The absence of Hck, Fgr, and Lyn tyrosine kinases augments lung innate immune responses to Pneumocystis murina

Abstract

Src family tyrosine kinases (SFKs) phosphorylate immunotyrosine activation motifs in the cytoplasmic tail of multiple immunoreceptors, leading to the initiation of cellular effector functions, such as phagocytosis, reactive oxygen species production, and cytokine production. SFKs also play important roles in regulating these responses through the activation of immunotyrosine inhibitory motif-containing inhibitory receptors. As myeloid cells preferentially express the SFKs Hck, Fgr, and Lyn, we questioned the role of these kinases in innate immune responses to Pneumocystis murina. Increased phosphorylation of Hck was readily detectable in alveolar macrophages after stimulation with P. murina. We further observed decreased phosphorylation of Lyn on its C-terminal inhibitory tyrosine in P. murina-stimulated alveolar macrophages, indicating that SFKs were activated in alveolar macrophages in response to P. murina. Mice deficient in Hck, Fgr, and Lyn exhibited augmented clearance 3 and 7 days after intratracheal administration of P. murina, which correlated with elevated levels of interleukin 1beta (IL-1beta), IL-6, CXCL1/KC, CCL2/monocyte chemoattractant protein 1, and granulocyte colony-stimulating factor in lung homogenates and a dramatic increase in macrophage and neutrophil recruitment. Augmented P. murina clearance was also observed in Lyn(-/-) mice 3 days postchallenge, although the level was less than that observed in Hck(-/-) Fgr(-/-) Lyn(-/-) mice. A correlate to augmented clearance of P. murina in Hck(-/-) Fgr(-/-) Lyn(-/-) mice was a greater ability of alveolar macrophages from these mice to kill P. murina in vitro, suggesting that SFKs regulate the alveolar macrophage effector function against P. murina. Mice deficient in paired immunoglobulin receptor B (PIR-B), an inhibitory receptor activated by SFKs, did not exhibit enhanced inflammatory responsiveness to or clearance of P. murina. Our results suggest that SFKs regulate innate lung responses to P. murina in a PIR-B-independent manner.

FIG. 1.

P. murina activates SFKs in alveolar macrophages. Alveolar macrophages were isolated from 6- to 8-week-old, male C57BL/6 mice and stimulated for 10 to 90 min with P. murina (PC) at a ratio of macrophages to total P. murina cells of 1:100. The controls included alveolar macrophages cultured in medium alone (Unstim). After this, cell lysates were extracted, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membranes, and immunoblotted with anti-pHck 411 or rabbit anti-pLyn 507 IgG. Positive bands were identified using an ECL Western blot detection kit and were subsequently analyzed using Image J software (NIH). The blots are representative blots for (A) p-Lyn and (B) p-Hck levels in unstimulated and P. murina-stimulated alveolar macrophages. Cumulative data from three independent studies employing Image J software were used to determine the area under the curve values for (C) p-Lyn and (D) p-Hck and their beta-actin controls for unstimulated and P. murina-stimulated alveolar macrophages. The data are expressed both as the ratio of p-Lyn or p-Hck to actin and as the change after unstimulated samples were normalized to a value of 1. Data are the means ± standard errors of the means. ** and ***, P < 0.01 and P < 0.001, respectively (paired two-tailed Student's t test).

FIG. 2.

Enhanced lung clearance of P. murina in Hck^−/− Fgr^−/− Lyn^−/− and Lyn^−/− mice. C57BL/6 (WT) and Hck^−/− Fgr^−/− Lyn^−/− (Src TKO) mice were inoculated with 2 × 10⁵ P. murina cysts via the intratracheal route. (A and B) At 3 days (A) and 7 days (B) postinoculation, lungs were collected, and the P. murina burden was determined by using real-time PCR to determine the P. murina rRNA copy number. The data are cumulative data from three independent studies with five mice per group. The bars and error bars indicate the mean P. murina rRNA copy numbers and the standard errors of the means. ** and ***, P < 0.01 and P < 0.001, respectively (unpaired two-tailed Student's t test). (C) C57BL/6 and Lyn^−/− mice were inoculated with 2 × 10⁵ P. murina cysts via the intratracheal route, and 3 days postinoculation lungs were collected and the P. murina burden was determined by using real-time PCR to determine the P. murina rRNA copy number. The data are cumulative data from three independent studies with five mice per group. The bars and error bars indicate the means and standard errors of the means. **, P < 0.01 (unpaired two-tailed Student's t test).

