
==== Front
Biochem Biophys Rep
Biochem Biophys Rep
Biochemistry and Biophysics Reports
2405-5808
Elsevier

S2405-5808(24)00173-0
10.1016/j.bbrep.2024.101809
101809
Research Article
Differential effects of TLR3 and TLR4 activation on MSC-mediated immune regulation
Kaundal Urvashi kaundal.urvashi@gmail.com
ab
Rakha Aruna aruna_pgi@yahoo.com
b⁎
a Department of Translational and Regenerative Medicine, Postgraduate Institute of Medical Education and Research, Sector-12, Chandigarh, 160012, India
b Scleroderma Genomics and Health Disparities Unit, NIAMS, NIH, Bethesda, USA
⁎ Corresponding author. Department of Translational and Regenerative Medicine, Research Block B, Level-5, Postgraduate Institute of Medical Education and Research, Sector-12, Chandigarh, 160012, India. aruna_pgi@yahoo.com
10 8 2024
9 2024
10 8 2024
39 10180914 12 2023
31 7 2024
5 8 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Mesenchymal stromal cells (MSCs) have evolved as an invaluable therapeutic cell type due to their broad therapeutic properties. Bone marrow-derived MSCs are currently being applied in numerous clinical trials, and the initial results have been encouraging. However, heterogeneous responsiveness amongst patients is also being experienced; therefore, the efficacy of MSCs in vivo is still debatable. Host microenvironment plays an essential role in determining the fate of MSCs in vivo. Recent studies have indicated the role of toll-like receptors (TLR) in modulating the biological properties of MSCs. TLRs are expressed by MSCs, and activation of TLR3 and TLR4 can alter the functionality of MSCs. While MSCs can suppress the effector and memory T cell function by promoting regulatory T cells, the effect of TLR activation on MSC-mediated immune cell induction is still not well understood. This study was performed to understand the TLR licensing of MSCs and its impact on MSC-mediated immunomodulation. We found that TLR3 mediated activation of MSCs (TLR3-MSCs) increased the expression of G-CSF & IL-10 while TLR4-mediated activation of MSCs led to an increase in CXCL-1, CXCL-10, and CXCL-12. To study the immunological aspect, an in vitro co-culture model was established-to imitate the brief in vivo interaction of MSCs and immune cells. We found that TLR3-MSCs led to increase in CD4 and CD8 naive T (TNAI) cells and vice versa for effector (TEFF) and memory T (TMEM) cells, while TLR4-MSCs did not show any effect.

Moreover, only TLR3-MSCs led to a non-significant increase in the regulatory T cells (TREGS) and Double negative regulatory cells. No change in B cell profile was evident while TLR3-MSCs depicted an increasing trend in regulatory B cells which was not statistically significant. TLR3 MSCs also inhibited the T cell proliferation in our setup. Our data indicate that TLR3 priming may regulate the function of MSCs through immunomodulation.

Understanding the role of TLRs and other microenvironmental factors causing subdued responses of MSCs in vivo would allow the uninhibited use of MSCs for many diseased conditions.

Highlights

• TLR3 activation increased the anti-inflammatory while TLR4-mediated activation of MSCs promotes pro-inflammatory cytokine production.

• TLR3-MSCs decreased the differentiation of naive T (TNAI) cells into effector (TEFF) and memory T (TMEM) cells, while TLR4-MSCs did not show any change.

• TLR3-MSCs led to reduced proliferation of T cells possibly through the expansion of the regulatory T cells compartment (TREGS).

• Overall, our data indicates that TLR3-MSCs can induce the immune regulatory functions.

Keywords

Toll-like receptors
Mesenchymal stromal cells
T cells
B cells
Cytokines
Conditioned medium
==== Body
pmc1 Introduction

Cell-based therapies specifically using mesenchymal stromal cells (MSCs), have gained the attention of the clinicians and researchers worldwide. Clinically, MSCs have been explored for a plethora of health conditions owing to their immunomodulatory and regenerative capacity [1]. However, the mechanisms underlying the regulatory effect of MSCs are not fully explored.

