A critical role for CARD9 in pneumocystis pneumonia host defence

Abstract

Caspase recruitment domains-containing protein 9 (CARD9) is an adaptor molecule critical for key signalling pathways initiated through C-type lectin receptors (CLRs). Previous studies demonstrated that Pneumocystis organisms are recognised through a variety of CLRs. However, the role of the downstream CARD9 adaptor signalling protein in host defence against Pneumocystis infection remains to be elucidated. Herein, we analysed the role of CARD9 in host defence against Pneumocystis both in CD4-depleted CARD9^-/- and immunocompetent hosts. Card9 gene-disrupted (CARD9^-/- ) mice were more susceptible to Pneumocystis, as evidenced by reduced fungal clearance in infected lungs compared to wild-type (WT) infected mice. Our data suggests that this defect was due to impaired proinflammatory responses. Furthermore, CARD9^-/- macrophages were severely compromised in their ability to differentiate and express M1 and M2 macrophage polarisation markers, to enhanced mRNA expression for Dectin-1 and Mincle, and most importantly, to kill Pneumocystis in vitro. Remarkably, compared to WT mice, and despite markedly increased organism burdens, CARD9^-/- animals did not exhibit worsened survival during pneumocystis pneumonia (PCP), perhaps related to decreased lung injury due to altered influx of inflammatory cells and decreased levels of proinflammatory cytokines in response to the organism. Finally, although innate phase cytokines were impaired in the CARD9^-/- animals during PCP, T-helper cell cytokines were normal in immunocompetent CARD9^-/- animals infected with Pneumocystis. Taken together, our data demonstrate that CARD9 has a critical function in innate immune responses against Pneumocystis.

Keywords: CARD9; Pneumocystis; host defence; inflammation; macrophage.

Figure 1.

CARD9−/− mice exhibit significantly increased organism burden during PCP. CARD9−/− and wildtype (WT) mice were immunosuppressed by depletion of CD4 cells and subsequently inoculated with P. murina organisms. (A) After 8 weeks, the P. murina organism burden was determined by P. murina 16S mitochondrial ribosomal DNA copy number and assessed using a standard curve. (Data are derived from 10 to 12 mice per group + SEM; *Denotes p < 0.05, comparing P. murina burden in the CARD9−/− and (WT) mice and are representative of 2 separate experiments.) (B) After 8 weeks of infection, the lungs were fixed in 10% phosphate-buffered formalin and 5-μm sections were obtained. Gomori Methenamine Silver stains (GMS) for P. murina organisms were performed, demonstrating clusters of stained organisms (subpanel 3, 4, and 5) whereas no organisms were noted in uninfected and CARD9−/− mice, respectively (subpanel 1 and 2). As indicated, for original magnification ×100, scale bars are 200 μm and for original magnification ×400, scale bars are 20 μm (C) GMS-stained slides were scanned using the Aperio ScanScope System at 100× magnification, typically scanning from 5 sections per slides and about 10 slides per condition. Pixel intensity was determined using the Aperio positive pixel count algorithm. The algorithm counts the number of positive (brown) and negative stains, the number of pixels, and the average intensity of staining using a hot spot approach. Data shown is the average pixel intensity + SEM from 50 sections per mouse lung and are representative of one experimental run with 12 total mice. ** Denotes p < 0.01 comparing silver stain pixel intensity in in the CARD9−/− and (WT) mice with P. murina pneumonia.

Figure 2.

Levels of inflammatory cytokines are reduced in the lungs of PCP-induced mice in absence of CARD9. WT or CARD9−/− mice were infected with P. murina. After 8 weeks of infection, the lungs were harvested and protein lysates obtained. Levels of the indicated inflammatory cytokines were measured by ELISA. ****p < 0.0001, comparing P. murina-infected CARD9−/− mice to P. murina infected WT mice and the data shown are derived from n>10 animals + SEM per group and are representative of 2 separate animal runs.

Figure 3.

