Our study aimed to determine the effects of hUC-MSC-EV in comparison to hUC-MSC for the treatment of COPD. The therapeutic potential of MSC and MSC derived secreted factors have been widely demonstrated in various diseases, including rheumatoid arthritis, asthma, and Crohn’s disease (Gonzalez-Rey et al., 2010; Song et al., 2015; Panes et al., 2016). In COPD, MSC capabilities to mitigate inflammation has been tested in the preclinical and clinical setting around the world (Weiss et al., 2013; Liu, Fang & Kim, 2016; Bich et al., 2020). However, little is known about the effect of extracellular vesicles isolated from MSC for the treatment of inflammation in COPD. hUC-MSC used in this study were positive for CD73, CD90, CD105, and CD166, and negative for CD34, CD45, CD31, and HLA DR DP DQ as previously described by Witwer et al., 2019. Meanwhile differentiation analysis showed the ability of hUC-MSC to differentiate into adipocyte, osteocyte, and chondrocyte. hUC-MSC-EV isolated from hUC-MSC showed a rounded morphology with the average of 153nm in diameter, and protein analysis showed a positive marker for CD63 exosomal marker. Following 12 weeks of CS exposure, the evidence of accumulation of inflammatory cell infiltrated in peribronchial and perivascular tissues as well as the parenchyma, goblet cell hyperplasia, expression of p65, and the development of emphysema was consistent to that of previously published studies (Nie et al., 2012; Zhang et al., 2014) indicating the development of COPD by CS inhalation. Two weeks of self-healing was significantly reduced the expression of p65, but did not reduce the inflammation and remoduelling the destruction of alveolar in the lung. The treatment of hUC-MSC-EV, hUC-MSC, as well as hUC-MSC-CM were significantly reversed the effect of sidestream CS on lung inflammation, expression of p65, and emphysema. Our study on microarray also revealed that CS was significantly regulated pathways related to COPD and upregulated genes related to inflammation including NFKB1, p65, and protein kinase Cζ (PRKCZ), whilst treatment with hUC-MSC-EV and hUC-MSC were observed to reverse these CS-induced gene expression effects.
Cigarette smoke is the main risk factor of COPD with over 80% of all COPD cases attributed to cigarette smoking, therefore cigarette smoke is widely employed by the researchers to develop the in vivo COPD model over other inducers such as biomass fuel, lipopolysaccharide, and elastase (Borzone et al., 2007; Al Faraj et al., 2014; He et al., 2017; Ghorani et al., 2017). To establish COPD model in animal, many studies had employed cigarette smoke exposure for 6 months period that showed much severe injury in the lung (Huh et al., 2011; Kim et al., 2016). However, there are studies which employed 12 weeks cigarette smoke exposure demonstrated characteristic of COPD including inflammation, airway remodelling, fibrosis, goblet cell hyperplasia, and emphysema (Gu et al., 2015; He et al., 2015). This method is more feasible for in vivo study as compared to 6 months period which is time-consuming. Our study is in agreement with the previous studies that showed 12 weeks of cigarette smoke exposure is sufficient to induce characteristics similar to COPD in SD rats. Importantly, our method of CS exposure for 2 times/day, 7 days/week for 12 weeks exposure induced the emphysema in rat lung, a characteristic of the chronic model of COPD (Leberl, Kratzer & Taraseviciene-Stewart, 2013). It should be noted that animal models do not fully mimic human condition, and regardless types of animal used and the duration of cigarette smoke exposure, the severity of the injury are only equivalent to the Global Initiative for Obstructive Lung Disease (GOLD) stage I or II diseases (Fricker et al., 2014).
COPD is characterized by airway and parenchymal inflammation that leads to mucus overproduction and emphysema, although these characteristic may not present in all patients, as the emphysematous lung only occurs in 20% of all COPD patients (Churg, Cosio & Wright, 2008; Akram et al., 2012). Nevertheless, in the animal model, the presence of emphysema is one of the important characteristics to confirm the development of COPD (de Oliveira, 2016). On the other hand, mucus overproduction is considered difficult to reproduce in the rat model due to the low number of goblet cells in the bronchi (Churg, Cosio & Wright, 2008). Our study using CS exposure for 12 weeks in SD rats successfully developed characteristic of COPD as we can observe the increased influx of immune cells indicating the development of inflammation in the lung, increased goblet cells count which shows increase mucus production, and increased mean linear intercept which shows the development of emphysema.
