Sex differences in the acute and subchronic lung inflammatory responses of mice to nickel nanoparticles

Abstract

Nickel nanoparticles (NiNPs) are increasingly used in nanotechnology applications, yet information on sex differences in NiNP-induced lung disease is lacking. The goal of this study was to explore mechanisms of susceptibility between male and female mice after acute or subchronic pulmonary exposure to NiNPs. For acute exposure, male and female mice received a single dose of NiNPs with or without LPS by oropharyngeal aspiration and were necropsied 24 h later. For subchronic exposure, mice received NiNPs with or without LPS six times over 3 weeks prior to necropsy. After acute exposure to NiNPs and LPS, male mice had elevated cytokines (CXCL1 and IL-6) and more neutrophils in bronchoalveolar lavage fluid (BALF), along with greater STAT3 phosphorylation in lung tissue. After subchronic exposure to NiNPs and LPS, male mice exhibited increased monocytes in BALF. Moreover, subchronic exposure of male mice to NiNP only induced higher CXCL1 and CCL2 in BALF along with increased alveolar infiltrates and CCL2 in lung tissue. STAT1 in lung tissue was induced by subchronic exposure to NiNPs in females but not males. Males had a greater induction of IL-6 mRNA in liver after acute exposure to NiNPs and LPS, and greater CCL2 mRNA in liver after subchronic NiNP exposure. These data indicate that susceptibility of males to acute lung inflammation involves enhanced neutrophilia with increased CXCL1 and IL-6/STAT3 signaling, whereas susceptibility to subchronic lung inflammation involves enhanced monocytic infiltration with increased CXCL1 and CCL2. STAT transcription factors appear to play a role in these sex differences. This study demonstrates sex differences in the lung inflammatory response of mice to NiNPs that has implications for human disease.

Keywords: Nickel nanoparticles; inflammation; lung; sex; susceptibility.

Figure 1.

Acute lung exposure of male and female mice by oropharyngeal aspiration (OPA) to LPS, NiNPs, or LPS and NiNPs. A) Transmission electron microscopy (TEM) of nickel nanoparticles used in this study (bar equals 100 nm). B) Illustration of experimental design. C57BL/6J mice were exposed to vehicle (0.1% Pluronic in DPBS), LPS (5 μg/kg), NiNPs (4 mg/kg), or LPS (5 μg/kg) and NiNPs (4 mg/kg) co-exposure via OPA delivery. Mice were euthanized and necropsy performed 24 hrs after the exposure. C) Protein concentration in BALF determined by Pierce BCA Protein Assay. D) Lactate dehydrogenase (LDH) activity in BALF measured using Pierce LDH Cytotoxicity Assay. (n=6 mice per group, ***P<0.001, ** P<0.01 or *P<0.05 compared to vehicle determined by one-way ANOVA, #P<0.05 between sexes as determined by two-way ANOVA).

Figure 2.

Inflammatory cells in the BALF from male and female mice 24 hrs after exposure to LPS, NiNPs, or LPS and NiNPs using the protocol illustrated in Fig. 1. A) Representative microscopic images of BALF cells from male and female mice on Diff-Quick stained Cytospin slides from each exposure group (Magnification 200X; inset 1000X oil immersion). B) Average number of total cells, neutrophils or macrophage per three microscopic fields on BALF Cytospin slides after acute (24 hr) exposure to NiNPs with or without LPS (Average number of cells were counted as described in the methods). (n=5–6 mice per group, ***P<0.001 or **P< 0.01 or *P<0.05 compared to vehicle determined by one-way ANOVA, ###P<0.001 or #P<0.05 between sexes determined by two-way ANOVA).

Figure 3.

Levels of pro-inflammatory cytokines in BALF and lung tissue. A) IL-6 protein in BALF measured by ELISA. B) IL-6 mRNA in lung tissue measured by qRT-PCR. C) CXCL1 protein in BALF measured by ELISA. D) CXCL1 mRNA in lung tissue measured by qRT-PCR. (n=5–6 mice per group, ***P<0.001 or **P< 0.01 or *P<0.05 compared to vehicle determined by one-way ANOVA, ###P<0.001 or ##P<0.01 between sexes determined by two-way ANOVA).

Figure 4.

Western blot analysis of STAT proteins from the lung tissue of male or female mice after acute exposure to NiNPs in the absence or presence of LPS A) Representative western blots of phosphorylated STAT3 (p-STAT3), total STAT3, and β-actin in lung tissue from each treatment group in male and female mice. B) Quantitative densitometry of the average expression of pSTAT3 normalized for total STAT3. C) Representative western blots of total STAT1 and β-actin in lung tissue from each treatment group in male and female mice. D) Quantitative densitometry of the average expression of total STAT1 normalized for β-actin. (n=4–6, **P< 0.01 or *P<0.05 compared to vehicle determined by one-way ANOVA, ^^P<0.01 compared to vehicle by paired Student’s t-test, #P<0.05 between sexes using Student’s t-test).

Figure 5.

