Inhalation of sulfur mustard causes long-term T cell-dependent inflammation: Possible role of Th17 cells in chronic lung pathology

Abstract

Sulfur mustard (SM) is a highly toxic chemical warfare agent that remains a threat to human health. The immediate symptoms of pulmonary distress may develop into chronic lung injury characterized by progressive lung fibrosis, the major cause of morbidity among the surviving SM victims. Although SM has been intensely investigated, little is known about the mechanism(s) by which SM induces chronic lung pathology. Increasing evidence suggests that IL-17⁺ cells are critical in fibrosis, including lung fibrotic diseases. In this study we exposed F344 rats and cynomolgus monkeys to SM via inhalation and determined the molecular and cellular milieu in their lungs at various times after SM exposure. In rats, SM induced a burst of pro-inflammatory cytokines/chemokines within 72 h, including IL-1β, TNF-α, IL-2, IL-6, CCL2, CCL3, CCL11, and CXCL1 that was associated with neutrophilic infiltration into the lung. At 2 wks and beyond (chronic phase), lymphocytic infiltration and continued elevated expression of cytokines/chemokines were sustained. TGF-β, which was undetectable in the acute phase, was strongly upregulated in the chronic phase; these conditions persisted until the animals were sacrificed. The chronic phase was also associated with myofibroblast proliferation, collagen deposition, and presence of IL-17⁺ cells. At ≥ 30 days, SM inhalation promoted the accumulation of IL-17⁺ cells in the inflamed areas of monkey lungs. Thus, SM inhalation causes acute and chronic inflammatory responses; the latter is characterized by the presence of TGF-β, fibrosis, and IL-17⁺ cells in the lung. IL-17⁺ cells likely play an important role in the pathogenesis of SM-induced lung injury.

Introduction

Sulfur mustard (SM; 2-bis-chloroethyl-sulfide) is a highly toxic vesicant that has been used in war settings, causing injuries and deaths to military and civilian personnel [1], [2]. Because of the ease of production and stability, SM represents a potential terrorist threat [3]. SM exposure occurs primarily through inhalation and absorption through skin and the anterior surface of the eye, making the lungs, the skin, and the eye as major targets of SM toxicity [4]. The severity of damage depends largely on the dose and duration of exposure. Acute effects may include skin blisters, eye irritation, and breathing discomfort (chest tightness, hacking cough, rhinorrhea); however, at sublethal doses these effects are relatively temporary. During the Iran–Iraq war, immediate mortality among the Iranian soldiers exposed to SM was only 3–4%, but decades later nearly half of the victims developed chronic respiratory complications (chronic bronchitis, airway hyperreactivity, lung fibrosis, and bronchopneumonia) — the primary cause of morbidity and mortality among these soldiers [2], [5], [6], [7], [8], [9].

The mechanism(s) of SM-induced respiratory toxicity are not clear. Altered immune/inflammatory responses such as decreased natural killer cell numbers [10], increased cytokine production [11], and possible changes in cytotoxic T cells [12], [13] have been reported in Iranian SM victims. Increased expression of proinflammatory cytokines has been observed in the rat skin [14] and lungs [15], and human lung cell cultures [16], [17] after SM exposure. A single lung exposure to the nitrogen mustard analog melphalan in mice induced an acute inflammatory response with increased IL-1 and IL-6 in the bronchoalveolar lavage (BAL) as well as a chronic respiratory impairment characterized by lymphocytic infiltration and lung fibrosis [18]. In a recent study exposure of mice to nitrogen mustard induced T cell-dependent long-term lung pathology [19].

The role of T cells in SM-induced lung injury is not well delineated. We have demonstrated that hairless euthymic guinea pigs dermally exposed to SM developed a SM-specific, delayed-type hypersensitivity response, suggesting that SM can activate specific T cell-mediated immune responses [20]. Th17, a subset of CD4⁺ T cells, has recently been implicated in a number of inflammatory, autoimmune, and chronic fibrotic lung diseases [21], and a strong relationship between the profibrotic cytokines and Th17 has been established [22]. In this communication, we show that SM exposure causes acute and delayed responses. The delayed lung responses include lung fibrosis and the presence of IL-17⁺ cells in the inflamed regions of the lung, suggesting that IL-17⁺ cells may have an important role in the development of chronic fibrosis in SM-exposed lungs.