Airway inflammation begins with the disruption of the airway and vascular function, allowing infiltration of immune cells in the lung (Schweitzer et al., 2011; Presson Jr et al., 2011). In the acute phase of CS exposure that lasts until the second week, increased of neutrophils was observed. After the second week, macrophage begins to increase, and neutrophils start to decrease but not fully resolve, indicating chronic inflammation began to develop (Stevenson et al., 2007). In our study, the increased in neutrophils, eosinophils, lymphocytes, and macrophages counts were observed, however, neutrophils and macrophages are the predominant immune cells infiltrating the lung. Our results also showed immune cells accumulation was observed more prominently in the alveolar area rather than the peribronchial and perivascular area which destroy the alveolar wall leading to the emphysematous lung.
The accumulation of immune cells in alveolar walls are prerequisite for the development of emphysema. Neutrophils elastase (NE) was reported to induce the epithelial apoptosis and emphysema, meanwhile excessive MMP-9 released by macrophage can result in permanent alveolar destruction (Atkinson et al., 2011; Hou et al., 2014). Shapiro et al., (2003) was demonstrated that crosstalk between these two cells is crucial in the development of emphysema. The presence of neutrophils is crucial as neutrophils release NE that is required to recruit more neutrophils and monocytes into the lung. The study was also reported that mice deficient of NE (NE-/-) has shown significantly protected from the development of emphysema. Shapiro and colleagues further proved that the synergistic effects of neutrophil and macrophage are required to enhance the potency of both cells. The absence of NE causes the tissue inhibitors of metalloproteinases (TIMPs) to inhibit the action of macrophage elastase. Likewise, the absence of macrophage elastase caused an increased in α-1 anti-trypsin, a major inhibitor of NE. Thus, the presence of both neutrophils and macrophages are an important factor in the development of emphysema (Shapiro et al., 2003).
CS exposure also causes mucus overproduction, although the symptoms may not present in all COPD patients (Burgel & Martin, 2010). The mechanism by which CS-induced the overproduction of mucus occurs through activation of TNF-α converting enzyme (TACE) which cleaved pro-TNF-α to release TNF-α that activates epidermal growth factor receptor (EGFR) which result in mucin production (Shao, Nakanaga & Nadel, 2004). The accumulation of neutrophils in the lung during CS exposure may also exacerbate the mucus overproduction as neutrophils are also in part responsible for the impaired mucociliary clearance, increased goblet cells count, and excessive mucus production. NE released by neutrophils increased the expression of MUC5AC by enhancing the mRNA stability via reactive oxygen species mechanism (Arai et al., 2010; Fischer & Voynow, 2002). Besides, activation of TNF-α and subsequent activation epidermal growth factor pathway can also stimulate NE to induce the expression of MUC5AC (Kohri, Ueki & Nadel, 2002).
MSC has been actively investigated as a potential therapy for COPD. Clinical studies measuring C-reactive protein in COPD patient revealed the benefit of MSC administration in mitigating the inflammation (Hayes et al., 2020). In the animal model, MSC alleviates the inflammation by reducing the alveolar macrophage, while at the same time promoting the expression of the anti-inflammatory cytokine, IL-10 in macrophages (Gu et al., 2015). MSC also reduced the neutrophil infiltration regardless of the route of administration (Antunes et al., 2014). This therapeutic effects of MSC are governed by the released of paracrine factors including growth factor, cytokine, and EV rather than cell-to-cell contact (Fontaine et al., 2016). Recently, research begins to unravel the therapeutic effects of MSC-EV and better understand the mechanism behind this ability. Several studies have shown anti-inflammatory effects of MSC-EV in mitigating the inflammation similar to MSC. Reduced number of eosinophils, lymphocytes, and airway remodelling were observed in the animal model of asthma when treated with adipose tissue MSC-EV (de Castro et al., 2017). In the rat model of hepatic ischemia-reperfusion injury, hUC-MSC-EV inhibited the activity of the neutrophils by attenuation of respiratory burst and oxidative stress, thus reducing the apoptosis of hepatocytes (Yao et al., 2019). Also, MSC-EV attenuated the pro-inflammatory cytokines such as IL-17, TNF-α, RANTES, MIP1α, MCP-1, CXCL1, HMGB1, while enhancing the production of IL-10, PGE2, and KGF (Stone et al., 2017). Our study demonstrated that hUC-MSC-EV possess anti-inflammatory similar to its cell counterpart, hUC-MSC. The treatment with hUC-MSC-EV significantly reduced immune cells accumulation in the lung especially neutrophils accumulation, reduced emphysema, reduced protein expression of p65, and downregulated DEG related to COPD.