MSCs possess immunomodulatory properties which can regulate the immune cell responses by hampering immune cell differentiation, maturation, and functional responses [[2], [3], [4], [5]]. MSCs can induce tolerogenic immune responses by activating regulatory cells and suppressing effector and memory immune cell subsets [[6], [7], [8]]. This property makes them suitable for application in solid organ transplantation (SOTx). Numerous studies in preclinical transplant models have successfully demonstrated the ability of MSCs to enhance and improve graft function [[9], [10], [11]]. Moreover, clinical trials conducted in transplant patients till date, have substantiated the safety of MSCs [12,13]. Nevertheless, efficacy of MSCs has not been completely reflected in the clinical trials. MSCs have been shown to affect both humoral and adaptive arms of immunity [3,7]. Our previous studies in kidney transplant (KTx) patients demonstrated the effect of bone marrow-derived MSCs on the interplay of B and T cell subsets. We found MSC infusion lead to an increase in regulatory B (BREGS) [14], and decreased differentiation of naive T (TNAI) cell into effector (TEFF) and memory (TMEM) cells [15]. However, MSCs did not produce the same response in all the patients and similar heterogeneity has been reported in other clinical trials [12,16].

Exogenously cultured MSCs after in vivo administration interact with the local cytokines or soluble proteins in that specific microenvironment that leads to MSC licensing towards specific functions [[17], [18], [19]].

Host microenvironment can therefore be held responsible for causing anomalies in MSC-responsiveness in heterogenous group of patients where MSC infusion is done. Toll-like receptors (TLRs) form an integral part of the microenvironment, and their importance in MSC-licensing has been highlighted in few studies [[19], [20], [21]]. Following stimulation, TLRs trigger several intracellular signalling cascades [22], and release of paracrine factors.

Out of all TLRs, both TLR3 and TLR4 are known to contribute to MSC licensing. TLR3 and TLR4 activation of MSCs has been shown to enhance immunosuppression by TREGS induction [20,23,24]. TLR3 preconditioning of MSCs promoted TREG differentiation in a trinitrobenzene sulfonate (TNBS)-induced mouse model of colitis [25]. Similarly, TLR3-MSCs proved to be therapeutically efficacious in a dextran sulfate sodium (DSS) induced mice model [26]. Tolstova et al. showed that TLR3 priming can enhance the immunosuppressive properties of MSCs [27]. TLR4-MSCs have also resulted in superior therapeutic neovascularisation and recovery of cardiac function in acute myocardial infarction model [28]. On the contrary to this, Liotta et al. showed that TLR3 and TLR4 priming inhibited the process of MSC-mediated T-cell immunomodulation [29]. TLR3 activation also failed to enhance the therapeutic effects of MSCs in B6.MRL-Fas(lpr) mice [30]. Another study by Pezzanite et al. demonstrated that TLR3-activated MSC treatment can induce T lymphocytes and suppress innate immune responses in synovium of septic arthritis equine model [31].

A study by Cassatella et al. suggested that TLR3 or TLR4 primed MSCs might trigger inflammatory disorders [32]. Interestingly, a study by Waterman et al. showed that TLR3 priming of MSCs could induce an anti-inflammatory phenotype (MSC2) while TLR4 priming induced a pro-inflammatory phenotype (MSC1) [19]. Supporting this view, a few studies have shown that TLR4 inhibition in MSCs can improve their therapeutic potential [33] and survival [34].

Keeping these findings in mind, the current in vitro study was designed to study the effect of TLR3/4 primed MSCs on the immune cell responsiveness. Ours is the first study to report the impact of TLR primed MSCs on the T and B cell profile of the responder cells.

Our study shows that TLR3 primed MSCs induce immunomodulation and anti-inflammatory microenvironment.

2 Materials and methods

2.1 Isolation and characterisation of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) were isolated from the bone marrow (BM) aspirate of a healthy kidney donor. This donor was recruited for another study to elucidate the effect of MSC infusion on immune cells of kidney transplant patients (KTx) in an allogeneic setting (NCT- NCT02409940). After the infusion of specific numbers in originally recruited KTx patients, the remaining MSCs were used for the current experimental setting. All protocols were approved by the Institutional Committee for Stem Cell Research of PGIMER (PGI-IC-SCRT-39-2013/1471), Chandigarh. Briefly, BM aspirate was diluted with 1X PBS (1:1) and was subjected to density gradient centrifugation at 550g for 30 min. The mononuclear cells were then separated and resuspended in α-minimal essential medium (α-MEM) (Sigma-Aldrich, USA) containing 7–10 % pooled human platelet lysate (pHPL), 5 IU/mL heparin (Caprin®, India), 1 % penicillin-streptomycin (Sigma-Aldrich, USA) and 2 mM l-glutamine (Sigma-Aldrich, USA). Cells were maintained under standard culture conditions (5 % CO2 and 37 °C). After 5–7 days non-adherent cells were washed off. MSCs were subsequently trypsinised at 70–80 % confluency and sub-cultured (10,000 cells per cm2) till passage-3.

