We investigated the impact of hAM-MSCs-CM on spleen cells, cytokines in the spleen culture supernatant, and lung α-SMA protein expression for the first time. Our findings indicated that the CM was capable of reducing α-SMA expression in the treatment group compared to the asthma group. Additionally, it was able to suppress inflammatory cytokines like IgE, IL-4, and TGF-β, which OVA elevated, and compensate for the reduction in IFN-γ and IL-10 in splenic cells caused by OVA. Our findings corroborated those of Koopmans et al., who demonstrated that when mice were exposed to OVA, IgE and Th2 cytokines such as IL-4 increased, and α-SMA protein expression increased in mice receiving OVA [7].
Bronchial wall remodeling and chronic inflammation occur as a result of the transformation of fibroblasts to myofibroblasts. In healing tissues, fibroblasts exhibit a de novo contractile phenotype of α-SMA expression, called myofibroblasts phenotype, linked with the secretion of significant substances, including ECM components such as collagen and fibronectin. These variables are all associated with pulmonary fibrosis. Myofibroblasts are expressed at high collagen I and III levels, α-SMA protein, and proteoglycans. Excessive myofibroblasts activity results in fibrosis and malfunction [19, 20].
According to studies, asthmatic patients have a greater level of α-SMA protein expression than the control group. The most significant discovery in myofibroblasts differentiation from fibroblasts is the expression of α-SMA protein. Its expression grows in proportion to the degree of differentiation. TGF-β is critical in fostering this differentiation [20]. As shown in Fig. 1, the findings of this research were linked with higher α-SMA protein expression in the asthma group relative to controls, which was substantially decreased by CM therapy. Ramos-Barbón et al. demonstrated a clear connection between T cell proliferation in ASM remodeling and α-SMA protein expression in asthmatic airway tissue, resulting in an increase in the airway and subepithelial smooth muscle mass [10].
Additionally, active TGF-β and collagen synthesis in fibrous tissues may be represented by α-SMA protein. Sun et al. demonstrated that bleomycin-induced fibrosis mice had higher levels of α-SMA protein in their lungs than controls [21]. Another research by Liu et al. demonstrated that pulmonary fibrosis had enhanced differentiation of fibroblasts to myofibroblasts, increased collagen production, and higher α-SMA protein expression. They demonstrated that decreasing TGF-β may inhibit fibroblast development into myofibroblasts and, therefore, fibrosis [22]. Additionally, α-SMA protein expression may be decreased in pulmonary fibroblasts by inactivating TGF-β. Hinz et al. established a clear correlation between α-SMA protein expression levels in cultured fibroblasts. Additionally, there is a direct connection between TGF-β and myofibroblasts, such that by lowering TGF-β, myofibroblast activity and α-SMA mass are decreased [23]. As a result, we hypothesized that the CM might decrease α-SMA protein production and fibroblast differentiation by influencing the immune system and ultimately decreasing TGF-β.
Allergic disorders have been linked to the spleen, according to reports. Spleen cells from OVA-sensitized mice were stimulated in vitro, resulting in the formation of mast cell populations. These cells formed in the spleen from hematopoietic stem cells and progenitor cells. Mast cells develop in the spleen during inflammatory diseases, including allergies, capable of releasing cytokines such as IL-4 in response to receptor-bound IgE. Mast cells are critical in eliciting the Th2 response, increasing IL-4 production, resulting in a Th1/Th2 imbalance. Because IFN-γ suppresses Th2 differentiation, a reduction in IFN-γ exacerbates the Th2 response, resulting in allergic inflammation [11]. Spleen cells of ovalbumin-sensitive mice produce significant amounts of IL-4, IL13 and IgE in response to OVA stimulation [24]. According to Li et al. research’s IL-4 expression was substantially higher in the asthma group's spleen than controls [5]. In the study by Yun et al., the asthma group had a greater level of spleen cell culture supernatant IL-4 than the control group, whereas the control group had a lower level of IFN-γ [4]. Our findings illustrate concerning this material. The asthma group's splenic culture supernatants included more IL4, IgE, and TGF-β, whereas the controls contained fewer IFN-γ and IL-10, and CM therapy reversed the findings.
Dendritic cells are found in lymphoid and non-lymphoid tissues and present antigens to T cells. Dendritic cells in the spleen (a secondary lymphoid tissue) are critical for T cell activation, which eventually results in a reduction in IFN-γ and increases IL-4 via the differentiation of Th cells. Indeed, dendritic cells may have a role in various illnesses, including asthma, by modulating the immune response [25]. On the other hand, research indicates that, in addition to Th2 and Th1 cells, other T cells are derived from Th cells that operate and produce cytokines differently from Th1 and Th2, and are referred to as T regulatory cells (Treg) [4]. Another principal action of asthma is a structural impairment of Treg. Asthma seems to be strongly linked to Treg. Reduced Treg numbers and function in asthma are significant contributors to immunological dysregulation [26]. There is evidence for the involvement of Treg in the suppression of airway allergic illnesses, which impair the spleen's activities and cause inflammation and allergic disease in OVA-sensitive mice [27]. Kianmehr et al. demonstrated that in the presence of OVA, IL-4 and TGF-β expression increased in the spleen cells of asthmatic mice, whereas IFN-γ and Treg expression reduced [28]. Tregs modulate the immune response by regulating cytokine production. Treg levels are lower in asthmatics than in controls, according to studies. Additionally, Jing's research has shown that Treg reduction in splenic lymphocytes may be beneficial in resolving Th1/Th2 imbalances [4].
Previous research has shown that MSC promotes Treg in mice and humans and that MSC-regulated immunity is linked with an increase in Treg quantity and function. Dai et al. demonstrate that the ratio of Treg to T lymphocytes in the spleen of asthma model mice is substantially lower than in the normal group and that MSC treatment increased the proportion of Treg while decreasing the rate of splenic cell infiltration [26]. According to Huang et al., a common characteristic of MSC and MSC-CM treatment is a substantial decrease in spleen macrophages. MSCs are therefore involved in the downregulation of pro-inflammatory macrophages. For instance, MSCs promote the transition of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages [29]. Thus, in this study, it can be asserted that increased mast cells, Th1/Th2 imbalance, and defect or decreased Treg function in the spleen increase inflammatory cytokines in the spleen culture supernatant, as altered levels of these cytokines in the splenic supernatant indicate an alteration in the immune system. Thus, based on prior research and the results of this investigation, hAM-MSC- CM likely reduced splenic cell infiltration and inflammatory cytokines while increasing IL-10 through an increase in Treg and macrophage M1 to M2 transition. As a result, the CM may have been able to increase Treg activity.