The mammalian immune system consists of innate and adaptive immunity, which cooperatively work for the elimination of exogenous pathogens or endogenous danger signals. The innate immune system eradicates the infective microbial pathogens upon their recognition and initiates further inflammatory responses (1-4). The innate immune cells possess various sensor proteins to distinguish self-molecules from foreign substances (5, 6). Pattern recognition receptors (PRRs) are sensors for pathogen-associated molecular patterns (PAMPs) (7). Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are representative families of PRRs. Previous studies have reported that TLRs and NLRs are functionally expressed in mesenchymal stem cells (MSCs) (8-12). In our previous study, the stimulation of nucleotide-binding oligomerization domain 2 (NOD2) resulted in the altered differentiation capabilities or immunoregulatory abilities of MSCs (11). Inflammasome is the recently reported cytosolic protein complex that is involved in the initiation of the inflammatory responses in response to exogenous microbial infection and endogenous danger signals. Inflammasome generally contains three components: a cytosolic PRR that senses stimuli, the enzyme caspase-1 which converts cytokine precursors into mature cytokines, and the adaptor protein such as apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) (13, 14). Assembly of the inflammasome leads to the activation of caspase-1 which subsequently transforms the pro-inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-18 (IL-18) to their mature and active forms (15).
The NLRP3 inflammasome has been well studied and most frequently implicated as a key player in the pathogenesis of various diseases. NLRP3 inflammasome is activated via two-step processes including priming step and activation step (16-18). Currently, little is known about the expression and role of the inflammasome in non-immune cells including adult stem cells.
MSCs have been reported to exhibit immunoregulatory properties against various autoimmune or allergic diseases, as well as differentiation potential into mesodermal lineages (19-25). This immunomodulatory effect of MSCs on immune cells is mostly exerted by the combination of soluble factors (26-32). In our previous findings, we showed that the priming of NOD2 receptors in MSCs before the administration led to the improved therapeutic efficacy against colitis and atopic dermatitis through regulation of T cells or mast cells, respectively, via the production of prostaglandin E2 (PGE2) (33, 34). Although few studies including the one by Oh
We first investigated the expression of inflammasome components, as well as final products of inflammasome activation including IL-1β in hUCB-MSCs. To activate NLRP3 inflammasome, LPS and ATP were treated for 4 hours and 45 minutes, respectively. In THP-1 cells, the expressions of NLRP3, cleaved caspase-1, and pro/mature IL-1β were elevated in the presence of LPS or LPS+ATP compared to the control group or ATP-treated group. hUCB-MSCs expressed NLRP3, ASC, caspase-1, and IL-1β. Interestingly, LPS+ATP treatment in hUCB-MSCs increased the expression of NLRP3, cleaved caspase-1, and pro IL-1β. However, the expression of mature IL-1β was not altered by LPS+ATP treatment (Fig. 1A). Subsequently, we measured the production of IL-1β from hUCB-MSCs after NLRP3 inflammasome activation. However, in hUCB-MSCs, NLRP3 activation did not induce the production of IL-1β and the basal level of IL-1β production was too low to exert physiological functions (Fig. 1B). Taken together, these findings indicate that hUCB-MSCs express the components of NLRP3 inflammasome and its activation is induced in a manner similar in the macrophages. However, the final product of the activation, mature IL-1β, was not robustly secreted from hUCB-MSCs in response to NLRP3 activation, indicating that inflammasome activation in mesenchymal non-immune cells might serve different functions as reported in innate immune cells.
Pyroptosis is a caspase-1-mediated cell death that commonly occurs after inflammasome activation in macrophages and dendritic cells. Therefore, we investigated whether the viability or proliferation of hUCB-MSCs is affected by NLRP3 inflammasome activation-mediated pyroptosis. NLRP3 activation did not affect the viability, proliferation, and death of hUCB-MSCs (Fig. 1C-E). Next, the surface marker expression in naïve and activated-hUCB-MSCs was analyzed. The expression pattern of all negative (CD14, CD31, CD34, and CD45) or positive (CD29, CD44, and CD73) markers characterizing MSCs was not altered after NLRP3 activation (Supplementary Table. 1, Supplementary Fig. 1A and 1B). Our results imply that NLRP3 inflammasome activation in hUCB-MSCs neither induces pyroptosis nor affects the surface marker expression.
