Differential polarization of alveolar macrophages and bone marrow-derived monocytes following chemically and pathogen-induced chronic lung inflammation

Abstract

Alveolar macrophages and BDMCs undergo sequential biochemical changes during the chronic inflammatory response to chemically induced lung carcinogenesis in mice. Herein, we examine two chronic lung inflammation models-repeated exposure to BHT and infection with Mycobacterium tuberculosis-to establish whether similar macrophage phenotype changes occur in non-neoplastic pulmonary disease. Exposure to BHT or M. tuberculosis results in pulmonary inflammation characterized by an influx of macrophages, followed by systemic effects on the BM and other organs. In both models, pulmonary IFN-gamma and IL-4 production coincided with altered polarization of alveolar macrophages. Soon after BHT administration or M. tuberculosis infection, IFN-gamma content in BALF increased, and BAL macrophages became classically (M1) polarized, as characterized by increased expression of iNOS. As inflammation progressed in both models, the amount of BALF IFN-gamma content and BAL macrophage iNOS expression decreased, and BALF IL-4 content and macrophage arginase I expression rose, indicating alternative/M2 polarization. Macrophages present in M. tuberculosis-induced granulomas remained M1-polarized, implying that these two pulmonary macrophage populations, alveolar and granuloma-associated, are exposed to different activating cytokines. BDMCs from BHT-treated mice displayed polarization profiles similar to alveolar macrophages, but BDMCs in M. tuberculosis-infected mice did not become polarized. Thus, only alveolar macrophages in these two models of chronic lung disease exhibit a similar progression of polarization changes; polarization of BDMCs was specific to BHT-induced pulmonary inflammation, and polarization of granuloma macrophages was specific to the M. tuberculosis infection.

Figure 1.

Cellular infiltration and cytokine contents in pulmonary BAL change after BHT administration. (A) BAL macrophage content increases 6 days following the last of four weekly BHT injections. By 30 days after the last injection, population size has returned to basal levels. (B) Lymphocytes display similar population kinetics, rising at the 6-day time-point and returning to control levels by Day 30. (C) IFN-γ levels rose above control (open bars) 6 days after chronic BHT exposure and then declined by 30 days. (D) IL-4 levels 6 days after BHT are similar to corn oil controls but increase by Day 30. Data represent the mean ± sem. *, P < 0.001, compared with control; n = 5. Mice treated with corn oil control (open bars); mice treated with BHT (solid bars). (E) BAL macrophages (F4/80, green) express iNOS (red) but not arginase I (blue) 6 days after chronic BHT (upper row). (F) BDMCs (CD68, green, white arrowheads) also express iNOS but not arginase I 6 days after BHT (E and F). BAL macrophages and BDMCs (white arrowheads) from mice 30 days after chronic BHT administration are argI^high-iNOS^high (lower rows). Final original magnification, ×630.

Figure 2.

Changes in BAL cellular composition and in the polarization of BAL macrophages and BDMCs upon exogenous pulmonary IL-4 and IFN-γ administration. Pulmonary macrophages (A) and lymphocytes (B) increase after IL-4 or IL-4 + IFN-γ administration by microspray into the trachea. Data represent the mean ± sem; n = 5; *, P < 0.001, compared with PBS- and IFN-γ-treated mice. (C) BAL macrophages (F4/80, green) were immunostained for arginase I and iNOS. Exposure to PBS resulted in an argI^lowiNOS^low phenotype. Exposure to IL-4 induced arginase I (blue) but not iNOS, indicating M2 polarization. Exposure to IFN-γ induced iNOS (red) but not arginase I, indicating M1 polarization. Intrapulmonary administration of IL-4 and IFN-γ resulted in an argI^highiNOS^high phenotype. (D) Co-localized images of BDMCs (CD68, green, white arrowheads) express iNOS after pulmonary IFN-γ instillation, arginase I after IL-4 instillation, and iNOS and arginase I when instilled with both cytokines.

Figure 3.

M. tuberculosis pathogenesis in mice. (A) Granuloma progression after M. tuberculosis infection. (B) BAL macrophage (▪) numbers rise and fall slightly, and lymphocytes (▴) and neutrophils (•) increase steadily. (C) The BM cell population decreases shortly after bacterial exposure but returns to normal levels by 60 days after infection. (D) The number of megakaryocytes increases during M. tuberculosis infection. (E) There are fewer megakaryocytes (black circles) in the marrow of naïve mice compared with BM from a mouse 60 days post-challenge. Data represent mean ± sem; n = 6; *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with control.

Figure 4.

Pulmonary macrophages, but not BDMCs, are polarized during M. tuberculosis infection. (A) Macrophages (F4/80, green) express no arginase or iNOS at Day 7 following M. tuberculosis challenge. At Day 21, macrophages express iNOS (red) but not arginase (blue), an M1 polarization. At Days 35 and 60, macrophages lose iNOS expression and gain arginase I expression, an M2 phenotype. (B) BDMCs (CD68, green, white arrowheads) from mice after M. tuberculosis are argI^lowiNOS^low. Final original magnification, ×630.

Figure 5.

IHC staining of granulomas for iNOS and arginase I. IHC analysis of arginase I and iNOS in M. tuberculosis infected lungs 21 and 60 days after infection. Alveolar macrophages are circled in red. Granuloma-associated macrophages and alveolar macrophages exhibit iNOS staining starting 21 days after infection. By Day 60, alveolar macrophages express arginase I and not iNOS. Alveolar and granuloma macrophage IHC staining was photographed in the same lung at each time-point, and alveolar staining was examined near and distal to granulomas and found to be similarly stained for arginase I and iNOS regardless of distance from the granulomas. Final original magnification, ×400.