2.2 Characterisation of mesenchymal stromal cells

Phenotypic and functional characterisation of MSCs was performed as described in the International Society for Cellular Therapy guidelines [35].

Phenotypic characterisation of MSCs was carried out by multi-parametric flow cytometry (BD FACS Aria II; BD Biosciences, USA) after staining with PE-conjugated negative cocktail antibody (anti-CD11b, anti-CD19, anti-CD34, anti-CD45 and anti-HLA-DR), APC conjugated anti-CD73, FITC conjugated anti-CD90, and PerCP Cy5.5 conjugated anti-CD105. Compensation was performed using single colour controls, and the unstained sample was used as a negative control. Data were analysed with FlowJo software (Tree Star, Inc, USA).

MSCs were functionally assessed by their potential to differentiate into different lineages as described in our previous study [36]. Briefly, MSCs at passage-4 were kept in the adipogenic, osteogenic and chondrogenic medium. After 21 days, cells were fixed, stained and observed microscopically for the presence of lipid droplets (adipocytes), calcium deposits (osteocytes) and aggrecans (chondrocytes).

Before using MSCs for the experiments, karyotyping was performed [37,38] to confirm chromosomal stability.

2.3 Priming of MSCs

The concentration and timings of TLR agonist and antagonist were decided upon by standards mention in the literature.

2.3.1 TLR-3 priming

MSCs (passage-3) at 70 % confluency were incubated with a TLR3 agonist (ago) - polyinosinic-polycytidylic acid (poly I:C) (100 μg/mL) [39,40], for 1 h in fresh complete α-MEM. MSCs treated with bafilomycin A1 (BafA1) (100 μM) for 1 h and then with poly I:C (100 μg/mL) for 1 h were used as control. TLR3-primed MSCs (TLR3-MSCs) were then washed and used for further assays.

2.3.2 TLR-4 priming

MSCs (passage-3) at 70 % confluency were incubated with a TLR4 agonist-lipopolysaccharide (LPS) (250 ng/mL), for 24 h in fresh complete α-MEM. MSCs treated with a polymyxin B (poly-B) (10 μg/mL) for 1 h and then with LPS (250 ng/mL) for 24 h were used as control. TLR4-primed MSCs (TLR4-MSCs) were then washed and used for further assays.

2.4 Flow cytometric analysis for TLR priming

Flow cytometry was performed for the primed MSCs to confirm the efficacy of the priming protocols. Primed MSCs were stained with PE-conjugated anti-TLR3, or APC conjugated anti-TLR-4 and were acquired on BD FACS Aria II (BD Biosciences, USA) and analysed by FlowJo software (Tree Star, Inc, USA). Unprimed-MSCs were used as a negative control, and gating was performed using unstained MSCs.

2.5 Human cytokine, chemokine array and analysis

The expression of different cytokines, chemokines and acute-phase proteins in the culture supernatant of primed-MSCs was determined using the Proteome Profiler Human Cytokine Array Kit (ARBY005B; R&D Systems) per the manufacturer instructions. Primed MSCs (TLR3/TLR4) were serum starved for 24h in α-MEM. MSC culture supernatant was then collected and incubated with the array membrane. Manufacturer's instructions were followed without any deviation. The change in expression of the cytokines was determined by volumetric analysis using ChemiDoc™ XRS+ (Bio-Rad, USA) and is expressed as fold change relative to the control sample (unprimed-MSCs).

