Toll-like receptor 2 mediates alveolar macrophage response to Pneumocystis murina

Abstract

The innate immune response to Pneumocystis infection is not well understood. In this study, normal C57BL/6 mouse alveolar macrophages were found to respond to Pneumocystis murina organisms through Toll-like receptor 2 (TLR2), leading to the nuclear translocation of NF-kappaB and the production of proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and chemokine macrophage inflammatory protein 2 (MIP-2). P. murina stimulation of normal alveolar macrophages from C57BL/6 mice resulted in increased TLR2 transcription but not increased TLR4 transcription. In gain-of-function studies with HEK293 cells expressing TLR2 or TLR4, only TLR2 was found to stimulate an NF-kappaB response to P. murina. TNF-alpha and MIP-2 production in response to P. murina by mouse alveolar macrophages was inhibited by a monoclonal antibody that specifically blocked the ligand-binding ability of TLR2. Alveolar macrophages from TLR2 knockout (TLR2-/-) mice showed little increase in TNF-alpha and MIP-2 mRNA levels upon P. murina stimulation. An in vivo study showed that TLR2-/- mice challenged with P. murina had reduced cytokine responses. These results indicate that TLR2 plays a major role in the innate immune response to P. murina.

FIG. 1.

Cytokine production in response to P. murina by alveolar macrophages. (A) Alveolar macrophages from C57BL/6 mice were stimulated with P. murina at an MOI of 6 for 8 h and then assessed for TNF-α and MIP-2 mRNA levels by real-time RT-PCR. Controls were aliquots of the same alveolar macrophages stimulated with saline. The mRNA of the RPS8 gene was coamplified with that of TNF-α or MIP-2 to serve as the internal PCR control in each assay. A total of 3 × 10⁵ alveolar macrophages from each mouse were used for each experiment; the cells were cultured in 1 ml/well RPMI 1640 medium in a 12-well plate. The severalfold increase in TNF-α or MIP-2 mRNA levels in P. murina-treated cells is relative to that of the saline-treated control cells, which was set to a value of 1. (B) Culture supernatants of the same P. murina-treated or saline-treated alveolar macrophages were analyzed for TNF-α or MIP-2 protein levels (picograms per milliliter) by ELISA. Bars represent means ± SD for five mice per group (*, P < 0.05 compared with the saline-treated group).

FIG. 2.

Alveolar macrophage NF-κB nuclear translocation in response to P. murina. Alveolar macrophages (3 × 10⁵) in 0.2 ml RPMI 1640 medium were stimulated with P. murina at an MOI of 6 for 1 h and then examined for NF-κB nuclear translocation by fluorescence microscopy. Positive controls were aliquots of the same alveolar macrophages stimulated with 1 μg/ml LPS, and negative controls were treated with saline. Cells were cytospun onto glass slides, fixed with 4% paraformaldehyde, and then reacted with rabbit anti-mouse NF-κB p65 antibody followed with Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (0.1 μg/ml). Magnification, ×400.

FIG. 3.

Effect of P. murina on the expression of TLR2 and TLR4. Alveolar macrophages from C57BL/6 mice were incubated with P. murina or normal saline (negative control) for 8 h as described in the Fig. 1 legend. Total RNA was isolated from the alveolar macrophages and subjected to Taqman real-time RT-PCR for TLR2 and TLR4 mRNA expression; RPS8 mRNA was coamplified as the internal PCR control. TLR2 and TLR4 real-time PCR results are normalized to those of the RPS8 internal control and are shown as severalfold increases relative to those of saline-treated control cells. Bars are means ± SD of five mice per group (*, P < 0.05 compared with the saline-treated group).

FIG. 4.

Response of TLR2- or TLR4-expressing HEK293 cells to P. murina. HEK293 cells were transiently transfected with plasmids encoding TLR2 and CD14 or with those encoding TLR4, MD2, and CD14. Cells transfected with the plasmid encoding CD14 alone were used as the negative control. All cells were cotransfected with pBIIX-Luc, which contains the firefly luciferase gene, and pRL-TK, which contains the Renilla luciferase gene. Cells were stimulated with the TLR2 ligand Pam3CSK4 (A), the TLR4 ligand E. coli LPS (B), or various numbers of P. murina organisms (C) for 8 h and then subjected to luciferase assay. Data from stimulated cells are shown as severalfold increases in luciferase activity relative to those of unstimulated control cells. Each time point represents means ± SD of results from three different sets of HEK293 cells transfected with the same plasmids.

FIG. 5.

Inhibition of P. murina-induced cytokine production by MAb T2.5. Alveolar macrophages from C57BL/6 mice were incubated with MAb T2.5 (Ab) for 30 min and then stimulated with purified P. murina for 8 h. Total RNA isolated from the alveolar macrophages was subjected to real-time RT-PCR analysis for TNF-α and MIP-2 mRNA expression. TNF-α and MIP-2 real-time PCR data are normalized to that of the RPS8 internal control and are shown as severalfold increases relative to those of unstimulated control cells. Bars are means ± SD of five mice per group (*, P < 0.05 compared with the saline group).

FIG. 6.

Diminished TNF-α and MIP-2 mRNA expression in alveolar macrophages from TLR2^−/− mice in response to P. murina stimulation. Alveolar macrophages from wild-type C57BL/6 and TLR2^−/− mice were stimulated with sterile saline or P. murina for 8 h. Total RNA isolated from the alveolar macrophages was subjected to real-time RT-PCR analysis for TNF-α (A) and MIP-2 (B) mRNA expression. TNF-α and MIP-2 real-time PCR data are normalized to that of the RPS8 internal control and are shown as severalfold increases relative to those of unstimulated control cells. Bars are means ± SD of five mice per group (*, P < 0.05 compared with the saline group).

FIG. 7.

Responses of alveolar macrophages to P. murina in vivo. Wild-type and TLR2^−/− mice were transtracheally inoculated with 5 × 10⁶ P. murina cells in 50 μl of sterile saline. Control mice were transtracheally inoculated with 50 μl of sterile saline. Eight hours after inoculation, alveolar macrophages were isolated, and total RNA isolated from the alveolar macrophages was subjected to real-time RT-PCR analysis for TNF-α and MIP-2 mRNA expression. TNF-α and MIP-2 real-time PCR data are normalized to that of the RPS8 internal control and are shown as severalfold increases relative to those of control mice that were inoculated with sterile saline. Bars are means ± SD of five mice per group (*, P < 0.05 compared with wild-type mice).