Several studies have shown that differentiation of MSCs can be modulated by triggering of innate immune receptors. Therefore, the differentiation capability of hUCB-MSCs into osteoblasts or adipocytes was determined after NLRP3 inflammasome activation. We observed that osteogenic differentiation was significantly down-regulated upon NLRP3 inflammasome activation compared with naïve hUCB-MSCs (Fig. 2A). On the other hand, NLRP3 inflammasome activation did not exert any influence on the potential of adipogenic differentiation (Fig. 2B). To confirm that this inhibitory effect of NLRP3 inflammasome activation on osteogenic differentiation is mediated via inflammasome assembly, siRNA targeting ASC, an essential component for inflammasome assembly, was employed (Fig. 2C). Interestingly, ASC suppression restored the inhibitory effect of NLRP3 inflammasome activation on osteogenic differentiation (Fig. 2D), suggesting that NLRP3 inflammasome is a negative regulator of osteogenesis in hUCB-MSCs.
MSCs have been reported to regulate the proliferation, activation, and maturation of various immune cells. Therefore, we evaluated
Given that MSCs can regulate macrophage activation, we next determined the regulatory function of hUCB-MSCs on M1 activation or NLRP3 activation of macrophages using THP-1-derived macrophage-like cells. When THP-1-derived M1 macrophages were co-cultured with NLRP3-activated hUCB-MSCs, TNF-α production was down-regulated to a greater extent compared to naïve MSC addition (Fig. 3F). THP-1-derived macrophages produced increased IL-1β in response to LPS and ATP treatment, and co-culture with hUCB-MSCs suppressed IL-1β production from macrophages. Interestingly, activated hUCB-MSCs down-regulated IL-1β production to a significantly greater extent (Fig. 3G). Taken together, these results suggest that NLRP3 inflammasome activation in hUCB-MSCs generally augments the immunoregulatory abilities including suppressive effects on T cell proliferation, Th1 cell differentiation and macrophage activation, as well as facilitating effects on Treg generation.
We then investigated whether NLRP3 activation in hUCB-MSCs can lead to any beneficial effects
MSCs have been reported to produce a variety of immunosuppressive soluble factors. To explore the key soluble factors in NLRP3 inflammasome activation, we measured more than 40 soluble factors in culture media from hUCB-MSCs. Based on the analysis of cytokine array, although there were slightly up-regulated factors including ALCAM and SDF-1α upon NLRP3 activation (Supplementary Fig. 2A and 2B), their mRNA expression after treatment with LPS or LPS+ATP was not increased (Supplementary Fig. 3A). We also investigated the expression of recently reported physiological anti-inflammatory antagonists such as IL-1RA, IL-17RA, and IL-18BP. There were no significant differences between naïve and activated hUCB-MSCs regarding the expression of these antagonists at the mRNA level (Supplementary Fig. 3A). We previously suggested PGE2 as one of the major anti-inflammatory mediators for the immunomodulatory function of MSCs. However, although LPS treatment slightly increased the secretion of PGE2, NLRP3 inflammasome activation by LPS+ATP treatment did not affect PGE2 production (Supplementary Fig. 3B). These findings propose that NLRP3 inflammasome activation potentiates the immunoregulatory functions of hUCB-MSCs presumably through the regulation of novel paracrine factors or other cell functions, which have not been reported previously.
The assembly of NLRP3 inflammasome leads to the secretion of IL-1β, followed by pyroptosis, an inflammatory programmed cell death by the microbial pathogens (15). However, in the present study, we could not observe the pyroptosis or significant production of IL-1β in MSC in response to NLRP3 inflammasome stimulation. Although the final phenotypes of inflammasome activation were not induced, osteogenic differentiation and immunomodulation of hUCB-MSCs were altered by NLRP3 inflammasome activation and were not affected by a single treatment with LPS or ATP. Several previous studies have shown that inflammasome or its components can regulate cell functions without classical activation. Wang
Here we have demonstrated that the osteogenic differentiation of hUCB-MSCs was impeded when NLRP3 inflammasome was activated. A recent study by Wang
More importantly, in the present study, for the first time, we demonstrated that NLRP3 inflammasome activation in hUCB-MSCs led to an increase in the immunosuppressive effects. Some studies reported the influence of PRR triggering on the immunomodulation of MSCs. In one study, TLR activation enhanced the immunosuppressive ability of hMSC by inducing an enzyme responsible for tryptophan catabolism (40). Also, Waterman
In conclusion, our findings clarified that NLRP3 stimulation decreased the differentiation potential of hUCB-MSCs into osteoblasts and increased immunosuppressive effects. We anticipate that these findings could provide a better understanding of the inflammasome-mediated regulation of non-immune cell functions, as well as a basis for the development of highly efficient cell therapy to treat several intractable inflammatory diseases.
The detailed methods are described in the “Supplementary Materials and Methods”.
This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2016R1C1B2016140, NRF-2018R1A5A2023879, and NRF-2018R1D1A1B07048035). We would like to thank Kangstem Biotech for providing hUCB-MSCs.