CSK 4 as the comparatively effective stimulant for enhancing regulatory activities

TLR ligands can polarize MSCs according to other reports. We thus examined TLR2, 3, 4, 5 ligands for their possibilities to polarize MCSs or for their abundance in MSCs under our system. According to our results from [³H]-thymidine incorporation assays, Pam3CSK4 at 1 µg/mL for 72 h further enhanced the suppressive function of BM-MSCs whereas a TLR3 ligand, poly (I:C) at 10 µg/mL; a TLR4 ligand, LPS at 10 µg/mL; and a TLR5 ligand, Rec-FLA-ST at 100 ng/mL for 72 h did not (Fig 6A). After we analyzed the regulatory factors expressed in BM-MSCs with different stimulation, we observed using qPCR that 1 µg/mL Pam3CSK4 and 10 µg/mL LPS substantially enhanced inducible nitric oxide synthase (iNOS) in BM-MSCs (Fig. 6B). The

expression of cox-2, il-1ra, il-10, tsg-6, hgf, ido, and tgf-b in BM-MSCs, on the other hand, was affected through neither TLR ligands we examined (data not shown). At the protein level, Pam3CSK4 at 1 µg/mL and LPS at 10 µg/mL for 96 h enhanced NO secretion in BM-MSCs, in parallel with the results at the mRNA level (Fig. 6C).

In addition to TLR ligands, proinflammatory cytokines, such as TNF-a and IFN-g, are well-known microenvironmental factors to stimulate MSCs. According to our results from [³H]-thymidine incorporation assays, Pam3CSK4-activated BM-MSCs performed the most effective immunomodulatory function among the BM-MSCs activated with different stimulants (Fig. 7A). After we analyzed the regulatory factors expressed in BM-MSCs with different stimulation, we observed using qPCR that iNOS could be substantially enhanced through Pam3CSK4 in BM-MSCs, and ido could be

enhanced through 200 ng/mL IFN-g with or without 10 ng/mL TNF-a (Fig. 7B). In addition, IL-1RA expression detected by ELISA assays could not be induced through IFN-g without TNF-a in BM-MSCs (Fig. 7C).

Deciding to treat BM-MSCs with 1 µg/mL Pam3CSK4 for 96 h

We subsequently examined the ideal Pam3CSK4 stimulation protocol for BM-MSCs. In OVA-induced asthma model, compared to 5 µg/mL (Mpam5), 1 µg/mL Pam3CSK4-treated BM-MSCs (Mpam1) alleviated airway hyper-responsiveness (Fig.

8A) and diminished IL-5 secretion in BALF (Fig. 8C) more effectively. Further, compared to 1 µg/mL Pam3CSK4 treated for 24 h, treating BM-MSCs with 1 µg/mL Pam3CSK4 for 96 h was a more effective regimen to enhance the immunosuppressive function of BM-MSCs (Fig. 9).

Enhanced immunosuppressive activities in Pam3CSK4-stimulated BM-MSCs To examine whether Pam3CSK4 could enhance the immunoregulatory function of BM-MSCs, we performed the [³H]-thymidine incorporation assays in vitro. The incorporated radioactivity increases proportionally with CD4⁺ T cell growth because CD4⁺ T cells use [³H]-thymidine as a nucleoside resource during mitosis. Consequently, CD4⁺ T cell proliferation was further inhibited significantly when they were cocultured with Mpam compared with when they were cocultured with Mctrl (Fig. 10).

Mpam as a more effective treatment in the asthma murine model

We further performed more experiments to compare the therapeutic effects of Mctrl and Mpam on OVA-induced allergic asthma murine model. The observations of

cytokine profiles on the BALF, eosinophil infiltration in BALF, airway resistance, and pathological changes in lung tissues are the main criteria for studying OVA-induced asthmatic mice. Based on our results, serum IgE level was significantly decreased by BM-MSCs, irrespective of the Pam3CSK4 treatment; however, serum IgG1/IgG2a level was further decreased in the Mpam-treated group, compared to that in the Mctrl-treated group (Fig. 11A). Th2-related IgG1 and Th1-related IgG2a represented whether