To date, there are no treatment options available to regenerate the lung damage in emphysema. However, stem cell-based therapy demonstrates a promising regenerative capability to restore the function of damaged lung. MSC and MSC-CM are shown to restore the lung function by mitigating the apoptosis in the emphysematous lung (Huh et al., 2011). This anti-apoptosis effect is in part is mediated by vascular endothelial growth factor (VEGF) and VEGF receptor (Guan et al., 2013). In addition, MSC reduced expression of cyclooxygenase-2 in alveolar macrophage, thereby mitigating the emphysema in rat model of COPD (Gu et al., 2015). On the other hand, relatively few studies were conducted to decipher the effects of MSC derived EV in the emphysematous lung. The study by Kim and colleague (2017) comparing the regenerative effects of nanovesicles generated from adipose stem cells (ASC) and ASC derived exosomes in elastase-induced emphysematous lung. The result showed that nanovesicles significantly reduced the emphysema via its cargo content, FGF2, while no significant reduction of emphysema was observed in ASC derived exosome (Kim et al., 2017). Our result provides the evidence of hUC-MSC-EV ability to reduce emphysema in CS-induced COPD in rat model. Considering the importance of neutrophils and macrophages accumulation in the pathogenesis of emphysematous lung, significant reduction in the accumulation of neutrophils when treated with hUC-MSC-EV and hUC-MSC in our study, in part might explain the reduction of emphysema. Decreased in macrophages accumulation were also observed when treated with hUC-MSC-EV, hUC-MSC, and hUC-MSC-CM although the reduction was not significantly different from the injury group. Recent studies also reported MSC derived microvesicles reduced the influx of neutrophils through the effects of KGF (Zhu et al., 2014). However, macrophages are shown to play an important role in MSC anti-inflammatory effects by changing from M1 to M2 phenotype which produces IL-10 that involve in the reduction of inflammation when treated with MSC and MSC-EV (Etzrodt et al., 2012; Gu et al., 2015; Sicco et al., 2017). Although the accumulation of macrophages is prerequisite for emphysema, however, in allergic asthma, depletion of alveolar macrophage reversed the immunosuppressive effect of MSC in which the production of IL-10 was dependent on the presence of alveolar macrophage (Mathias et al., 2013). The macrophages role might explain why macrophages in our study did not significantly reduce as it aids in MSC anti-inflammatory response.
To date relatively few studies examining the effect of MSC in reducing the mucus overproduction. Although there are reports stated that mucus can be mitigated with the administration of MSC, however, in-depth study on the mechanism involve remain unknown (Lee et al., 2011; Mohammadian et al., 2016). In addition, there is no report on the ability of MSC-EV to reduce mucus overproduction. Our study showed a significant reduction of goblet cells count in hUC-MSC. Reduction of goblet cells can be observed in hUC-MSC-EV and hUC-MSC-CM, however, the reduction was not significant. Nevertheless, the details mechanism on how hUC-MSC, hUC-MSC-EV, and hUC-MSC-CM effects on goblet cells are remained unknown and was not elucidated in the current study.
