Plasma Membrane Damage by Environmental Materials Enhances Cell-Cell Fusion and Impairs Immune Functions of Macrophages

Abstract

Macrophages are the most abundant phagocytes and play an essential role in host defense. Previous studies have shown that many environmental materials can activate macrophages and trigger inflammatory responses. However, whether these exposures alter macrophage function in host defense remains unclear. This study found that many environmental materials (such as carbon nanotubes, tungsten carbide [WC], and detergents) can damage the plasma membranes of macrophages. This damage leads to decreased reactive oxygen species (ROS) production and phagocytosis but elevated cell-cell fusion. In vivo, airway exposure to laundry detergent impaired the recruitment of macrophages and other myeloid cells to the lung and dramatically dampened protective TH1 and TH17 cell responses, leading to increased susceptibility to Candida infection in mice. Overall, our data indicate that exposure to environmental materials compromises macrophage membrane integrity and impairs host defense. These findings may aid in the development of effective preventive and therapeutic strategies.

Keywords: Environmental Materials; Macrophages; Membrane Damage; Phagocytosis.

Figure 1.. Environmental materials damage macrophage plasma membranes.

(A) BMDMs were treated with saponin, detergent, or a vehicle. (B) BMDMs were treated with carbon nanotubes or a vehicle. Plasma membrane damage was assessed by Annexin V staining, and cell death was indicated by PI staining. Horizontal scale bar, 50 μm.

Figure 2.. Environmental material-caused membrane damage promotes macrophage fusion.

BMDMs were treated with carbon nanotubes (A), saponin (B), detergent (C), or a vehicle and then cultured with or without IL-4. Macrophage fusion was assessed by H&E staining. Horizontal scale bar, 100 μm. FBGC, foreign body giant cells; Veh, vehicle. Data (mean ± SD) shown are representative of 2independent experiments (n = 3 per group). Student’s t-test, *p < 0.05; **p < 0.005.

Figure 3.. Saponin enhances macrophage fusion via upregulation of CD44.

(A) Flow cytometry of CD44 expression on BMDMs after treatment with or without saponin. (B) Representative fluorescent images of Cd44-WT (Upper) or Cd44-deficient (Lower) BMDMs treated with saponin or a vehicle in the presence of IL-4. Images were acquired by using Cellomics. MGCs are indicated by arrowheads. (C) Statistical analysis of macrophage fusion in (B), respectively. One hundred cells (n = 100) were assessed in each sample. Percentages of cells with 2 or ≥3 nuclei were depicted. Veh, vehicle; Sap, saponin. Data shown are representative of 2 independent experiments. Student’s t-test, *p < 0.05.

Figure 4.. Environmental materials (saponin and detergent) decrease the production of ROS and pro-inflammatory cytokines in macrophages.

BMDMs were treated with saponin (A-C), detergent (D-F), or a vehicle and stained with DCFH-DA and Hoechst. Veh, vehicle; Det, detergent. (A, D) Representative fluorescent images. Horizontal scale bar, 50 μm. (B, E) Mean fluorescent intensity (MFI) of DCFH-DA. (C, F) RT-qPCR of mRNA expression of indicated genes. Actb was used as an internal loading control. Data (mean ± SD) shown are representative of 2 independent experiments (n = 3 per group). Student’s t-test, *p < 0.05; **p < 0.005.

Figure 5.. Environmental material-caused membrane damage impairs the phagocytic function of macrophages.

BMDMs were cocultured with CFSE-labelled E. coli (A-C) or C. albicans (D, E). After fixation, the cells were stained with CD11b (red) and DAPI (blue). (A, C) Phagocytosis of E. coli (A) or C. albicans (C) by BMDMs treated with/without detergent or saponin, respectively. Horizontal scale bar, 20 μm. (C, D) Statistical analysis of (A, C). One hundred cells (n = 100) were assessed in each sample. Det, detergent. Data shown are representative of 3 independent experiments. Student’s t-test, **p < 0.005.

Figure 6.. Laundry detergent increases susceptibility to Candida airway infection in mice

(A) C57BL/6 mice were intratracheally (i.t.) administered live Candida following i.t. receiving a laundry detergent or PBS once daily for four consecutive days, and sacrificed on the next day after the last dose of Candida. (B) Colony-forming unit (CFU) assays of Candida burden in the bronchoalveolar lavage fluids (BALF). (C) Body weights of the two groups of mice. (D) Profiles of myeloid cells in BALF. (E) Flow cytometry of IL-17- and IFNγ-expressing CD4⁺ TH cells in BALF. (F) ELISA of the levels of IL-17 and IFNγ in BALF. (G) Flow cytometry of MHC-II and CD68 on macrophages on a CD11b⁺Cd11c^lo gate. (H) Mean fluorescence intensity of MHC-II and CD68 in (G). (I) Flow cytometry of MHC-II and CD68 on dendritic cells on a CD11b⁺Cd11c^hi gate. (J) Mean fluorescence intensity of MHC-II and CD68 in (I). Veh, vehicle; Det, detergent; Iso, isotype control. Data (mean ± SD) are representative of two experiments. n = 5-6 per group. Student’s t-test, *p< 0.05; **p < 0.005.