The poly(I:C)-primed MSCs were more effective than unstimulated MSCs to ameliorate the DSS-induced colitis in mice
To identify the immunomodulatory effects of the poly(I:C)-primed MSC, we used the murine DSS-induced colitis model (Fig. 1A). MSCs were primed with poly(I:C) as described before (26) and injected i.p. to mice on days 1 and 3 (DSS + primed MSCs group). Other mice were treated with the same volume of saline (DSS group) or unstimulated MSC (DSS + MSCs group) on days 1 and 3. Compared to the DSS group and DSS + unstimulated MSC group, the DSS + primed MSCs group had reduced DAI scores and less weight loss (Fig. 1B-C). Additionally, the primed MSC treatment shortened the colon length less, which is the anatomic marker of colonic inflammation (Fig. 1D). A pathologic examination confirmed the symptomatic improvement of the DSS-induced colitis, which was caused by the primed MSC. Figure 1E shows that the typical pathologic findings of the DSS-induced colitis (epithelial loss, crypt destruction, and inflammatory cell infiltration) were significantly ameliorated by the unstimulated MSC and further by the primed MSC. These results demonstrated the improved anti-inflammatory effects of the primed MSC compared to the unstimulated MSC.
The poly(I:C)-primed MSCs significantly stimulated the intestinal stem cell (ISC) proliferation, enterocyte differentiation, and epithelial regeneration
We did the IHC staining of a cell proliferation marker, Ki-67, using colon tissues, which were harvested on day 9. The Ki-67 expression was prominently increased in the DSS + primed MSC group more than in other groups, suggesting the intestinal epithelial layer was recovered significantly faster (Fig. 2A). Next, we investigated the ISC proliferation and Wnt/β-catenin signaling pathway, since they play a pivotal role in maintaining intestinal homeostasis (21). Total RNA was isolated from colon tissues, and the mRNA of target genes was quantified by a real-time RT-PCR method. Markers of active ISC (Lgr5 and OLFM4) and quiescent ‘4+’ ISC (Bmi1) were significantly increased in the primed MSC group more than in the other groups. The expression of Axin2, a target gene of the Wnt/β-catenin signaling pathway, was increased in the primed MSC group (Fig. 2B). The mRNA levels of Muc2 (goblet cells), Alpi (enterocytes), Chga (enteroendocrine cells), and occludin (epithelial tight junction) were prominently upregulated in the primed MSC group more than in the other groups. (Fig. 2C). The IHC staining of lysozyme revealed that the primed MSC treatment significantly increased Paneth cells and preserved crypt structures better than the unstimulated MSC did (Fig. 2D). These results confirmed that the poly(I:C) priming, i.e., TLR3 stimulation, increased the MSC’s efficacy in promoting intestinal epithelial regeneration and maintain homeostasis.
The poly(I:C)-primed MSC decreased inflammatory response in the spleen, mLN, and colon tissue
IBD is a chronic inflammatory state of the gastrointestinal tract, which involves effector T cells and inflammatory cytokines (1). Therefore, we studied the anti-inflammatory and immunomodulatory effects of the primed MSC. Spleen, mLN, and colon tissues were harvested on day 9, as described in Fig. 1A. Like other findings, the primed MSC treatment reduced the inflammatory cell infiltration into lymphoid organs more effectively than the unstimulated MSC did. The numbers of monocytes (CD11b+), dendritic cells (CD11c+), and CD4+ T cells significantly decreased in the spleen and mLN from mice that had received the primed MSC treatment (Fig. 3A-C). The mRNA levels of inflammatory cytokines (IL-1β, TNF-α, and IL-6) and a chemotactic factor (MCP-1) in colon tissue decreased more in the primed MSC group than in other groups (Fig. 3D). The mRNA expression of immune-suppressive cytokine (IL-10) in colon tissue was elevated in the primed MSC group (Fig. 3F). These findings can account for the increased efficacy of the poly(I:C)-primed MSC on the IBD.
