Macrophage polarization and activation at the interface of multi-walled carbon nanotube-induced pulmonary inflammation and fibrosis

Abstract

Pulmonary exposure to carbon nanotubes (CNTs) induces fibrosing lesions in the lungs that manifest rapid-onset inflammatory and fibrotic responses, leading to chronic fibrosis in animals and health concerns in exposed humans. The mechanisms underlying CNT-induced fibrogenic effects remain undefined. Macrophages are known to play important roles in immune regulation and fibrosis development through their distinct subsets. Here we investigated macrophage polarization and activation in mouse lungs exposed to multi-walled CNTs (MWCNTs). Male C57BL/6J mice were treated with MWCNTs (XNRI MWNT-7) at 40 μg per mouse (∼1.86 mg/kg body weight) by oropharyngeal aspiration. The treatment stimulated prominent acute inflammatory and fibrotic responses. Moreover, it induced pronounced enrichment and polarization of macrophages with significantly increased M1 and M2 populations in a time-dependent manner. Induction of M1 polarization was apparent on day 1 with a peak on day 3, but declined rapidly thereafter. On the other hand, the M2 polarization was induced on day 1 modestly, but was remarkably elevated on day 3 and maintained at a high level through day 7. M1 and M2 macrophages were functionally activated by MWCNTs as indicated by the expression of their distinctive functional markers, such as iNOS and ARG1, with time courses parallel to M1 and M2 polarization, respectively. Molecular analysis revealed MWCNTs boosted specific STAT and IRF signaling pathways to regulate M1 and M2 polarization in the lungs. These findings suggest a new mechanistic connection between inflammation and fibrosis induced by MWCNTs through the polarization and activation of macrophages during MWCNT-induced lung pathologic response.

Keywords: M1–M2 polarization; fibrosis; inflammation; macrophage; multi-walled carbon nanotube.

Figure 1.

Lung fibrosis and macrophage enrichment. Wild-type mice treated with DM or MWCNTs (40 μg per mouse) were sacrificed on day 1, 3, or 7 post-exposure. (A) Fibrillar collagens detected by Sirius Red/Fast Green staining in lung tissues from mice exposed for 7 days. Collagen fibers are in red and non-collagenous proteins in green (scale bar: 20 μm). (B) Accumulation of macrophages in fibrotic foci in the lungs on day 7 post-exposure examined by double immunofluorescence staining of F4/80 (green) and Collagen I (red). Blue indicates nuclear staining (scale bar: 20 μm). (C) and (D) Time-dependent induction of macrophages by MWCNTs in the lungs on days 1, 3, and 7 post-exposure determined by immunohistochemistry of F4/80 (C) and CD68 (D). Red indicates positive staining and blue nuclear counterstaining (scale bar: 50 μm). Quantification of positively stained cells is shown as mean ± SD (n = 4).

Figure 2.

M1–M2 macrophage polarization. Wild-type mice treated with DM or MWCNTs (40 μg per mouse) were sacrificed on day 1, 3, or 7 post-exposure. (A) Immunoblotting. Macrophage surface proteins CD86 and MHC II were detected as markers for M1 macrophages, and CD206 and CD163 for M2 macrophages (n = 2). (B) M1 polarization examined by double immunofluorescence staining of CD86 (red) and F4/80 (green). (C) M2 polarization examined by double immunofluorescence staining of CD206 (red) and F4/80 (green). Blue indicates nuclear staining (scale bar: 20 μm). Representative double positive cells are marked with arrows. Quantification of double positive cells is shown as mean ± SD (n = 4).

Figure 3.

Activation of M1 and M2 macrophages. Wild-type mice were exposed to DM or 40 μg MWCNTs for 1, 3, or 7 days. (A) Immunoblotting. iNOS was detected as a functional marker for M1 macrophage activation and ARG1 for M2 macrophage activation (n = 2). (B) M1 activation examined by double immunofluorescence staining of iNOS (red) and F4/80 (green). (C) M2 activation examined by double immunofluorescence staining of ARG1 (red) and F4/80 (green). Blue indicates nuclear staining (scale bar: 20 μm). Representative double positive cells are marked with arrows. Quantification of double positive cells is shown as mean ± SD (n = 4).

Figure 4.

Induction of FIZZ1 and YM1. Wild-type mice received DM or MWCNTs (40 μg per mouse) and were sacrificed on day 1, 3, or 7 post-exposure. (A) qRT-PCR. Levels of Fizz1 and Ym1 mRNA from lung tissues were determined, with Gapdh as internal control (mean ± SD, n = 5). (B) Immunoblotting. Levels of FIZZ1 and YM1 proteins in lung tissue lysates were determined (n = 2). (C) and (D) Time-dependent induction of FIZZ1 (C) and YM1 (D) detected by immunohistochemistry on lung tissue sections. Red indicates positive staining and blue nuclear counterstaining (scale bar: 50 μm). Quantification of positively stained cells is shown as mean ± SD (n = 4).

Figure 5.

Activation of STAT signaling. Wild-type mice were exposed to DM or 40 μg MWCNTs for 1, 3, or 7 days. (A) Immunoblotting. Phosphorylated STAT1 was detected as a marker for activation of M1 signaling, whereas phosphorylated STAT6 and phosphorylated STAT3 were examined for M2 signaling activation (n = 2). In addition to the Actin control, STAT1, STAT6, and STAT3 levels were examined as controls for the phosphorylated proteins, respectively. (B) Time-dependent activation of M1 signaling examined by double immunofluorescence staining of p-STAT1 (red) and F4/80 (green). (C) Time-dependent activation of M2 signaling determined by double immunofluorescence staining of p-STAT6 (red) and F4/80 (green). Blue indicates nuclear staining (scale bar: 20 μm). Representative double positive cells are marked with arrows. Quantification of double positive cells is shown as mean ± SD (n = 4).

Figure 6.

Activation of IRF signaling. Wild-type mice received DM or MWCNTs (40 μg per mouse) and were sacrificed on day 1, 3, or 7 post-exposure. (A) Activation of IRF signaling examined by immunoblotting. IRF5 was detected as a marker for M1 signaling activation and IRF4 for M2 signaling activation (n = 2). (B) and (C) Time-dependent induction of IRF5 (B) and IRF4 (C) detected by immunohistochemistry. Red indicates positive staining and blue nuclear counterstaining (scale bar: 50 μm). Quantification of positively stained cells is shown as mean ± SD (n = 4).