Mechanisms of Action of Vitamin D as Supplemental Therapy for Pneumocystis Pneumonia - PubMed (original) (raw)

Mechanisms of Action of Vitamin D as Supplemental Therapy for Pneumocystis Pneumonia

Guang-Sheng Lei et al. Antimicrob Agents Chemother. 2017.

Abstract

The combination of trimethoprim and sulfamethoxazole (TMP-SMX) is the most effective regimen for therapy of Pneumocystis pneumonia (PCP). As many patients with PCP are allergic or do not respond to it, efforts have been devoted to develop alternative therapies for PCP. We have found that the combination of vitamin D3 (VitD3) (300 IU/kg/day) and primaquine (PMQ) (5 mg/kg/day) was as effective as TMP-SMX for therapy of PCP. In this study, we investigated the mechanisms by which vitamin D enhances the efficacy of PMQ. C57BL/6 mice were immunosuppressed by CD4+ cell depletion, infected with Pneumocystis murina for 8 weeks, and then treated for 9 days with the combination of VitD3 and PMQ (VitD3-PMQ) or with TMP-SMX or PMQ to serve as controls. The results showed that vitamin D supplementation increased the number of CD11c+ cells, suppressed the production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], and interleukin-6 [IL-6]) and inducible nitric oxide synthase (iNOS), and enhanced the expression of genes related to antioxidation (glutathione reductase and glutamate-cysteine ligase modifier subunit), antimicrobial peptides (cathelicidin), and autophagy (ATG5 and beclin-1). These results suggest that the main action of vitamin D is enhancing the ability of the host to defend against Pneumocystis infection.

Keywords: Pneumocystis pneumonia; alveolar macrophages; vitamin D3.

Copyright © 2017 American Society for Microbiology.

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Figures

FIG 1

FIG 1

Changes in body weight during therapy. Mice infected with P. murina for 8 weeks were treated with TMP-SMX, PMQ, or VitD3-PMQ. Each mouse was weighed before and every 3 days after initiation of treatment for 9 days. Each dot represents the average body weight of 5 mice in various treatment groups.

FIG 2

FIG 2

Lung histopathology. H&E- and methenamine silver-stained lung sections of the following mouse groups are shown: immunosuppressed mice with PCP for 8 weeks (Untreated) and mice with PCP for 8 weeks treated with TMP-SMX, PMQ, or VitD3-PMQ for 9 days. (A) H&E-stained lung sections (magnification, ×100). Arrows indicate infiltrated inflammatory cells. (B) Methenamine silver-stained lung sections (magnification, ×200). Pneumocystis cysts are seen as black dots or clusters.

FIG 3

FIG 3

Cytokine levels in BAL fluids. The first 2 ml of BAL fluid was used for ELISA. (A) TNF-α levels in BAL fluids of immunosuppressed uninfected mice, mice with PCP, and mice with PCP treated with TMP-SMX, PMQ, or VitD3-PMQ. (B) IFN-γ levels in BAL fluids of various mouse groups. (C) IL-6 levels in BAL fluids of various mouse groups. ND, not detected. *, P < 0.05.

FIG 4

FIG 4

Histograms of CD11c+ cells from BAL fluids. Immunosuppressed mice were infected with P. murina for 8 weeks and then treated with TMP-SMX, PMQ, or VitD3-PMQ for 9 days. BAL fluid cells of mice in various groups were collected, stained with Alexa Fluor 647-labeled anti-CD11c antibodies, and then analyzed by flow cytometry. The percentage of CD11c+ cells in each group of mice is indicated.

FIG 5

FIG 5

Morphology of isolated CD11c+ cells. BAL fluid cells from mice with PCP treated with VitD3-PMQ for 9 days were collected. CD11c+ cells were isolated using biotin–anti-mouse CD11c antibody and antibiotin magnetic microbeads. Isolated cells were cytospun on slides, stained with Giemsa stain, and examined by light microscopy.

FIG 6

FIG 6

Expression levels of stress genes. Thirty milligrams of lung tissue from each mouse in various treatment groups was placed in 600 μl RLT buffer (RNeasy minikit; Qiagen) and homogenized. Total RNA was isolated and reverse transcribed to cDNA. mRNA levels of iNOS (A), GR (B), GCLM (C), CAMP (D), TLR2 (E), TLR4 (F), and NF-κB (G) were determined by SYBR green qRT-PCR. Data are presented as fold change (mean ± SD). *, P < 0.05.

FIG 7

FIG 7

Expression levels of autophagy markers. The converted cDNA as described for Fig. 6 was used to determine mRNA levels of ATG5 (A) and BCN1 (B). Data are presented as fold change (mean ± SD). *, P < 0.05.

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