2.6 Preparation of conditioned medium

1X10 [6] MSCs (unprimed, TLR3-primed and TLR4-primed) were cultured in α-MEM for 24 h (without supplements) in a CO2 incubator at 37 °C. The culture supernatant was then collected and concentrated 20 times by using centrifugal filters (cut-off 3  kDa MW) (Millipore, Germany) at 4000g for 20–25 min. The concentrated supernatant or conditioned medium (CM-MSC) was then snap-freezed and stored at −80 °C till further use.

2.7 Isolation of peripheral blood mononuclear cells

Peripheral blood was collected from healthy donors (n = 6). Peripheral blood mononuclear cells (PBMCs) were then separated using Lymphoprep (Stem cell technologies, Canada) by density gradient centrifugation [41]. The samples collected from healthy donors were randomly assigned to two different groups, namely, responder cells (n = 3) and stimulator cells (n = 3).

2.8 Labelling of responder cells and inactivation of stimulator cells

Responder cells were labelled with 5 μM carboxyfluorescein succinimidyl ester (CFSE) [42]. Untreated CFSE-labelled responder cells were used as controls. Stimulator cells were inactivated using mitomycin-c at a concentration of 30 μg/mL for 2 h.

2.9 Mixed lymphocyte reaction

CFSE labelled responder cells were co cultured with inactivated stimulator cells (MLR) at a number of 0.3 × 106 for each to induce lymphocyte proliferation. Either TLR3/TLR4 primed or unprimed MSCs or CM-MSC were added to the MLR at a 1:10 ratio; MSC:PBMC ratio. Co-cultures were incubated for four days at 37 °C in a CO2 incubator, before analysing the immune cell population & proliferation in the cultures.

2.10 Immune cell profiling of responder cells

On the 5th day, immune cell subsets were determined for responder cells. For this, CFSE labelled responder cells were assessed for T cell (CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CD25, CD127 and FoxP3), B cell (CD19, CD5, CD1d, CD24, CD27 and CD38) and cell viability marker (7-amino actinomycin D (7-AAD)). Lymphocyte subsets (Table S1) were analysed on a flow cytometer using fluorochrome-conjugated monoclonal antibodies according to previously published protocols [36]. Cells were acquired on a flow cytometer and analysed on FlowJo software. Gating strategy for the subsets is provided in Supplementary Figs. S2–S5.

2.11 Proliferation of responder cells

T and B cell proliferation was measured by CFSE dilution assay. For this, CFSE labelled responder cells were stained for T cell markers (CD3, CD4, CD8), B cell (CD19) and cell viability marker (7-amino actinomycin D (7-AAD)). Cells were acquired on a flow cytometer and analysed on FlowJo software. Gating strategy for the subsets is provided in Supplementary Figure S5 (A-E) and S6.

2.12 Statistical analysis

All experiments were performed in duplicates and at least twice. The analysis was performed using GraphPad Prism software (GraphPad, USA). Statistical analysis for multiple group comparison was performed using one-way analysis of variance (ANOVA). Comparison between two groups was performed by two-tailed Student's t-test or Mann-Whitney test and p < 0.05 was considered statistically significant. All data are expressed as mean ± SD.

3 Results

3.1 Phenotypic and functional characterisation of mesenchymal stromal cells

MSCs stained with fluorochrome labelled antibodies showed >95 % positivity for CD73, CD90 and CD105 and < 2 % positivity for negative markers (CD11b, CD19, CD34, CD45 and HLA-DR) (Supplementary Fig. S7). MSCs differentiated into adipocytes, osteocytes and chondrocytes following standard protocols (Supplementary Fig. S8). Moreover, cultured MSCs demonstrated genetic stability as analysed by karyotyping (Supplementary Fig. S9).