CD4 depleted CARD9−/− PCP-infected mice had significantly reduced inflammation and lung injury but similar survival rates compared to CD4 depleted controls with PCP. (A) Data shown are Kaplan-Meier survival curves of WT and CARD9−/− mice that were immunosuppressed by depletion of CD4 cells and subsequently inoculated with P. murina organisms for a course of 65 days. Despite the enhanced organism burden in the CARD9−/− mice with PCP, we did not observe any worsened mortality compared with that of the WT mice with PCP. ns=not significant, p =0.7884, by Log-rank (Mantel-Cox) test with n = 9 mice total inoculated in each group. After 8 weeks of infection WT and CARD9−/− mice that were immunosuppressed by depletion of CD4 cells, the lungs were harvested and (B) levels of the lung injury marker, MPO, and the neutrophil recruitment cytokine, G-CSF, were measured by ELISA using protein lysates and (C) inflammatory cell influx was assessed by flow cytometry and shown are the average percentages of different leukocytes in the lungs. Data in (B-C) are derived from n>10 animals per group + SEM and are representative of 2-3 separate experimental runs; *p < 0.05; ****p < 0.0001 comparing P. murina-infected CARD9−/− mice to P. murina infected WT mice. (D) H&E staining of CARD9−/− animals infected with Pneumocystis compared to that of WT animals shows that overall lung inflammation was lower in the lungs of CARD9−/− animals infected with Pneumocystis compared to that of WT animals.

Figure 4.

CARD9 is not required for T-helper responses during Pneumocystis infection. WT or CARD9−/− mice were infected with P. murina. After 20 days of infection, the lungs were harvested and, (A) Organism burden was determined by 16S mitochondrial ribosomal DNA–targeted qPCR and copy number assessed using a standard curve. Protein lysates prepared and levels of (B) T-helper cell secreted-cytokines as measured by ELISA and, (C) IL-12, Th-1 stimulating cytokine were measured by ELISA. Data in (A-C) are derived from n=13-14 animals per group + SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 comparing P. murina–infected CARD9−/− mice to P. murina–infected WT mice.

Figure 5.

CARD9 is a critical factor in macrophages for the production of inflammatory response during Pneumocystis infection. BMDM were stimulated with P. carinii (10 P. carinii:1 cell) for 24 h after overnight priming with 10 ng/ml of GM-CSF or 25 U/ml IFNγ. After stimulation, supernatants were harvested and analyzed by ELISA for protein levels of different cytokines as indicated. The data shown are integrated means ± the SEM of 4-5 independent experiments; *p < 0.05, **p < 0.01.

Figure 6.

Macrophages require CARD9 for differentiation and induction of Dectin-1 and Mincle in response to Pneumocystis. BMDM were stimulated with P. carinii (10 P. carinii:1 cell) for 24 h. After stimulation, macrophage mRNA samples were harvested and analyzed by qPCR for mRNA expression levels of (A) M1 (iNOS) and M2 (Arg-1) markers and (B) Dectin-1 and Mincle, respectively. The mRNA expression levels were normalized to GAPDH and data shown are integrated means ± the SEM of at least three independent experiments; *p < 0.05, p** < 0.01. (C) BMDMs were stimulated with P. carinii for 24 hours and total DNA was extracted from the contents of each well and qPCR for P. carinii 16S mitochondrial ribosomal DNA copy number was performed. The data are integrated means ± the SEM from five independent experiments; *p < 0.05.

Figure 7.. CARD9 triggers MAPK and NF-κB activation in macrophages following challenge with Pneumocystis.

(A). Macrophage lysates from BMDM stimulated with P. carinii were prepared at the indicated times and expression levels of total and phospho-proteins as indicated were analyzed by Western blot to assess the activation of the different signaling events in the presence and absence of CARD9. GAPDH is used as a loading control for p-IκB-α, since the total IκB-α degrades with activation. Data are representative of three independent experiments. (B). Quantification of phosphorylation of signaling proteins was quantified with Image Studio Lite software and normalized against the total protein amount or GAPDH levels as denoted. (* Denotes p<0.05 compared to control).

Figure 8.

Overexpression of CARD9 in macrophages enhances the TNF-α secretion in response to Pneumocystis. RAW264.7 macrophages stably expressing full length CARD9, CARD domain, or coiled-coil domain were stimulated with P. carinii (10 P. carinii:1 cell) for 24 h. After stimulation, supernatants were harvested and analyzed by (A) ELISA for TNF-α protein. Data shown are integrated means ± the SEM of three independent experiments; *p < 0.05, **p < 0.01 compared with the cells expressing the empty control vector or comparing full length CARD9 to the respective CARD9 mutants.