The poly(I:C)-primed MSC expanded Treg cells in an IDO1-dependent manner
The Foxp3+ Treg cells were functionally defective or its frequency was significantly lower in active IBD patients (22). Interestingly, the antiTNFα therapy, such as infliximab, significantly increased the frequency of functional Foxp3+ Treg cells in patients with active IBD (23). In this study, we investigated the change of Foxp3+ Treg cell frequencies in lymphoid organs and colon tissue after treatments of the poly(I:C)-primed or unstimulated MSCs for DSS-induced colitis. The proportion of Treg (CD25+Foxp3+CD4+) to CD4+ T cells in spleen was significantly higher in the primed MSC group than in the unstimulated MSC group and the DSS control group. Difference of Treg proportion between the primed and unstimulated MSC treatments was not prominent in mLN (Fig. 4A). The mRNA of Foxp3 in colon tissue increased considerably more in the primed MSC group than in the other groups. The unstimulated MSC could not increase the Foxp3 expression compared to the DSS control group, although they showed some anti-inflammatory effects on the IBD. We suppose that the ability of MSCs to induce the Treg cell expansion is the most important improvements induced by the poly(I:C) priming. To further confirm the capacity of the primed MSCs for expanding Treg, we did in vitro experiments. In brief, T cells were isolated from splenocytes of B6 mice and co-cultured with the primed or unstimulated MSC. The primed MSC caused a significantly higher increase of Treg (CD25+Foxp3+) proportion to CD4+ T cells than the unstimulated MSCs did (Fig. 4C). Since we demonstrated that the poly(I:C) priming of MSC induced the considerable IDO1 up-regulation (19), we tried to find out whether the primed MSC promoted Treg cells in an IDO1-dependent manner. Figure 4D shows that a competitive IDO1 inhibitor, L-1-MT, diminished the increment of Treg (CD25+Foxp3+) proportion induced by the primed MSCs, confirming our hypothesis.
The poly(I:C)-primed MSCs increased the expression of colonic IDO1 and COX2
We explored how the IDO1 and PGE2 pathways were affected by the MSC treatment, since they promote intestinal homeostasis by limiting inflammatory responses and protecting the epithelium (24). We did the IHC staining of IDO1 using colon tissues, which were harvested on day 9, as described in Fig. 1A. The primed MSC treatment induced a significant increment of IDO1-expressing cells in intestinal crypts than the unstimulated MSCs did, suggesting the role of IDO1 in maintaining the intestinal epithelial layer (Fig. 5A). The real-time RT PCR confirmed the same finding for the IDO1 expression (Fig. 5B). We found that the poly(I:C)-primed MSCs induced a significantly higher expression of Cyclooxygenase 2 (COX2) and Prostaglandin E synthase 3 (PTGES3) genes in colon tissue than did the unstimulated MSC, suggesting that the local PGE2 level may be increased in the inflamed site (Fig. 5B).
Pharmacologic IDO1 inhibition decreased the therapeutic efficacy of the poly(I:C)-primed MSCs on the DSS-induced colitis.
Next, we tried to find out whether IDO1 is essential for the protective effects of the primed MSCs. We induced experimental colitis and injected MSCs, as described in Fig. 1A. In addition, a competitive IDO inhibitor, L-1-MT (200 mg/kg), was administered by oral gavage from day 1 to day 5. Figure 6 suggests clearly that the pharmacologic IDO1 inhibition abolished the beneficial effects of the primed-MSC treatment, such as less weight loss, reduced DAI scores, and preserved colon length (Fig. 5B-D). Importantly, the mRNA expressions of Foxp3 and IDO1, which were increased by the primed-MSC treatment, were down-regulated by the IDO1 inhibition. In contrast, the expression of IL-10 was not affected by the IDO1 inhibition, implying that IL-10 was possibly secreted from monocytes rather than from Treg cells in the colon tissue of the experimental IBD, or that other mediators besides IDO1 might be involved in the IL-10 production (Fig. 5E). These results altogether suggest that IDO1 is an important mediator of the poly(I:C)-primed MSCs in promoting intestinal epithelial recovery and suppressing inflammatory responses.