Innate immune processes are sufficient for driving silicosis in mice

Abstract

The lung is constantly exposed to potentially pathogenic particles and microorganisms. It has become evident recently that not only innate but also adaptive immune responses to particulates, such as SiO(2) entering the respiratory tract, are complex and dynamic events. Although the cellular mechanisms and anatomical consequences involved in the development of silicosis have been studied extensively, they still remain poorly understood. Based on their capacity for immune regulation, lymphocytes may play a key role in the respiratory response to environmental challenge by SiO(2). The objective of this study was to characterize the impact of SiO(2) exposure on respiratory immune processes, with particular emphasis on evaluating the importance of lymphocytes in the murine silicosis model. Therefore, lymphopenic mice, including NK-deficient, Rag1(-/-), or a combination (Rag1(-/-) NK-depleted), were used and demonstrated that SiO(2)-induced fibrosis and inflammation can occur independently of T, B, NK T, and NK cells. Studies in Rag1(-/-) mice suggest further that lymphocytes may participate in the regulation of SiO(2)-induced inflammation through modulation of the Nalp3 inflammasome. This observation may have clinical relevance in the treatment of inflammatory and fibrotic lung diseases that are refractory or respond suboptimally to current therapeutics.

Figure 1.

Interstitial cell changes over time following instillation with SiO₂. Following an initial loss in cell number, exposure to SiO₂ increased the number of interstitial leukocytes recovered from the lungs over time compared with saline control (A). Within the interstitial immune cells recovered at 14 days post- SiO₂ exposure, each of the four subpopulations of lymphocytes examined was augmented compared with saline control, although the ration amongst the cell populations remained unchanged (B). Values are means ± sem; n = 5 mice/group; *P < 0.05 compared with saline control. The percent (C) and absolute number (D) of NK1.1⁺ CD3^– NK cells increased at 3, 7, and 28 days but decreased at 16 weeks post-SiO₂ exposure compared with saline. No change was observed at Day 1 in the percent or the absolute number. Values are means ± sem; n = 8–13 mice/group; *P < 0.05 compared with saline control.

Figure 2.

SiO₂-induced changes in pulmonary NK subsets over time. Analysis of the NK cell subsets demonstrates changes in the percent and absolute number of immature (R1), double-positive (R2), and mature (R3) NK cell subsets in response to SiO₂ exposure over time. Values are means ± sem; n = 8–13 mice/group; *P < 0.05 compared with cumulative saline control average.

Figure 3.

Cytokine expression following SiO₂ exposure. SiO₂ increased the levels of IFN-γ, TNF-α, IL-10, and IL-13, present in whole lung homogenates over time, compared with saline control (dashed line; n=5 mice/group; *P < 0.05 compared with saline control; A). Sterile-sorted NK1.1⁺ cells from 7 days post-saline- or -SiO₂-exposed mice contributed to increased levels of TNF-α and IL-13, present in the whole lung homogenates. In contrast, SiO₂ exposure resulted in decreased production of IFN-γ and no change in IL-10 production by NK1.1⁺ cells (B). Values are means ± sem; n = 6–12 wells/group; *P < 0.05 compared with saline control.

Figure 4.

Collagen deposition following instillation with SiO₂. SiO₂ exposure results in fibrosis in lymphopenic mice. All SiO₂-exposed mice, regardless of strain, have significantly higher levels of hydroxyproline compared with their respective saline controls. In contrast, TiO₂ (nonfibrogenic control particle) showed no change from saline control values (A); n = 7–17 mice/group. Values are means ± sem; *P < 0.05 compared with saline controls. Lung sections from C57Bl/6 and lymphocyte-deficient mice exposed to SiO₂ show increased collagen deposition, as measured by quantification of LY fluorescence using LSC techniques (B); n = 5–6 mice/group. Values are means ± sem; *P < 0.05 compared with saline controls.

Figure 5.

Histological comparison of lymphocyte-deficient mice compared with controls following instillation with SiO₂. Lung sections from C57Bl/6 and lymphopenic mice 16 weeks following exposure were subjected to histochemical analysis for Gomori’s trichrome. A comparison of the gross architecture of the lung sections from saline-exposed mice revealed no difference between lymphocyte-deficient strains and C57Bl/6 mice (data not shown). In contrast, SiO₂-exposed mice develop features of pulmonary inflammation and fibrosis, characteristic of murine silicosis. Representative images from C57Bl/6 mice show increased staining for collagen (green), as well as increased inflammation and granuloma formation typical of chronic silicosis (A). Similar results were obtained from NK-deficient mice (B). In contrast, although Rag1^−/− (C) and Rag1^−/− NK-depleted (D) mice showed increased collagen deposition, inflammation was diffuse throughout the parenchyma, and granulomas were not visible; n = 5–6 mice/group; 20× original magnification.

Figure 6.

IL-1β and IL-18 expression in the whole lung lavage and lung homogenates of SiO₂-exposed mice. C57Bl/6 and Rag1^−/− mice were exposed to saline or SiO₂ and whole lung lavage (A and C) and lung homogenates (B and D) analyzed for the expression of IL-1β and IL-18. SiO₂-exposed C57Bl/6 and Rag1^−/− mice showed elevated levels of IL-1β and IL-18 at Day 28, although Rag1^−/− mice showed greater increases in cytokine expression relative to saline control values; n = 7–17 mice/group. Values are means ± sem; *P < 0.05 compared with saline controls.