Deciphering the pathways of death of Histoplasma capsulatum-infected macrophages: implications for the immunopathogenesis of early infection

Abstract

Apoptosis of leukocytes is known to strongly influence the immunopathogenesis of infection. In this study, we dissected the death pathways of murine macrophages (MΦs) infected with the intracellular pathogen Histoplasma capsulatum. Yeast cells caused apoptosis of MΦs at a wide range of multiplicity of infection, but smaller inocula resulted in delayed detection of apoptosis. Upon infection, caspases 3 and 1 were activated, and both contributed to cell death; however, only the former was involved in apoptosis. The principal driving force for apoptosis involved the extrinsic pathway via engagement of TNFR1 by TNF-α. Infected MΦs produced IL-10 that dampened apoptosis. The chronology of TNF-α and IL-10 release differed in vitro. The former was detected by 2 h postinfection, and the latter was not detected until 8 h postinfection. In vivo, the lungs of TNFR1(-/-) mice infected for 1 d contained fewer apoptotic MΦs than wild-type mice, whereas the lungs of IL-10(-/-) mice exhibited more. Blockade of apoptosis by a pan-caspase inhibitor or by simvastatin sharply reduced the release of TNF-α but enhanced IL-10. However, these treatments did not modify the fungal burden in vitro over 72 h. Thus, suppressing cell death modulated cytokine release but did not alter the fungal burden. These findings provide a framework for the early pathogenesis of histoplasmosis in which yeast cell invasion of lung MΦs engenders apoptosis, triggered in part in an autocrine TNF-α-dependent manner, followed by release of IL-10 that likely prevents apoptosis of newly infected neighboring phagocytes.

Figure 1

Apoptosis of Mφ infected with H. capsulatum. Panel A represents studies done using the Cell Death ELISA kit, Panel B is the response by BMDMφ differentiated with M-CSF, Panel C is the apoptotic response of BMDMφ using a kit from Millipore Corp., Panel D examines necrosis, and Panel E illustrates the apoptotic response by alveolar Mφ. The enrichment factor was calculated using the formula: absorbance of cells incubated with yeast cells/absorbance of cells incubated in medium alone. For graphic purposes, the enrichment factor for cells incubated in medium alone was assigned a value of 1. The heavy dashed line is the enrichment factor of the uninfected cells. The data represent the mean ± SEM of 6–12 experiments. The enrichment factor for H. capsulatum incubated in the absence of Mφ was < 0.01 for each ratio of yeast:Mφ. Hc= H. capsulatum. * = p < 0.05; ** = p < 0.01.

Figure 2

Photomicrographs of Mφ and H. capsulatum. Phase contrast photomicrographs of uninfected and infected Mφ at 24 h and 72 h post exposure to yeast cells. Hc= H. capsulatum. Magnification of 200x.

Figure 3

Analysis of cytokines and SOCS3 in Mφ infected with H. capsulatum. TNF-α and IL-10 production as assessed by ELISA (Panel A). The data represent the mean ± SEM of 6 experiments. SOCS3 expression as assessed by qRT-PCR in BMDMφ prepared from IL-10^+/+ and IL-10^−/− mice (Panel B). RQ = relative quantification. Panels C and D demonstrate chronology of TNF-α and IL-10 generation in infected BMDMφ. Cytokines concentrations were determined in supernatants of cells incubated with medium alone or with yeast cells by ELISA. ND = Not detected. Hc= H. capsulatum. Data indicate the mean ± SEM of 5 experiments.

Figure 4

Caspase inhibitors and simvastatin suppress apoptosis. BMDMφ were incubated with DMSO, z-FA-FMK, Boc-D-FMK, QVD-OPh, or simvastatin (Sim) and infected with H. capsulatum yeast cells for 24 h. The heavy dashed line is the enrichment factor of the uninfected cells. Hc= H. capsulatum. The data represent mean ± SEM of 6 experiments. * = p < 0.05 compared to cells treated with Boc-D-FMK, QVD-OPh, or simvastatin; ** p < 0.01 compared to cells treated with Boc-D-FMK, QVD-OPh, or simvastatin.

Figure 5

Caspase activity and inhibition of apoptosis. BMDMφ were incubated with H. capsulatum for 24 h or 72 h and caspase 3 activity was assessed (A). In panel B, caspase 1 activity was assessed at 24 h post-infection. Apoptosis was assessed in cells exposed to the caspase 3 inhibitor Ac-DEVD or the caspase 1 inhibitors YVAD or WEHD 24 h post-infection (C). In panel D, caspase 3 activity was assessed after exposure to z-FA-FMK, QVD-OPh, or simvastatin (Sim). Cell death analysis in the presence or absence of the caspase 1 inhibitors (E), and IL-1β generation in infected cells in the presence or absence of caspase 1 inhibitors (F). The heavy dashed line is the enrichment factor of the uninfected cells. Data represent the mean ± SEM of 5–7 experiments. ND = Not detected. Hc= H. capsulatum. * = p < 0.05; ** = p < 0.01.

Figure 6

TNF-α and IL-10 modulation of apoptosis. Apoptosis of BMDMφ in the presence of isotype control mAb, anti- TNF-α, or anti-IL-10R. Cells were incubated with the mAb and infected for 24 h and assessed for apoptosis (A &B). In panel C is the apoptotic response by BMDMφ from TNFR1^−/− and WT mice. BMDMφ from WT and TNFR1^−/− mice produce TNF-α (D). The heavy dashed line is the enrichment factor of the uninfected cells. The data represent the mean ± SEM of 5 experiments. Hc = H. capsulatum. * = p < 0.05; ** = p < 0.01. In panels A and B, statistical analysis was performed by comparing antibody treated cells to isotype controls.

Figure 7

TNF-α and IL-10 production in the presence of inhibitors of apoptosis. BMDMφ were incubated with z-FA-FMK, QVD-OPh, or simvastatin (Sim) and infected with H. capsulatum for 24 h. Supernatants were removed and assayed for cytokines by ELISA. Data represent the mean ± SEM of 6–7 experiments. Hc= H. capsulatum. TNF-α and IL-10 levels were not detected in uninfected Mφ treated with z-FA-FMK, QVD-OPh, or simvastatin. ** = p < 0.01 compared to unstimulated cells or cells treated with QVD-OPh or simvastatin for Panel A or compared to unstimulated cells or cells incubated with medium alone or z-FA-FMK for Panel B.

Figure 8

Recovery of H. capsulatum from BMDMφ. BMDMφ infected with H. capsulatum and exposed to medium only, z-FA-FMK, QVD-OPh, or simvastatin (Sim) were lysed at 24 and 48 h post-infection and plated. Data represent the mean CFU ± SEM of 5 experiments. Hc= H. capsulatum.