Type I interferon signaling regulates the composition of inflammatory infiltrates upon infection with Listeria monocytogenes

Abstract

Type I IFN is key to the immune response to viral pathogens, however its role in bacterial infections is less well understood. Mice lacking the type I IFN receptor (IFNAR-/-) demonstrate enhanced resistance to infection with Listeriamonocytogenes. We have now determined that following infection with Listeria, the composition of innate cells recruited to the peritoneal cavity of IFNAR-/- mice reflects an increase in the frequency of neutrophils and a decrease in monocyte frequency compared to WT controls. These differences in inflammatory infiltrates could not be attributed to distinct bone marrow composition prior to infection or to level of apoptosis. We also observed no differences in neutrophil oxidative burst. However, blocking CXCR2 prevented enhanced neutrophil influx and hampered bacterial clearance. Taken together, these studies highlight a novel mechanism by which type I interferon signaling regulates the immune response to Listeria, through negative regulation of chemokines driving neutrophil recruitment.

Figure 1. IFNAR regulates chemokine/cytokine profile and morphology of cellular infiltrates in the peritoneal cavity upon Lm infection

WT and IFNAR−/− mice were infected with 10⁵ Lm i.p. and at 24h post infection, peritoneal exudates were harvested and separated into fluid and cellular fractions by centrifugation. A) Peritoneal fluid was subjected to dot blot cytokine array analysis and signals from 62 cytokines were quantified based on pixel intensity. Those that were markedly different between WT and IFNAR−/− are featured. Intensity of IFNAR−/− was compared to WT and expressed as fold induction over WT. B) Cytospin preparations of PEC collected at 24 hours post infection following Diffquick staining. Cells with segmented or ring-shaped nuclei were (characteristic of neutrophils) or rounded and uniform (typical of monocytes) were identified at different frequencies in WT vs. IFNAR−/−.

Figure 2. Recruitment of CD11b⁺ and Gr-1⁺ into the peritoneal cavity following Listeria infection is enhanced in the absence of IFNAR

WT and IFNAR−/− mice were infected i.p. with 10⁵ Lm. At 24h post infection, peritoneal lavage was collected and peritoneal exudate cells (PEC) were stained for flow cytometry. A) Total number of CD11b+ cells found prior to and following Lm infection. B) Frequencies of macrophages, neutrophils, monocytes, and other cells in PEC illustrated as pie charts. Macrophages were defined as CD11b^hi, Gr-1⁻, and F4/80⁺, neutrophils as CD11b⁺, Gr-1⁺, Ly6C^lo and monocytes as CD11b⁺, Gr-1⁺, Ly6C^hi. All CD11b+ cells that did not express these markers were labeled as other. Numbers indicate mean frequencies of 5–7 animals per group. C) Representative dot plots of Gr-1 and Ly6C expression on PEC collected from WT and IFNAR−/− mice following Lm infection. Numbers in the gates indicate the percent of the CD11b+ PEC that are either Ly6C^lo or Ly6C^hi indicative of neutrophils or monocytes, respectively. Number of D) Gr-1+ cells in PEC recovered from WT and IFNAR−/− mice at 24h post infection. Data represent mean +/− SD from the indicated number of animals per group- Mock: 5–7 animals per group, infected: 7–9 animals/group. Statistics were calculated using a student’s unpaired t test with Welch’s correction. No significant differences (p< 0.05) in numbers were observed between WT and IFNAR−/− mice prior to infection for any cell type examined.

Figure 3. IFNAR negatively regulates the influx of neutrophils to the peritoneal cavity following Lm infection, over a 72h time course

WT and IFNAR−/− mice were infected i.p. with 10⁵ Lm. At 24, 48, and 72h post infection, peritoneal lavage and spleens were collected, processed, stained for flow cytometry. Neutrophils were defined as CD11b⁺, Gr-1⁺, Ly6C^lo and monocytes as CD11b⁺, Gr-1⁺, Ly6C^hi. A) Number of peritoneal neutrophils; B) Number of splenic neutrophils; C) Number of peritoneal monocytes; and D) Number of splenic monocytes. Data represent mean +/− SD from the indicated number of animals per group- Mock: 4–7 animals per group, infected: 6–10 animals/group. Statistics were calculated using a student’s t test.