immune homeostasis was achieved in our asthma murine model. In our results, the Mpam treatment decreased the serum IgG1/IgG2a levels more effectively than those did the Mctrl treatment, suggesting that the imbalanced phenomenon in asthmatic mice was corrected more effectively with the Mpam than with that of the Mctrl treatment. IL-13 secretion of the OVA restimulated splenocytes was significantly downregulated only by Mpam treatment (Fig. 11B). The decreased serum IgG1/IgG2a ratio and IL-13 secretion of the restimulated splenocytes might suggest the advanced systematic therapeutic effects of Mpam on asthmatic mice. For the cytokine profiles and the infiltrated cell compositions from BALF, less Th2 cytokines (such as IL-4 and IL-5) were secreted (Fig. 12A) and less eosinophil was accumulated (Fig. 12B) in the Mpam-treated group compared with those in the Mctrl-treated group. In addition, the cell compositions in BALF were confirmed through flow cytometry (Fig. 12C). The results supported that the pulmonary inflammation attributed to OVA sensitization was alleviated more

effectively by Mpam than that by Mctrl. Evidenced by a plethysmograph with paralyzed and tracheotomized mice, the increased airway resistance in OVA-sensitized mice revealed that the recurrent inflammation made their airways susceptible to stimulants.

Our results demonstrated that the airway resistance of the OVA-induced asthmatic mice could be ameliorated further with the Mpam treatment than with the Mctrl treatment (Fig. 13A). The susceptible airways of asthmatic mice aggravated the pathological

effects of the recurrent inflammation, accordingly leading to the remodeled

parenchymal lung tissues as the disease progressed. Among the lung histopathological sections of all groups, both Mctrl and Mpam apparently diminished the inflammatory cell infiltration and the bronchial epithelial thickness (Fig. 13B). Moreover, mucin 5AC, which is one of the major components of the mucus secreted by goblet cells in the respiratory tracts, was also measured (76). Further downregulated muc5ac expression in the lung tissues was observed in the Mpam-treated group, compared to that in the Mctrl-treated group. This result implied that the Mpam treatment might be able to abolish mucin hypersecretion more effectively than did Mctrl treatment (Fig. 13C). In summary, the therapeutic effectiveness of BM-MSCs on asthmatic mice can be enhanced with Pam3CSK4 preactivation.

Pam3CSK4 changed the multipotent stem cell properties of BM-MSCs Mpam was found to form more colonies than Mctrl according to our colony formation assays (Fig 14A). In other words, Pam3CSK4 might help enhancing the self-renewal properties of BM-MSCs. In addition, the expression of the stem cell factor, Sca-1, was slightly enhanced in Mpam, compared to that in Mctrl (Fig 14B). Further, under the adipogenic condition, while Pam3CSK4 seemed to impair the adipogenic ability of normal mice-derived BM-MSCs, BM-MSCs isolated from TLR2 knockout mice were more prone to differentiate into adipocytes (Fig 14C).

STAT3 signals of the immunosuppression-prone BM-MSCs

To determine whether the STAT3 signaling pathway was involved in the Pam3CSK4-enhanced immunoregulatory function of BM-MSCs, we used S3I-201, a

assays revealed that the CD4⁺ T cell proliferative reduction affected by Mpam was reversed when the STAT3 activating process of Mpam was disturbed. This result supported that the STAT3 signaling pathway was involved in the enhanced regulatory function of BM-MSCs induced by Pam3CSK4. However, neither Pam3CSK4 nor S3I-201 affected the immunosuppressive function of BM-MSCs in the transwell system (Fig. 15A). It additionally suggested that the enhanced immunosuppressive function of Mpam through STAT3 signaling was cell-cell contact-dependent.

The role of STAT3 activation in enhancing the regulatory function of Mpam was also suggested in vivo. Compared to the Mctrl-treated group, less eosinophils infiltrated to lungs in the Mpam-treated asthmatic mice. However, such diminished eosinophil infiltration in the Mpam-treated group was reversed in the Ms&p-treated group. It might imply that the enhanced ameliorating effect of Mpam in asthma murine model was abolished by STAT3 inhibition (Fig 15B).

