D) In some cases, cells were pretreated with IFN-γ for 30 min at the indicated concentrations prior to infection. (E–F) For antibody neutralization, cells were pretreated with indicated neutralizing antibody (anti-IL-10 or anti-IFN-β) or isotype control at a concentration of 250 ng/ml prior to infection or IFN-γ treatment (10 U/ml). Data is shown as the mean ± SEM from triplicate wells. Significance: NS, No significant difference; *, p<0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001. The results are representative of at least 3 independent experiments.

Figure 4. C. pneumoniae induces IFN-β and IL-10 production in BMDM from B6.C3H-sst1 via TBK1/IKKξ dependent pathway and is independent of MyD88.

BMDM were prepared from B6.C3H-sst1 mice as described in the Methods. Prior to infection with C. pneumoniae (Cp) or treatment with LPS, cells were pretreated with IFN-γ (5 U/ml) and the following inhibitors for 30 min at the indicated concentrations; supernatant was harvested at 24 hpi for assay of IL-10 and IFN-β. A–B: TBK1/IKKξ inhibitor BX795 or vehicle control (DMSO); or the PKR inhibitor 2-aminopurine (2-AP) or vehicle control PBS∶Glacial acetic acid at a ratio of 200∶1 (Glacial AA). C–D: MYD88 inhibitor Pepinh-MYD (Pep-MYD), or Pepinh-control (Pep-Ctr) Significance: *, p≤0.05; **, p≤0.01; ***, p≤0.001 compared to corresponding control. The results are representative of 2 independent experiments.

Figure 5. In vitro C. pneumoniae growth.

BMDMs prepared from C57BL/6 (B6) or B6.C3H-sst1 mice were infected with C. pneumoniae at an MOI of 3∶1, in the absence or presence of IFN-γ (5 U/ml). A–B: Cells were lysed at the indicated time points and recovered EBs were quantitatively cultured, as described in the Methods. Each time point was run in triplicate and data is reported as the mean recovered bacteria per well ± SEM. C: Cells were harvested at 69 hpi and processed for electron microscopy, as described in the Methods. Shown above are representative images from triplicate infected wells. Original magnification: 20,000×, scale bars: 1 µm. High magnification images (upper right inset of each panel): 80,000×, scale bars: 500 nm.

Figure 6. Neutralization of IFN-β protects cells from death and enhances C. pneumoniae replication.

BMDMs prepared from C57BL/6 (B6) or B6.C3H-sst1 mice were infected with C. pneumoniae (MOI 3∶1) in the presence or absence of IFN-γ (5 U/ml). The ratio of dead cells to total cell number was calculated using propidium iodide and Hoechst staining, as described in the Methods section, and is reported as the percent cytotoxicity. Where indicated, antibody neutralization was carried out using neutralizing antibody against IL-10 or IFN-β, or isotype control at a concentration of 250 ng/ml prior to infection or IFN-γ treatment. A: Cytotoxicity was determined over time at 24, 48 and 72 hpi in Cp infected BMDM, and is reported as percent cell death. B: Effect of IL-10 and IFN-β neutralization on Cp-induced cytotoxicity at 72 hpi. C: Effect of IL-10 and IFN-β neutralization on Cp growth. Infected BMDM were lysed at 69 hpi, and recovered EBs were quantitatively cultured, as described in the Methods. All data shown above represents the mean ± SEM from triplicate wells, and is representative of at least two independent experiments. Significance: *, p<0.05; **, p≤0.01; ***, p≤0.001.

Figure 7. Neutralization of IFN-β protects cells from apoptosis.

BMDM were prepared from B6.C3H-sst1 mice. Where indicated (panel a, c and d), cells were primed with IFN-γ (10 U/ml). Cells were incubated with neutralizing Ab against IFN-β (panel d) or isotype control (panel c) at a final concentration of 250 ng/ml 6 hr prior to infection with Cp (MOI 3∶1). At 10 hpi, cells were fixed and stained using the TUNEL assay. A: fluorescent images showing TUNEL positive cells (green); cells are counterstained with DAPI (blue). B: Quantified data from fluorescent images shown in A are graphed as the percent TUNEL positive cells. The small letters (a–d) in image A correspond to the bars shown in graph B below. Significance: ***, p≤0.001.

Figure 8. C. pneumoniae infected B6.C3H-sst1 mice show evidence of increased apoptosis in lung tissue.

C57BL/6 (B6) or B6.C3H-sst1 congenic mice were infected with C. pneumoniae, and at day 3 and day 6 post infection, lungs were removed for processing, as described in Figure 1. Immunohistochemistry was carried out using monoclonal Ab against cleaved caspase-3. Panels A–B, day 3; E–F, day 6. Panels C–D and G–H are taken from the rectangle area in panels A–B, E–F, respectively. Original magnification: A–B, E–F: 100×; C–D, G–H: 400×. No caspase-3 staining in the lung was observed by immunohistochemistry in mock infected mice (data not shown).