Our microarray analysis was aimed to determine the pathways associated with COPD and gene expression profile in our COPD model. We also seek to understand how the treatment with hUC-MSC-EV and hUC-MSC can change the gene expression profile and pathways in COPD model. Our on DEG analysis of microarray data revealed the importance of p50, p65, and PRKCZ in our animal model. 12 weeks CS exposure significantly upregulated p50, p65, and PRKCZ and the treatment with hUC-MSC-EV were significantly downregulated the expression of these genes. Immunohistochemistry staining on p65 confirms the significant upregulation of p65 protein in the CS group, and significant downregulation of p65 when treated with hUC-MSC-EV, hUC-MSC, and hUC-MSC-CM. Our study was also revealed that p50, p65, and PRKCZ involved in many pathway regulations that includes TNF-α NF-κβ signaling pathway, IL-2 signaling pathway, oxidative stress, estrogen signaling pathway, and IL-4 signaling pathway.
The expression of PRKCZ and NF-κβ play a vital role in inflammation and thus the pathogenesis of COPD. PRKCZ is upstream of NF-κβ, phosphorylating p65 at serine 311 to promote the acetylation of Lysine 310, thus activating the κβ transcription (Diaz‐Meco & Moscat, 2012). Mice deficient of PRKCZ was found to reduce myeloperoxidase and influx of neutrophils, and reduced pro-inflammatory cytokines such as IL-13, IL-17, IL-18, IL-1β, TNF-α, MCP-1, MIP-2, and IFN-γ, while the use of PRKCZ inhibitors blocked the activation of NF-κβ by TNF-α, thus reducing the pro-inflammatory IL-8 expression (Yao et al., 2010; Aveleira et al., 2010). Meanwhile, NF-κβ composed of five members, NF-κβ1 (p50), NF-κβ2 (p52), RelA (p65), RelB, and c-Rel, that regulate a multitude of genes involved in inflammatory responses (Liu et al., 2017). Among all heterodimers of NF-κβ, p50/p65 heterodimer represents the most abundant NF-κβ activated by the canonical pathway (Giridharan & Srinivasan, 2018).
Cigarette smoke activates NF-κβ within one hour of exposure to the lung thus causing inflammatory reactions which increase white blood cell count, lymphocyte count, and granulocyte count (Churg et al., 2003; Flouris et al., 2012). Data from the pre-clinical study showed 4 weeks of CS exposure significantly increased p65 and Iκβα in mice lung as compared to control group (Yu et al., 2018). NF-κβ is also required by IL-1β and IL-17A to induce the expression of MUC5B in bronchial epithelial cells that cause goblet hyperplasia in COPD (Fujisawa et al., 2011). Besides, various studies demonstrated the upregulation of p65 and p50 expression in COPD patients (Di Stefano et al., 2002; Caramori et al., 2003; Tan et al., 2016; Zhou et al., 2018). Microarray study conducted by Yang et al., (2013) revealed the important role of p50 in regulating many pathways of COPD including toll-like receptor signalling pathway, cytokine-cytokine receptor interactions, chemokine signalling pathway, and apoptosis (Yang et al., 2013).
Our study revealed the downregulation of PRKCZ, p65, and p50 expression when treated with hUC-MSC-EV. p50 regulated 18 pathways in hUC-MSC-EV group, while PRKCZ and p65 regulated 10 pathways suggesting the important role of NF-κβ pathway in hUC-MSC-EV therapeutic effects in our model. Downregulation NF-κβ subunit by hUC-MSC-EV can affect multiple pathways in our model thus reducing the inflammation. MSC-EV has been shown to decrease the expression of NF-κβ in in vitro model of cystic fibrosis and experimental colitis (Yang et al., 2015; Zulueta et al., 2018). However, much is still unknown about how MSC-EV regulates the NF-κβ pathway. In our study, we did not elucidate the cargo content of EV that is responsible for anti-inflammatory effects on CS-induced lung inflammation. Nevertheless, the study demonstrated that micro-RNA content of MSC derived exosome can reduce p50 NF-κβ pathway in macrophage thus preventing the Toll-like receptor-induced macrophage activation (Phinney et al., 2015). In addition, CCR2 in MSC derived exosomes abolished the ability of CCL2 to induce p65 phosphorylation in macrophages (Shen et al., 2016). Meanwhile, knockdown of GPX-1 in human MSC, reverse the effect of MSC derived exosomes in reducing the phosphorylation of p65 (Yan et al., 2017). These results proved that multiple cargo contents of MSC-EV play a vital role in mediating the inflammation.