Nitric oxide as the dominant immunoregulatory factor of Mpam To evaluate which regulatory molecules determined the enhanced

immunosuppressive effects of Mpam, we screened the well-known candidates through qPCR. The mRNA upregulation of iNOS, interleukin-1 receptor antagonist (il-1ra), tumor necrosis factor-stimulated gene sequence (tsg)-6, and hepatocyte growth factor (hgf) observed in the TLR2 ligand-activated BM-MSCs was downregulated with STAT3 inhibition (Fig. 15C). By contrast, the mRNA expression of cyclooxygenase (cox)-2, indoleamine 2,3-dioxygenase (ido), and transforming growth factor (tgf)-β in BM-MSCs was not influenced by TLR2/STAT3 signaling (data not shown). The high expression of iNOS and il-1ra in Mpam was further confirmed at the protein level. In coherence with the patterns at the mRNA level, the increased NO secretion detected

using Griess assays and the increased IL-1RA secretion detected using ELISA in Mpam were diminished in Ms&p (Fig. 15D, E). Therefore, because the NO and IL-1RA production in the Pam3CSK4-activated BM-MSCs were upregulated without STAT3 inhibition but were downregulated with STAT3 inhibition, STAT3 signaling pathway was involved in Pam3CSK4-induced NO and IL-1RA production. Although both iNOS and il-1ra were overexpressed in BM-MSCs through the TLR2/STAT3 signaling pathway, NO worked in a cell-cell contact-dependent manner, whereas IL-1RA worked as a soluble factor. The Pam3CSK4-enhanced immunosuppressive function in BM-MSCs and the diminished effects on Mpam through S3I-201 inhibition were observed using [³H]-thymidine incorporation assays without transwell inserts; however, neither TLR2 activation nor STAT3 inhibition induced any differences in the

immunosuppressive properties of BM-MSCs in the transwell system (Fig. 15A). The results suggested that cell contact dominated the enhanced regulatory effects of Mpam through the STAT3 pathway. Therefore, NO, rather than IL-1RA was the plausible Pam3CSK4-induced regulatory factor in BM-MSCs.

The mobility of BM-MSCs might be increased with Pam3CSK4 treatment

It is easy for MSCs to recognize and then migrate to inflammatory sites in hosts. It is therefore believed that MSCs are able to exert their proinflammatory/regulatory function because of such property. We thus examined whether Pam3CSK4 increased any chemokines and/or adhesion molecules in BM-MSCs. Among the chemoattractant which is induced in MSCs by TLR ligation, we found that the expression of ccl7 (detected by qPCR), mcp-1 (detected by qPCR), CCL3 (detected by ELISA), CCL5 (detected by ELISA), and VCAM-1 (detected by flow cytometry) was upregulated in

when we treated Mpam with either STAT3 or iNOS inhibitor. The upregulated expression of VCAM-1 in Mpam was reduced when we treated Mpam with STAT3 inhibitor. (Fig 16).

Unchanged immunoprivileged properties of the conditioned BM-MSCs

To determine the mechanism by which Mpam advanced its immunoregulatory function, we examined the immunoprivileged properties of the conditioned BM-MSCs.

The unchanged expression of major histocompatibility complex and costimulatory molecules indicated that TLR2 activation did not significantly affect the

immunoprivileged properties of BM-MSCs (Fig. 17).

The molecules indicating apoptosis and anergy in CD4⁺ T cells were not induced by Mpam

To find out whether Pam3CSK4 enhanced the regulatory function of BM-MSCs through inducing T cell death, we examined molecules which are known to be related to apoptosis and anergy in conditioned CD4⁺ T cells. The expression of FAS and PD-1 detected by flow cytometry showed no differences between all the different conditioned CD4⁺ T cells (Fig 18A). The expression of caspase-3 and itch detected by qPCR also showed no differences between all the different conditioned CD4⁺ T cells (Fig 18B).

Regulatory T cells induced by nitric oxide as the immunosuppressive operators The generation of regulatory T cells is one method by which MSCs control the T cell reactivity. Other reports have also shown that more regulatory T cells are induced through enhanced NO secretion (77). We next examined whether the excessively secreted NO of Mpam performed its immunoregulatory function by inducing regulatory

T cells. To determine the role of NO as one of the downstream signals of TLR2 activation in BM-MSCs, we treated the Mpam with an iNOS inhibitor (Ml&p), L-NMMA. Furthermore, because STAT3 in the downstream of TLR2 was involved in the NO production in Mpam, we examined whether the STAT3 pathway was involved in the regulatory T cell induction. Through [³H]-thymidine incorporation assays, the 24 h preactivated CD4⁺ T cells were cocultured with the conditioned BM-MSCs (Fig. 1C).

