All relevant data are within the manuscript and its Supporting Information files. Introduction {#sec001} ============ Sulfur mustard (SM) or mustard gas, 1,2-bis(chloroethyl)-sulfide, is a blistering chemical warfare agent that is listed by the Chemical Weapons Convention (CWC) as a toxic chemical warfare agent \[[@pone.0211171.ref001]\]. SM is a potent alkylating agent that attacks DNA, RNA, and proteins. Long-term adverse health outcomes of SM exposure include skin, eye, and respiratory system disorders. SM also causes liver, kidney, and hematopoietic system damage \[[@pone.0211171.ref002]\]. In the late 1930s, SM was deployed in the chemical warfare in the battlefields of World War I. During World War II, Germany and Japan used SM in bombs and grenades and in gas masks for attacks. Recently, the use of this chemical agent as a weapon has been reported in Iran-Iraq war \[[@pone.0211171.ref003]\]. SM is a highly volatile chemical agent. Thus, its accidental release can cause severe toxicity. Moreover, because of its toxic characteristics, it is a major environmental pollutant and remains a potent terrorist threat \[[@pone.0211171.ref004]\]. The toxicity of SM is dependent on the dose. In acute exposures, SM causes symptoms of irritation and inflammation. The acute lethal dosage for rats exposed to SM is considered to be about 2--10 mg/kg body weight \[[@pone.0211171.ref005]\]. Based on this lethal dose, the safe therapeutic dose of SM is considered to be 5% of the acute lethal dose. In humans, SM exerts toxic effects on different cell types. The eye is considered to be a highly susceptible target to SM injury. Eye injuries include conjunctivitis and keratitis and may result in blindness in exposed individuals \[[@pone.0211171.ref006]\]. In addition to this, SM causes skin burns, and mucous membrane injuries. An inhalation exposure of rats to SM results in mucous membrane and lung damage \[[@pone.0211171.ref007]\]. In addition to SM, mustard-fiber (MF), a long-chained polyfunctional chemical residue, is also released during SM deployment. Similar to SM, MF is highly toxic. Therefore, this chemical species may cause severe damage to the respiratory system \[[@pone.0211171.ref008]\]. Since the United Nations (UN) recognized the persistent threat posed by this chemical species, in 2006, the UN and the Organisation for the Prohibition of Chemical Weapons (OPCW) agreed on an export ban on MF for any nonmilitary purpose \[[@pone.0211171.ref009]\]. Many efforts have been made to find effective therapies for SM toxicity in human beings. These efforts include the use of vitamin B12 to reduce the severity of SM exposure symptoms \[[@pone.0211171.ref010]\]. However, this approach has some limitations. This chemical species also causes damage to the central nervous system (CNS) and may result in neurotoxic effects in the nervous system. Moreover, vitamin B12 has side effects that limit its use. Therefore, there is a critical need for new treatments for SM toxicity in human beings. Recently, several studies have reported that MSCs reduce SM toxicity \[[@pone.0211171.ref011],[@pone.0211171.ref012]\]. MSCs are multipotent adult stem cells that can be found in bone marrow, dental pulp, and umbilical cord blood \[[@pone.0211171.ref013]\]. MSCs are well-characterized stromal cells that have a self-renewal ability and can differentiate into multiple cell types. Thus, these cells can be used as tools to treat several diseases \[[@pone.0211171.ref014]\]. In addition, MSCs have been shown to inhibit wound repair in burn patients by reducing necrosis in the wound site \[[@pone.0211171.ref015]\]. Moreover, MSCs have anti-inflammatory and immunomodulatory properties and can be considered as therapeutic agents for inflammatory and autoimmune diseases such as type 1 diabetes mellitus, multiple sclerosis, and graft-versus-host disease (GVHD) \[[@pone.0211171.ref016]\]. Moreover, studies have shown that MSCs can inhibit acute lung injury and improve the course of the disease \[[@pone.0211171.ref017]\]. The therapeutic effects of MSCs are associated with secreted factors in their secretome \[[@pone.0211171.ref018],[@pone.0211171.ref019]\]. The MSC secretome comprises more than 30% of the protein content of MSCs, including growth factors and cytokines \[[@pone.0211171.ref020]\]. This pool of secreted factors has anti-inflammatory and immunomodulatory properties and is involved in the wound healing process. MSCs produce paracrine