3.2 Patterns of TLR3/4 expression on mesenchymal stromal cells

Flow cytometric analysis revealed that MSCs in their native form express both TLR3 (intracellular) and TLR4 (on the cell surface) (Fig. 1A). Upon exposure to poly I:C (TLR3 agonist), there was an increase in the percentage of TLR3 expressing MSCs (TLR3-MSCs) in comparison to the unprimed MSCs (un-MSCs) (20 ± 5.9 % Vs 6.08 ± 1.66 %; p = 0.01; Fig. 1A). Similarly, MSCs exposed to LPS (TLR-4 agonist) showed higher percentage of TLR4 expressing MSCs (TLR4-MSCs) than un-MSCs (27.07 ± 5.57 % Vs 7.06 ± 3.91 %; p = 0.007; Fig. 1A).Fig. 1 Comparison of TLR expression on MSCs and their secretory cytokine/chemokine under different culture conditions. Flow cytometric analysis plots indicating the percentage of MSCs expressing (A) TLR3 and TLR4. Volumetric analysis indicating the fold change in expression of (B) CCl-2, CCl-5, CXCL-1, CXCL-10, CXCL-12, GCS-F, ICAM-1, IL-6, IL-8, MIF and Serpin. Data are represented as mean ± SD. * represents the statistical difference between the respective groups (*p < 00.05; **p < 00.005). TLR3 antagonist- BafA1 (bafilomycin A1); TLR3 agonist-poly I:C (polyinosinic-polycytidylic acid); TLR4 antagonist - poly-B (polymyxin B); TLR4 agonist - LPS (lipopolysaccharide).

Fig. 1

3.3 Paracrine factor secretion patterns of TLR3/4 primed mesenchymal stromal cells

Supernatant from TLR3-MSCs and TLR4-MSCs was tested for expression of 36 chemokines, cytokines and acute phase proteins (C5a, CD40L, G-CSF, GM-CSF, CXCL1, CCL1, ICAM-1, IFN-γ, IL-1 α, IL-1β, IL-1RA, IL-2, IL-4 IL-5, IL-6, IL-8, IL-10, IL-12 p70, IL-13, IL-16, IL-17, IL-17E, IL-18, IL-21, IL-27, IL-32α, CXCL-10, CXCL-11, CCL-2, MIF, MIP-1, CCL-5, CXCL12, Serpin E1, TNF-alpha, TREM-1). Out of 36, only 11 factors were detectable in the MSC samples. We found that TLR4-MSCs showed an increased expression of CXCL-1(1.1 ± 0.07 vs 0.8 ± 0.06; p = 0.001; Fig. 1B), CXCL-10(2 ± 0.2 vs 0.9 ± 0.04; p = 0.0007; Fig. 1B), CXCL-12 (1 ± 0.1 vs 0.8 ± 0.1; p = 0.02; Fig. 1B) while TLR3-MSCs showed an increase in expression of G-CSF (1.7 ± 0.1 vs 0.9 ± 0.08; p = 0.001; Fig. 1B).

3.4 TLR-3 primed MSCs modulate the proliferation of T lymphocytes

TLR3-MSC or TLR4-MSC treatment led to no change in frequency of CD3, CD4 or CD8 responder cells in a MLR reaction in comparison to un-MSCs (Fig. 2A).Fig. 2 Comparison of responder T lymphocyte subsets and their proliferation in response to primed/unprimed MSCs. Flow cytometric analysis plots indicating the percentage of (A) Responder T cells and (B) Proliferation index of Responder T cells, flow cytometry was performed on Day-5 post-co-culture. Data are represented as mean ± SD. * represents the statistical difference between the respective groups (*p < 00.05; **p < 00.005, ns = not significant).

Fig. 2

Proliferated responder cells were identified by CFSE dilution [42]. The proliferation index of CD4 T cells was significantly reduced in TLR3 group when compared to TLR4 or unprimed group (Fig. 2B).

3.5 TLR-3 primed MSCs favour the survival naïve T cells

TNAI cells are metabolically inactive until they encounter a cognate antigen, which results in their activation and differentiation into TEFF/TREG and TMEM cells. Effect of TLR-primed MSCs was assessed to identify alterations in the T-cell profile of responder cells. We compared the ratios of TNAI, TEFF and TMEM cells for both helper and cytotoxic T cells.

