Boosting efferocytosis in alveolar space using BCG vaccine to protect host against influenza pneumonia

Abstract

Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia.

Fig 1. Delivery of BCG to alveolar space enhances efferocytosis by APs.

Staurosporine-induced apoptotic mouse lung epithelial (MLE) cells were labeled with CFSE and intranasally transferred to mice treated intranasally with BCG or PBS. Bronchoalveolar lavage (BAL) cells were collected 1 h later and stained with F4/80. (A) Flow cytometry analysis of individual mice is shown. (B) Average of free CFSE+apoptotic MLE cells and efferocytosis efficiency from panel A. Efferocytosis efficiency was calculated as percent of efferocytosed CFSE+ apoptotic cells (CFSE+ and F4/80+F4/80+double-positive cells)/total percent of CSFE^+ve cells. A representative of two to four independent experiments with 3–5 mice per group are shown. Error bars show SEM. (C) Localization of apoptotic cells insideAPs. WT mice were treated as in panel A. Apoptotic MLE cells were stained with red pHrodo® and administered intranasally to PBS-treated (upper row) control and BCG-treated (lower row). BAL cells were collected and subjected to confocal microscopy.60x Magnification. A representative of two independent experiments with 3–4 mice per group is depicted. (D-F) Pulmonary treatment with BCG rapidly and efficiently boosts efferocytosis by APs in disease condition. WT mice were treated intranasally with BCG or PBS. Two days later, all mice were infected with PR8 strain of influenza virus. Two days after IAV infection, all mice received CFSE-labeled apoptotic MLE cells intranasally and the apoptotic cell clearance was measured by flow cytometry, as described above. (D) Flow cytometry analysis of each individual mouse either treated with PBS (top row) or BCG (bottom row) is shown. (E)Average percent of free CFSE+ apoptotic MLE cells and efferocytosis efficiency from panel (D) are shown. Efferocytosis efficiency of APs was calculated using the formula from panel B. A representative of three to four independent experiments shown with 3–5 mice per group. Error bars show SEM. (F) Visualization of the engulfed apoptotic cells inside the APs in BCG vaccinated and control mice after IAV infection. WT mice were treated as in panel D. Two days after IAV infection, all mice received pHrodo®-labeled apoptotic MLE cells intranasally and the fate of apoptotic cells inside the AMs of PBS-treated (upper row) control and BCG-treated (lower row) mice was studied using confocal microscopy, as described for panel1C. 60x magnification. A representative of two independent experiments with 3 mice per group is depicted.

Fig 2. Intranasal treatment with BCG protects mice against lethal influenza infection.

WT mice (9 per group) were treated with either PBS or BCG (either intranasally or subcutaneously), and all mice were infected with a lethal dose (2 LD50) of influenza PR8 virus. Weight loss (A) and mortality (B) were recorded daily. (C)Different groups of mice were treated as for panels A and B. Three and 7 days after influenza infection, mice were sacrificed, lung homogenates were generated and virus load was measured. Depicted data are average of 5 mice per group and error bars show SEM. NS = Not significant. (D) BAL cells from a group of BCG- and PBS-treated mice prior to and after IAV infection were quantified. Depicted data are mean of 5 mice per group. Error bars show SEM. (E-F) Depletion of APs abrogated BCG-mediated protection against lethal influenza infection. Mice were intranasally treated with BCG and 24 hrs prior to influenza PR8 infection (2LD50) they were treated with either liposomal clodronate to deplete APs or PBS-liposome as control. Infected mice were monitored for (E) weight loss and (F) mortality. Mean of weight loss with SEMs are depicted. n = 5. DPI: Days Post Infection.

Fig 3. BCG vaccination increases Tim4 expression in APs.

(A) Expression of Tim-4 was determined in BCG vaccinated and control PBS treated mice before and after influenza infection using western blotting. WT mice were treated with either BCG or PBS and infected with lethal PR8 as described in the legend of Figs 1D and 2. Two days after influenza infection, BAL cells were collected, pooled and lysed as delineated in materials and methods. Each lane is pool of BALs from 5 mice. Equal amount of protein extracts from each group were loaded into each lane. (B) Densitometry analysis of band-intensity using Image Lab software (Molecular Imager Gel Doc system from Bio-Rad) is shown. Data is representation of band-intensities of 3 independent blots. Error bars show SEM. DPI: Days Post Infection.

Fig 4. Neutralizing Tim4 reduces BCG mediated efferocytosis by APs.

WT micewere treated intranasally with BCG and 24 hours later were treated with 200μg of either anti-TIM4 blocking antibody or isotype control. Next day, all mice were infected with lethal dose of PR8 influenza virus (2LD50). Antibody treatment continued with 3 days intervals at days 2,5,and 7 post PR8infection. (A) Apoptotic MLE cells labeled with CFSE were intranasally transferred to isotype (top row) or anti-Tim4 (bottom row) treated mice, BALs were collected an hour later andefferocytosis efficiency of AMs was evaluated using flow cytometry, as described for Figs 1 and 3. (B) Average percent of free CFSE+ apoptotic MLE cells and efferocytosis efficiency from panel (A) are shown. Efferocytosis efficiency of AMs was calculated as described for Fig 1C and 1F. A representative of two independent experiments is shown with 3 mice per group. Error bars show SEM. (C and D)Weight loss and mortality were recorded daily. A representative of two independent experiments with similar results is shown. n = 10.

Fig 5. Effects of BCG on expression of small GTPases in APs.

APs from WT mice treated with either PBS or BCG were collected prior to and 2 days after influenza infection. Total RNA was extracted, cDNA was synthesized, and quantitative reverse transcription-PCR was carried out. Beta Actin was used as a housekeeping gene and expression levels of (A) Rab5 and (B) Rab7 were normalized against the Beta Actin mRNA level. (C) Rho A and (D) Rac1activity of AMs from BCG- or PBS-treated mice prior to and 2 days after influenza infection was measured using G-LISA RhoA and Rac 1 activation assay protocol, respectively. A representative of two independent experiments with 4–6 mice per group are shown with error bars as SEM. DPI: Days Post Infection.

Fig 6. Effects of BCG on TNF and IL10 cytokines in the lungs.

WT mice were treated as in Fig5. mRNA expression of TNF (A) and IL10 (B) in the BAL cells and protein levels in the BAL supernatants (C and D) were measured by real time PCR and ELISA, respectively. Mean of 6 mice/group is shown with error bars showing SEM. DPI: Days Post Infection.

Fig 7. Expression of PPAR nuclear receptor in BAL samples of BCG- and PBS-treated mice.

Expressions of PPAR- α/β/δ in mouse alveolar phagocytes were measured using western blotting. Each lane is pool of BALs from either 3 BCG- or 3 PBS-treated mice that were collected prior to and 2 (A) and 7 (B) days after influenza PR8 infection. Cell lysates were subjected to western blot analyses. β-actin was used as control. Representative of 2 independent experiments with similar results is shown. DPI: Days Post Infection.