Subchronic exposure of male and female mice by repeated oropharyngeal aspiration (OPA) to LPS, NiNP, or LPS and NiNP over a period of three weeks. A) Illustration of experimental design. C57BL/6J mice were exposed to vehicle (0.1% Pluronic in DPBS), LPS (0.83 μg/kg), NiNPs (0.67 mg/kg), or combination of (0.83 μg/kg) LPS and (0.67 mg/kg) NiNPs on days 1, 3 and 5, then allowed to rest for week and exposed again on days 15, 17 and 19. Mice were euthanized and necropsy performed 24 days after the first exposure. B) Total protein concentration in BALF was determined by Pierce BCA Protein Assay. C) Lactate dehydrogenase (LDH) activity in BALF was measured using Pierce LDH Cytotoxicity Assay. (n=5–6 mice per group, ***P<0.001 or **P<0.01 or *P<0.05 compared to vehicle determined by one-way ANOVA).

Figure 6.

Inflammatory cells in the BALF of male and female mice 24 days after repeated subchronic exposure to LPS, NiNPs, or LPS and NiNPs. A) Microscopic images of representative cell types on BALF Cytospin slides used for identification (Magnification, 1000X oil immersion, taken from a male mouse exposed to NiNPs and LPS). B) Representative microscopic images of BALF cells from male and female mice on Diff-Quick stained Cytospin slides from each exposure group (Magnification 200X; inset 1000X oil immersion). C) Average number of total cells, monocytes, macrophages, neutrophils or lymphocytes per three microscopic fields on BALF Cytospin slides after repeated subchronic exposure to NiNPs with or without LPS. Average number of cells were counted as described in the methods. (n=5–6 mice per group, ***P<0.001 or **P< 0.01 or *P<0.05 compared to vehicle determined by one-way ANOVA, ###P<0.001, ##P<0.01 or #P<0.05 between sexes determined by two-way ANOVA).

Figure 7.

Hexagonal crystals in the lungs of male and female mice after repeated subchronic exposure to NiNPs. A) Representative microscopic image of Diff-Quick stained Cytospin slide from a male mouse 24 days after repeated exposure to NiNPs showing a crystal with BALF cells (Magnification 200X; inset 1000X oil immersion with arrowheads indicating NiNPs). B) Hematoxylin and eosin stained lung section from a male mouse after repeated subchronic exposure to NiNPs showing side view of a crystal (arrow) in the bronchoalveolar region associated with a macrophage containing NiNPs (arrowheads). C) Average number of crystals in the BALF of male and female mice after exposure to NiNPs in the absence or presence of LPS. The data represent the total numbers of crystals in Cytospin slides from each animal. (n=5–6 mice per group, *P<0.05 compared to vehicle determined by one-way ANOVA).

Figure 8.

Lung pathology of male and female mice after repeated subchronic exposure to NiNPs in the absence or presence of LPS. A) Representative hematoxylin and eosin stained lung sections from mice treated with NiNPs or LPS and NiNPs. Inflammatory lesions are indicated by arrows. ‘A’ and ‘V’ indicate airways and vessels, respectively. B) Results of pathology scoring of inflammatory patterns showing relative scores for cellular infiltrates in alveolar lumina, perivascular and peribronchiolar lymphocyte aggregates and dense epithelioid macrophage aggregates. See methods and Supplementary File 1 for scoring system. (n=5–6 mice per group, ***P<0.001 or **P< 0.01 compared to vehicle determined by one-way ANOVA, ##P<0.01 between sexes determined by two-way ANOVA).

Figure 9.

Protein levels of cytokines measured by ELISA in the lungs of male and female mice after repeated subchronic exposure to NiNPs in the absence or presence of LPS. A) CXCL1 protein in BALF. B) IL-6 protein in BALF. C) CCL2 protein in BALF and lung lysates. (n=5–6 mice per group, ***P<0.001 or **P< 0.01 or *P<0.05 compared to vehicle determined by one-way ANOVA, ##P<0.01 or ###P<0.001 between sexes determined by two-way ANOVA).

Figure 10.

Western blot analysis of STAT proteins from the lung tissue of male or female mice after repeated subchronic exposure to NiNPs in the absence or presence of LPS. A) Representative western blots of p-STAT3, total STAT3, and β-actin in lung tissue from each treatment group in male and female mice. B) Quantitative densitometry of the average expression of p-STAT3 normalized for total STAT3. C) Representative western blot of total STAT1 and β-actin in lung tissue from each treatment group in male and female mice. D) Quantitative densitometry of the average expression of total STAT1 normalized for β-actin. (n=4–6 mice per group, **P< 0.01 or *P<0.05 compared to vehicle determined by one-way ANOVA, ##P<0.01 or #P<0.05 between sexes determined by using Student’s t-test).

Figure 11.

Hypothetical illustration of mechanisms underlying the susceptibility of male mice to a single acute or repeated subchronic exposure to NiNPs in the absence or presence of LPS. Red and green arrows indicate an increase or decrease in males compared to females.