FIG. 3.

Increased lung proinflammatory response in P. murina-exposed Hck^−/− Fgr^−/− Lyn^−/− mice. C57BL/6 (WT) and Hck^−/− Fgr^−/− Lyn^−/− (TKO) mice were inoculated with 2 × 10⁵ P. murina cysts via the intratracheal route. (A and B) At 3 days (A) and 7 days (B) postinoculation, lungs were collected, and clarified supernatants from lung homogenates were analyzed for G-CSF, CXCL1/KC, CCL2/MCP-1, IL-1β, and IL-6 levels by Bio-Plex. The data are cumulative data from three independent studies with five mice per group. The bars and error bars indicate the means and standard errors of the means. * and ***, P < 0.05 and P <0.001, respectively (unpaired two-tailed Student's t test). (C) Three days postinoculation, lungs were collected from Lyn^−/− mice, and clarified supernatants from lung homogenates were analyzed for G-CSF, CXCL1/KC, CCL2/MCP-1, IL-1β, and IL-6 levels by Bio-Plex. The data are cumulative data from three independent studies with five mice per group. The bars and error bars indicate the means and standard errors of the means. **, P < 0.01 (unpaired two-tailed Student's t test). (D) C57BL/6 and Hck^−/− Fgr^−/− Lyn^−/− (Src TKO) mice were inoculated with 2 × 10⁵ P. murina cysts via the intratracheal route. Three days postinoculation, bronchoalveolar lavage was performed, and cells were stained for F4/80 (macrophages), Gr-1 (neutrophils), and CD3 (T cells) and assessed by flow cytometry. The data are representative data from one of two independent studies. BALF, bronchoalveolar lavage fluid.

FIG. 4.

Histological evidence for inflammatory differences in the lungs of P. murina-exposed Hck^−/− Fgr^−/− Lyn^−/− mice: representative hematoxylin- and eosin-stained lung sections from (A) Hck^−/− Fgr^−/− Lyn^−/− (Src TKO) mice and (B) wild-type (WT) mice challenged intratracheally with 2 × 10⁵ P. murina cysts for 3 days. Original magnification, ×40. (Insets) Magnification, ×200.

FIG. 5.

Hck^−/− Fgr^−/− Lyn^−/− alveolar macrophages are more efficient at killing P. murina. Alveolar macrophages were isolated from 6- to 8-week-old, male C57BL/6 (WT) or Hck^−/− Fgr^−/− Lyn^−/− (Src TKO) mice and cocultured overnight with P. murina at a ratio of macrophages to P. murina cysts of 100:1. The controls included P. murina cultured in the absence of macrophages. After this, RNA was isolated from the contents of each well, and quantitative real-time PCR to determine the P. murina rRNA copy number was performed. The data are the cumulative results from three independent studies. The bars and error bars indicate the means and standard errors of the means. **, P <0.01 (unpaired two-tailed Student's t test).

FIG. 6.

Enhanced responses to P. murina in Hck^−/− Fgr^−/− Lyn^−/− mice are independent of PIR-B. (A) C57BL/6 (WT) and PIR-B^−/− mice (PIR-B KO) were inoculated with 2 × 10⁵ P. murina cysts via the intratracheal route, and 3 days postinoculation lungs were collected and the P. murina burden was determined by using real-time PCR to determine the P. murina rRNA copy number. The data are cumulative data from two independent studies with five mice per group. The bars and error bars indicate the means and standard errors of the means. (B) Three days postinoculation, lungs were collected, and clarified supernatants from lung homogenates were analyzed for CXCL1/KC, CCL2/MCP-1, IL-1β, and IL-6 levels by Bio-Plex. The data are cumulative data from two independent studies with five mice per group. The bars and error bars indicate the means and standard errors of the means. *, P < 0.05 (unpaired two-tailed Student's t test).