Alveolar Macrophages Treated With Bacillus subtilis Spore Protect Mice Infected With Respiratory Syncytial Virus A2

Abstract

Respiratory syncytial virus (RSV) is a major pathogen that infects lower respiratory tract and causes a common respiratory disease. Despite serious pathological consequences with this virus, effective treatments for controlling RSV infection remain unsolved, along with poor innate immune responses induced at the initial stage of RSV infection. Such a poor innate defense mechanism against RSV leads us to study the role of alveolar macrophage (AM) that is one of the primary innate immune cell types in the respiratory tract and may contribute to protective responses against RSV infection. As an effective strategy for enhancing anti-viral function of AM, this study suggests the intranasal administration of Bacillus subtilis spore which induces expansion of AM in the lung with activation and enhanced production of inflammatory cytokines along with several genes associated with M1 macrophage differentiation. Such effect by spore on AM was largely dependent on TLR-MyD88 signaling and, most importantly, resulted in a profound reduction of viral titers and pathological lung injury upon RSV infection. Taken together, our results suggest a protective role of AM in RSV infection and its functional modulation by B. subtilis spore, which may be a useful and potential therapeutic approach against RSV.

Keywords: Bacillus subtilis; MyD88; alveolar macrophage activation; respiratory syncytial virus; spore.

Figures

FIGURE 1

Pre-treatment with spore through intranasal route reduces the disease severity following RSV infection and induces the population change of alveolar macrophages (AM) and enhances antiviral effector molecules. Mice were administered with 1 × 109 CFU of spore via i.n. route at 5 days prior to RSV infection with 2 × 106 PFU per mouse (n = 3). (A) Body weight was monitored daily after the infection and (B) viral load in the lungs was analyzed by plaque assay at 4 days post-infection (DPI). Change of various innate immune cells in the (C) post-lavaged lung and (D) BAL fluid was analyzed by flow cytometry at 0 to 4 DPI. Empty and filled circles indicate PBS and spore pre-treated mice, respectively. (E) IFN-β and IL-12p40 in BAL fluid were measured by ELISA. Data are expressed as mean ± S.E.M. for the group. Significant differences from results with the PBS control are ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001, respectively.

FIGURE 2

Mice selectively depleted with AMs fail to protect RSV infection. Mice were injected i.t. with clodronate-encapsulated liposome twice on days 1 and 3 prior to RSV infection. (A) Body weight was monitored daily after the infection and (B) viral load in the lungs was analyzed by plaque assay at 4 DPI (n = 3). At 4 DPI, perfused lungs were stained with H&E (C) for histological examination by microscopy at 200 × magnifications and (D–G) scored for histopathology. “Cont” indicates the mice injected with control liposome and “Clod” indicates the mice injected with clodronate-encapsulated liposome. Arrows indicated as follows; orange, epithelium thickness and destruction; green, pulmonary edema; red, inflammatory cells; and black, cell death. Data are expressed as mean ± S.E.M. for the group. Significant differences from results with the PBS control are ∗P < 0.05 and ∗∗P < 0.01, respectively.

FIGURE 3

Alveolar macrophages pre-treated with spore extend their antiviral effects. MH-S cells were stimulated with spore for 24 h, with a ratio of MH-S:Spore at = 1:0, 1:1, 1:10, and 1:50. Then, the cells were washed and infected with 1 MOI RSV for 12 h. (A) The viral load was analyzed by using plaque assay and (B) the production of IL-12p40 and IL-6 was measured by ELISA. Data are expressed as mean ± S.E.M. of three replicates. ∗ and ∗∗ indicate significant differences at P < 0.05 and P < 0.01, respectively.

FIGURE 4

Alveolar macrophages in mice intranasally administered with spore play a key role in RSV infection. Mice were intranasally administered with spore at 5 days prior to RSV infection and injected with control or clodronate-encapsulated liposome through intratracheal on days 3 and 1 before the infection and sacrifice on 4 DPI. (A) Body weight was monitored daily after the infection and (B) viral load in the lung was examined at 4 DPI, respectively (n = 3). (C) At 4 DPI, perfused lungs were stained with H&E for histological examination by microscopy at 200 × magnifications and (D–G) scored for histopathology. Arrows indicated are as follows; orange, epithelium thickness and destruction; green, pulmonary edema; red, inflammatory cells; and black, cell death. Significant differences from results with the PBS control are ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001, respectively.

FIGURE 5

Protective mechanism of spore pre-treated mice infected with RSV is dependent on MyD88 signaling in AMs. Wild type or MyD88 knockout mice were administered i.n. with spore at 5 days prior to RSV infection. (A) Body weight was monitored daily after the infection (n = 3). At 4 DPI, (B) viral load in the lungs was analyzed by plaque assay and absolute number of AMs in (C) post lavaged lungs, and (D) BAL cells were acquired by flow cytometry. Values with different letters (a, b, c, d) are significantly different one from another (P < 0.05). (E) Blood-perfused lungs were stained with H&E for histological examination by microscopy at 200× magnification and (F–I) scored for histopathology at 4 DPI. Arrows indicated are as follow; orange, epithelium thickness and destruction; green, pulmonary edema; red, inflammatory cells; and black, cell death. Significant differences from results with the PBS control are ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001, respectively.

FIGURE 6

Bone marrow-derived macrophages pre-treated with spore develop antiviral effects through MyD88-dependent pathway. Wild type or MyD88 knockout BMMs were treated with spore for 24 h at ratio of spore per number of cells for 0, 1, 10, and 50. Then, the cells were washed and infected with 1 MOI virus for additional 12 h. (A) Viral titers from wild type or MyD88 knockout BMMs were measured by plaque assay and (B) the production of IL-12p40 was measured by ELISA. Data are expressed as mean ± S.E.M. for 3 independent experiments. ∗, ∗∗, and ∗∗∗ indicate significant differences at P < 0.05, P < 0.01, and P < 0.001, respectively.