Modulation of paired immunoglobulin-like type 2 receptor signaling alters the host response to Staphylococcus aureus-induced pneumonia

Abstract

Paired immunoglobulin-like type 2 receptors (PILRs) inhibitory PILRalpha and activating PILRbeta are predominantly expressed on myeloid cells. Their functions in host defense and inflammation are largely unknown, and in this study, we evaluated their roles in an acute Staphylococcus aureus pneumonia model. Compared to their respective controls, Pilrb(-/-) mice or mice in which PILRalpha was activated with an agonistic antibody showed improved clearance of pulmonary staphylococci and improved survival. These mice had reduced serum or bronchoalveolar lavage fluid levels of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and IL-6 and elevated levels of gamma interferon (IFN-gamma), IL-12, and IL-10. In contrast, mice in which PILRbeta was activated had increased lung bacterial burdens and higher mortality coupled with an intense proinflammatory response with highly elevated levels of IL-1beta, TNF-alpha, and IL-6. Treatment groups with reduced bacterial burdens had higher levels of Keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), and MIP-1alpha in bronchoalveolar lavage fluid and an increased influx of neutrophils and macrophages to the lungs. Consistent with our in vivo findings, bone marrow-derived macrophages from Pilrb(-/-) mice released significantly less IL-1beta and TNF-alpha and more IFN-gamma and IL-12 than did the wild-type macrophages when directly stimulated with heat-killed S. aureus. To our knowledge, this is the first evidence that S. aureus directly interacts with PILRbeta. It provides a mechanism by which manipulating the balance in favor of an inhibitory PILR signal, by activation of PILRalpha or deletion of PILRbeta, helps to control acute S. aureus-mediated pneumonia and attenuate the inflammatory response. These results highlight the importance of PILRs in innate immunity and the control of inflammation.

FIG. 1.

Specificity and agonist activity of anti-PILR antibodies. (A) Anti-PILRβ (DX266) bound only to the mouse mast cell transfectant DT754, which expresses PILRβ, and not to DT755, which expresses PILRα. Conversely, anti-PILRα (DX276) bound only to DT755 and not to DT754. (B) DX266, but not DX276 or the isotype control, triggered degranulation in a concentration-dependent manner in mast cell transfectant DT865, which also expresses only PILRβ. (C) DX276, but not the isotype control, blocked anti-CD200RLa-triggered degranulation in DT866, which expresses only PILRα. OD, optical density; conc, concentration.

FIG. 2.

Expression levels of Dap12, Pilra, and Pilrb in lungs from control and S. aureus-infected mice. Transcription levels of Dap12, Pilra, and Pilrb in lungs from infected and uninfected WT mice were analyzed by qRT-PCR. Infected lungs were removed 48 h postinoculation. Levels are expressed relative to that of ubiquitin mRNA. *, P ≤ 0.05.

FIG. 3.

Effect of anti-PILRα or anti-PILRβ treatment on S. aureus-mediated pneumonia. C57BL/6J mice were challenged intranasally with 1 × 10⁸ CFU of S. aureus followed by an intravenous dose of anti-PILRα, anti-PILRβ, or isotype control antibody 2 h postinfection. Mice were monitored for lung bacterial burden (A), mortality (B), MPO levels in the lung at 6 and 24 h postinfection (C), and serum cytokine levels at 24 and 48 h (D). Data points in panel A represent the bacterial load of lungs harvested from individual mice at 48 h postinfection. Data for each of the panels are representative of at least two individual experiments, with 8 to 12 mice per group in each experiment. *, P ≤ 0.05; **, P ≤ 0.002.

FIG. 4.

Phenotypic characterization of Pilrb^−/− mice. (A) Transcription of Pilra and Pilrb genes was analyzed by qRT-PCR in various organs of WT mice (black bars) and Pilrb^−/− mice (gray bars). An asterisk indicates a lack of Pilrb expression in the knockout mice. (B) Following depletion of red blood cells, the remaining blood cells were stained with anti-PILRβ or anti-PILRα antibodies followed by PE-conjugated secondary antibody, as described in the text. Cells from WT and Pilrb^−/− mice were gated on the granulocyte population as shown in the dot plots. The histograms show cellular expression of PILRα and PILRβ in leukocytes from WT (shaded) and Pilrb^−/− (solid line) mice. The isotype control rIgG1 (anti-hIL-4) is indicated by dashed lines for both WT and Pilrb^−/− cells.

FIG. 5.

Deletion of PILRβ protects mice from S. aureus-mediated pneumonia. C57BL/6J WT and Pilrb^−/− mice were infected intranasally with 1 × 10⁸ CFU of S. aureus. Mice were monitored for lung bacterial burden at 6, 24, and 48 h postinfection (n = 8 mice/group in three different experiments) (A); survival (cumulative data from three experiments) (B); serum cytokine levels (mean ± standard error of the mean [SEM] from three experiments each with five to six mice per group (C); and lung mRNA levels for IL-1β, IFN-γ, and IL-12p40 at 48 h postinfection (D). *, P ≤ 0.05; **, P ≤ 0.002; ***, P ≤ 0.001.

FIG. 6.

Chemokine and cytokine levels in BAL fluid of WT and Pilrb^−/− mice following pulmonary infection with S. aureus. Levels of various cytokines and chemokines in BAL fluid samples collected by lavage at 6, 24, and 48 h postinfection were measured as described in the text. Data are expressed as means ± SEM of five to six mice per time point from at least three different experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG. 7.

Dynamics of neutrophil and macrophage infiltration during acute S. aureus pulmonary infection. (A) Cells isolated from the lungs of naïve and S. aureus-infected WT and Pilrb^−/− mice were divided into two main populations. Cells defined as FSC^loSSC^lo were gated as R2 (primarily comprising the lymphocyte and monocyte populations), and those defined as FSC^mid-hiSSC^mid-hi were gated as R3 (primarily comprising the granulocyte population). The R3 population was significantly increased in infected mice compared to that in naïve mice. (B and D) Plots showing a significant increase in the CD11b⁺Gr1^lo-int (macrophage) population in the Pilrb^−/− mice (white squares) compared to that in the WT mice (gray squares) at 24 h in the R2 gate and at both 24 and 48 h in the R3 gate. (C and E) Plots showing cellular infiltration in R2 and R3 gates, with a significant increase in the CD11b⁺Gr1^+hi (neutrophil) population in Pilrb^−/− mice at 24 h postinfection. The plots are representative of three to four separate experiments with five to six mice per group at each time point. Data are expressed as means ± SEM. *, P ≤ 0.05; **, P ≤ 0.001.

FIG. 8.

Cytokine and chemokine release from bone marrow-derived macrophages stimulated with heat-killed S. aureus. Bone marrow-derived macrophages from WT and Pilrb^−/− mice were stimulated for 6 h or 24 h with heat-killed S. aureus, and the supernatants were assayed for various key cytokines and chemokines. Results represent the means ± standard deviation (SD) of three experiments. *, P ≤ 0.05; **, P ≤ 0.001.