Our results demonstrated that Mpam diminished more proliferative CD4⁺ T cells than did Mctrl, whereas Ms&p and Ml&p did not (Fig. 19A). The NO secretion of the conditioned BM-MSCs was confirmed using Griess assays. With TLR2 activation, more than ten times the amount of NO was secreted by the BM-MSCs. By contrast, both STAT3 and iNOS inhibitors diminished the NO secretion of Mpam (Fig. 19B). To determine whether the regulatory T cells educated by Mpam caused the differences, the 24 h preactivated responder CD4⁺ T cells were cocultured with the CD4⁺CD25⁺ T cells, which were respectively preeducated by the different conditioned BM-MSCs

(conditioned CD4⁺CD25⁺ T cells) (Fig. 1C). The results revealed that the Mpam-educated CD4⁺CD25⁺ T cells (Tpam) were able to inhibit the responder CD4⁺ T cell proliferation more effectively than were Mctrl-educated CD4⁺CD25⁺ T cells (Tctrl).

Furthermore, this phenomenon was reversed when the Mpam was treated with either the STAT3 inhibitor or the iNOS inhibitor (Fig. 19C). In conclusion, our results suggested that more immunoregulatory CD4⁺CD25⁺ T cells tended to be induced by Mpam than by Mctrl, and the STAT3 and iNOS inhibition appeared to diminish the

immunoregulatory CD4⁺CD25⁺ T cell induction of Mpam.

Increased CD4⁺CD25⁺Foxp3⁺ T cells induced by Mpam

Next, to determine the phenotypes of the conditioned CD4⁺CD25⁺ T cells, we investigated the mRNA expression of regulatory molecules in the conditioned CD4⁺CD25⁺ T cells through qPCR and flow cytometry. Compared with the

noneducated CD4⁺CD25⁺ T cells, the expression of tgf-b, il-10 (Fig 20A), GITR, and CTLA-4 (Fig 20B) was not significantly changed in Tctrl, Tpam, Ts&p, and Tl&p.

Compared with the noneducated CD4⁺CD25⁺ T cells, foxp3 was upregulated only in Tpam (Fig. 20C). The upregulated Foxp3 expression of Tpam compared with that of Tctrl was also observed at the protein level through flow cytometry. Moreover, the increased CD4⁺CD25⁺Foxp3⁺ T cells were induced by Mpam but not by Ms&p or Ml&p (Fig. 20D). Mononuclear cells were also examined in lungs of asthmatic murine model. With the Mpam treatment, less mononuclear cells were recruited, however, a higher percentage of CD4⁺CD25⁺Foxp3⁺ cells was observed in the lung homogenate (Fig. 20E).

Sequentially activated NF-κB/STAT3/iNOS signaling in Mpam

The mediation of the seemingly independent mechanisms between TLR2 and STAT3 was examined. TLR2 ligation is known to lead to inhibitor of κB (IκB) kinase degradation then nuclear factor (NF)-κB transcriptional activation. In addition, our data suggested that the NO excessively generated by iNOS was the downstream product of Pam3CSK4-activated BM-MSCs through the STAT3 pathway. The sequential

correlation between TLR2 stimulation and the downstream NF-κB/STAT3/iNOS activation was finally demonstrated using Western blot. The Western blot results

suggested that the expression of pSTAT3 and iNOS in BM-MSCs was upregulated after Pam3CSK4 activation for 30 min and 24 h, respectively. In addition, the diminished expression of pSTAT3 and iNOS in Ms&p indicated that S3I-201 inhibited the STAT3

activation and thus its downstream iNOS expression. The diminished expression of iNOS in Ml&p, however, was mainly attributed to the translation of iNOS mRNA, which was regulated by the availability of its substrate, l-arginine (Fig. 21).

Mediation between NF-κB and STAT3 through IL-6

Because STAT3 signaling is mainly induced by cytokine-mediated activation, we examined the cytokine profiles on BM-MSCs with different treatments (conditioned BM-MSCs). We observed that IL-6 production was increased by Pam3CSK4 induction irrespective of the inhibition of S3I-201 and L-N^G-monomethyl arginine citrate (L-NMMA), an iNOS inhibitor, suggesting that IL-6 secretion might occur upstream of STAT3 and iNOS activation (Fig. 22A). Moreover, detected using Western blot, TLR2 ligation no longer stimulated iNOS production in MSCs after being treated with IL-6 neutralizing antibody (M6&p), but not with IgG1 k isotype control antibody (Mic&p) (Fig. 22B). The augmented immunosuppressive function of Mpam was then abolished by IL-6, but not by IgG1 k isotype control neutralization (Fig. 22C). We thus suggested that IL-6 might play a critical role through the stimulation of iNOS to enhance the induction of regulatory T cells.