Figure 4. The impact of bacterial dose on the relative composition of innate cells recruited upon Lm infection

WT and IFNAR−/− mice either mock treated of infected with Lm i.p. with the indicated dose. Twenty four hours post infection, PEC were collected by lavage and splenocytes were harvested. Cells were stained and flow cytometry was used to assess the expression of Ly6C on Gr-1⁺ recruited cells. A) Number of neutrophils in PEC at the indicated dose of Lm. B) Number of PEC that are monocytes. Data represent mean +/− SEM from the indicated number of animals per group- Mock: 6 animals per group, infected: 5–10 animals/group. Statistics were calculated using a student’s unpaired t test.

Figure 5. WT and IFNAR−/− mice have a similar composition of neutrophils and monocytes in the bone marrow

Bone marrow was harvested from WT and IFNAR−/− mice that were either mock treated or infected for 24 hours with Lm. The cells were then stained and expression of Ly6C on Gr-1⁺ cells was determined by flow cytometry. A) Percent of neutrophils in the bone marrow of WT or IFNAR−/− mice following either mock treatment, or 24 hours Lm infection. B) Percent of monocytes in bone marrow. Data are compiled from 4–6 mice per group.

Figure 6. ROS production by neutrophils from WT vs. IFNAR−/− mice

Bone marrow was harvested from WT and IFNAR−/− mice and neutrophils were enriched with an isolymph density gradient. Neutrophil oxidase function was tested by adding 5mM DHR to neutrophils concurrently stimulated with 100ng PMA or Lm. Twenty minutes post stimulation, DHR fluorescence, as a measure of oxidase activity, was measured by flow cytometry. A) Histograms of DHR fluorescence from WT (left) or IFNAR−/− (right) neutrophils. Gray shaded histograms show background level of untreated cells (NT) or and black lines indicate level after stimulation with Lm. B) DHR fluorescence depicted as a fold increase over control for WT and IFNAR−/− neutrophils stimulated as indicated. Values are compiled from 3 independent experiments. Responses between WT and IFNAR−/− were determined to be not significantly different using a 2-way ANOVA with bonferroni post test.

Figure 7. Enhanced presence of neutrophils observed in IFNAR−/− mice was not due to decreased apoptosis

WT or IFNAR−/− mice were either mock treated or infected with 10⁵ Lm for 24 or 72 hours. At the indicated times post infection, PEC were collected by lavage and stained with Annexin V and 7-AAD to assess levels of apoptosis. A) Representative dot plots of Annexin-V and 7-AAD staining on PEC at 72 hours post infection. Numbers in upper right quadrants represent the percent of PEC that are Annexin-V and 7-AAD double positive indicative of cells in late stage apoptosis. B) Percent of PEC at the indicated timepoint post infection that are Annexin-V and 7-AAD double positive. Data represent mean +/− SEM from 3–6 animals per group.

Figure 8. Inhibition of CXCR2 reduced neutrophil and monocyte infiltrates in the spleen and rendered IFNAR−/− mice more susceptible to Lm infection

WT and IFNAR−/− mice were infected with 10⁴ Lm. Thirty minutes prior to infection, a group of WT and IFNAR−/− mice was injected with 1 mg of antileukinate i.p. to inhibit CXC chemokine activity. Twenty-four hours post infection, splenocytes were harvested and stained. Flow cytometry was used to assess the numbers of neutrophils and monocytes. A) Number of splenic neutrophils from either WT or IFNAR−/− mice in the presence or absence of antileukinate 24 hours post infection. B) Number of splenic monocytes. C) WT or INFAR−/− mice were infected with 10⁴ Lm thirty minutes prior to infection, mice were treated with 1 mg antileukinate i.p. or mock treated. The drug was re-administered every 24 hours. Seventy two hours post infection spleens were homogenized and bacterial burdens were determined. Each symbol represents one experimental animal. Lines indicate mean for that group. Statistics were calculated using and unpaired student’s t test with Welch’s correction.