factors that enhance tissue repair by suppressing local inflammation and fibrosis. In addition to this, MSCs produce pro-angiogenic, anti-apoptotic, and immunomodulatory factors that lead to the generation of new blood vessels, enhanced extracellular matrix (ECM) remodeling, and the inhibition of the immune responses. Thus, MSCs can be used as a promising cell source for the treatment of many diseases such as wound healing and GVHD \[[@pone.0211171.ref021],[@pone.0211171.ref022]\]. Furthermore, MSCs play a role in tissue repair and have been shown to be therapeutic agents for several diseases \[[@pone.0211171.ref023]\]. One of the most important features of MSCs is that they are used as immunomodulatory and anti-inflammatory factors by regulating the immune cells and avoiding their recognition and attack by immune system cells. Previous studies have reported that MSCs are capable of migration and migration to injured areas and their effect in acute inflammation and tissue repair in the treatment of chemical injuries such as mustard gas, cisplatin, and lipopolysaccharide \[[@pone.0211171.ref024]--[@pone.0211171.ref027]\]. We hypothesized that MSCs would modulate the immune response by secreting soluble factors in SM-exposed tissues, which in turn, promotes tissue regeneration. This hypothesis was based on several previous reports \[[@pone.0211171.ref028]--[@pone.0211171.ref030]\]. However, although MSCs have been shown to be beneficial in various types of chemical injuries \[[@pone.0211171.ref031]--[@pone.0211171.ref033]\], the therapeutic effect of MSCs on SM-induced tissue damage has not yet been demonstrated. Therefore, the present study evaluated the therapeutic effect of MSCs on mice exposed to SM. Moreover, the anti-inflammatory effects of MSCs on injured mouse tissues were evaluated. The study evaluated the use of MSCs to reduce the severity of chemical agent damage in different tissues in order to develop a novel treatment for chemical agents. Materials and methods {#sec002} ===================== Preparation of chemical mustard {#sec003} ------------------------------- A 30% SM in 70% ethanol was used for animal exposure. This chemical was purchased from the Iranian Biological and Technical Research Center (Tehran, Iran). Animal model of SM-induced tissue damage {#sec004} ---------------------------------------- All animal experiments were approved by the Animal Research Ethics Committee of Ahvaz Jundishapur University of Medical Sciences (AJUMS) (approval no.: IR.AJUMS.REC.1395.985). All of the animals were treated according to the Animal Care Guidelines of AJUMS. In this study, 60 Swiss mice (21 ± 2 g in weight) were used. These mice were purchased from the Pasteur Institute (Tehran, Iran). The mice were kept at the animal facility of the School of Public Health (Ahvaz, Iran) for one week under laboratory conditions (temperature of 22--24°C, relative humidity of 45--65%, 12-h light/dark cycle, and a standard rodent diet). SM-induced toxicity was evaluated in mice via an intraperitoneal (i.p.) injection of 30% SM diluted in 70% ethanol (1 mL). Male Swiss mice (n = 60, 21 ± 2 g in weight) were divided into 4 groups as follows: control mice were given normal saline, the sham group was exposed to ethanol only, and the SM-treated group received 30% SM in 70% ethanol for 1 h. In the MSC treatment group, 30% SM in 70% ethanol was injected (i.p.) to the mice for 1 h, followed by an i.p. injection of the MSCs (1 × 10^6^ cells/mice) for 2 weeks. Vitamin B12 as an anti-inflammatory therapy was used as a positive control. Vitamin B12 was diluted in normal saline and injected (i.p.) to the mice for 1 h. The i.p. injection of vitamin B12 was performed at 1 mg/kg dose after exposure to SM. Vitamin B12 was purchased from Sigma-Aldrich (USA). Histopathological analysis {#sec005} -------------------------- After two weeks, mice were euthanized and samples of the lungs and testes were collected and fixed in 10% formalin for 24 h. These samples were processed and embedded in paraffin according to the standard methods. Paraffin-embedded tissue samples were prepared, sliced, and stained with hematoxylin and eosin (H&E) using standard procedures. Tissue preparation and flow cytometry analysis {#sec006} ---------------------------------------------- The inguinal lymph nodes (n = 3) and thymus (n = 3) were removed from each mouse. These organs were added to 2-mL Eppendorf tubes and centrifuged at 1,000 rpm for 5 min. After