Chapter IV: Discussion

This study established an appropriate priming protocol to enhance the immunosuppressive function of BM-MSCs.

Although MSCs have been proposed for the treatment of inflammatory lung diseases (17), the sustainable translation of MSC therapies to clinical settings has been hampered by the heterogeneity of MSCs and nonstandardized in vitro culture

technologies (78). Therefore, the present study focused on enhancing the

immunoregulatory function of mouse BM-MSCs by using the Pam3CSK4-polarizing protocol.

Heterogeneous MSCs can mediate their effects by sensing fluctuating

proinflammatory cytokines and TLR ligands in the microenvironment (79). At the beginning of infectious challenges, TLR ligations link innate and adaptive immunity and trigger positive feedback of the immune system by activating proinflammatory cytokine and chemokine production. To maintain the health status of the host, proinflammation-prone MSCs help to eliminate pathogens by recruiting immune effector cells to the sites of infection. However, at the end of the challenges, proinflammatory damage and oxygen stress gradients are accumulated by resultant pathogen cell necrosis and tissue damage debris. To preserve host integrity, anti-inflammation-prone MSCs reduce effector immune cell infiltration but increase

regulatory immune cell induction (48). Hence, plasticity in immunomodulatory function causes MSCs to be both advantageous and disadvantageous in the context of

inflammation.

The polarization of BM-MSCs is apparently susceptible to the stimuli conditions.

This is why the discrepancies in the experimental settings both in vitro and in vivo led to several inconsistent and partially contradictory results related to TLR-polarized MSCs (35, 80). The differences in the source of MSCs, the concentration and timing of the stimuli used, and the culture conditions might explain the conflicting results (81). Given the uncertain effects of TLR polarization on MSCs, consistent therapeutic outcomes and immunoregulatory mechanism-based validations are required to establish the

effectiveness of modified MSCs in asthma therapy.

We demonstrated that the immunomodulatory BM-MSCs were polarized by a TLR2 agonist, Pam3CSK4. According to both our in vitro and in vivo functional assays conducted using [³H]-thymidine incorporation assays and acute OVA-induced asthmatic murine models, respectively, Pam3CSK4 augmented the immunosuppressive function of BM-MSCs. The results of our immunoregulation-prone BM-MSCs stimulated by Pam3CSK4 are consistent with the findings of Pevsner-Fischer et al. (82) but inconsistent with the findings of Lei et al (83). In those two studies, the mouse BM-MSCs were incubated with different concentrations (1 vs. 2 µg/mL) of Pam3CSK4 for the same period (48 h) before they were cocultured with different responder cells (T cell line specific to MOG p35-55 vs. splenocytes isolated from C57BL/6 and BALB/c spleens) in different MSC to responder cell ratios (1:20 vs. 1:10). The discrepant protocols might have caused the distinct results.

Based on our study, we observed that the immunosuppressive BM-MSCs should be administered in post-treatment instead of pretreatment settings. In other words, they should be cocultured with 24 h preactivated CD4⁺ T cells in vitro and infused after asthmatic mice were OVA-challenged in vivo. The suggested modification of

BM-asthma by using BM-MSCs after the disease had developed. The ideal BM-MSC to responder CD4⁺ T cell ratio was 1:2 to 1:200 for acting immunosuppressive function in vitro, and the ideal infused cell number of BM-MSCs was 5 × 10⁵ cells per mouse. BM-MSCs treated with 1 µg/mL Pam3CSK4 exerted better immunosuppressive effects than those with 5 µg/mL Pam3CSK4 in vivo. Moreover, BM-MSCs treated with 1 µg/mL Pam3CSK4 for 96 h were more effective than those treated for 24 h in vitro.

According to our data, it was in contrast to the instinct that neither more BM-MSC administration (1 × 10⁶ cells per mouse) nor BM-MSCs pretreated with higher

According to our data, it was in contrast to the instinct that neither more BM-MSC administration (1 × 10⁶ cells per mouse) nor BM-MSCs pretreated with higher