An increase in the responder CD4 TNAI:TEFF cells was observed for TLR3-MSC group in comparison to the un-MSC group (0.4 ± 0.1 Vs 0.2 ± 0.06; p = 0.01; Fig. 3A). Responder CD4 TNAI:TMEM cells in TLR3-MSC group showed slight increase in comparison to un-MSC, though statistically not significant (0.4 ± 0.1 Vs 0.2 ± 0.06; p = 0.06; Fig. 3A). Further, the responder CD4 TNAI cells were compared against the TMEM cell subsets, i.e. TMEM-EM and TMEM-CM. An increase in responder CD4 TNAI: TMEM-EM cells was evident in TLR3-MSC group in comparison to un-MSC (5 ± 6.2 Vs 2.3 ± 2.1; p = 0.02; Fig. 3A) while no change was observed for CD4 TNAI: TMEM-CM cells (Fig. 3A).Fig. 3 Comparative analysis of responder T lymphocyte distribution under different culture conditions. Flow cytometric analysis plots indicating (A) Responder CD4 T cell subset ratio (CD4 TNAI: TEFF cells, CD4 TNAI: TMEM cells, CD4 TNAI: TMEM-EM cells and CD4 TNAI: TMEM-CM cells) and (B) Responder CD8 T cell subset ratio (CD8 TNAI: TEFF cells, CD8 TNAI: TMEM cells, CD8 TNAI: TMEM-EM cells and CD8 TNAI: TMEM-CM cells) and percentage of (C) TREGS. Responder cells in a MLR setup were cultured with TLR3 primed MSCs or TLR4 primed MSCs, or unprimed MSCs. Flow cytometry was performed on Day-5 post-co-culture. Data is represented as mean ± SD. * represents the statistical difference between the respective groups (*p < 00.05; **<0.005, ns = not significant).

Fig. 3

Further, the analysis of CD8 subset ratios was performed which revealed a slightly higher responder CD8 TNAI:TEFF and CD8 TNAI:TMEM cells for TLR3-MSC group in comparison to the un-MSC group, however, this increase statistically insignificant (Fig. 3B). Subsequent analysis of CD8 TNAI cells against memory cell subsets showed no difference in CD8 TNAI:TMEM-CM for TLR3-MSC or TLR4-MSC group in comparison to un-MSC group (Fig. 3B).

TREGS have been identified as important mediators of immune tolerance [43,44]. A small but slightly significant increase in TREGS was observed for responder TREGS of TLR3-MSC group in comparison to the un-MSC group (1 ± 0.8 % Vs 0.4 ± 0.2 %; p = 0.06; Fig. 3C).

3.6 TLR-primed mesenchymal stromal cell pre-treatment does not modulate the responder B lymphocytes

Similar to T cells, B cells are also considered imperative mediators of the immune system. We found that treatment of responder cells with TLR-3,4 or unprimed MSCs in a MLR setup did not result in any change in the frequency (Fig. 4A) or proliferation (Fig. 4B) of B cells.Fig. 4 Comparison of responder B cell distribution under different culture conditions. Flow cytometric analysis plots indicating the percentage of responder (A) CD19 B cells, (B) proliferated CD19 B cells and (C) BREGS (Bregs, B10 cells and BIM cells). Responder cells in a MLR setup were cultured with TLR3 primed MSCs or TLR4 primed MSCs, or unprimed MSCs were co-cultured with inactivated stimulator cells. Flow cytometry was performed on Day-5 post-co-culture. Data are represented as mean ± SD. * represents the statistical difference between the respective groups (*p < 00.05; **p < 00.005).

Fig. 4

We analysed all relevant regulatory B cell subsets including Bregs, BIM and B10 cells as well, which are known to contribute to immunotolerance.

A slight increase in Bregs, B10 and BIM cells (Fig. 4C) was evident for responder cells in TLR3-MSC group. However, these changes were statistically insignificant. A minor increase in the percentage of responder Bregs was also observed in TLR4-MSC group.

3.7 Conditioned medium from TLR-primed mesenchymal stromal cells failed to modulate the T and B lymphocytes

Conditioned medium derived from MSCs has been suggested as alternate cell therapy in many studies. However, we found CM to be ineffective in modulating the response of T and B cells in our setup (Figs. S10–12).

4 Discussion

MSCs display unique immunomodulatory properties both in vitro and in vivo, which makes them desirable as a therapy for organ transplantation [45,46]. Paracrine factors secreted by MSCs [47,48] that aid in tolerance induction makes their use even more appealing. However, the mechanisms involved in the MSC immunomodulation in vivo are still not clear, thus limiting their use.

TLR3 and TLR4, have been reported to influence the biological properties of MSCs [49], including their immunomodulation [19,29]. This study was designed to understand the effect of TLR3 and TLR4 primed MSCs on the immune profile of responder cells upon stimulation. Taken together, the results from our study indicate three significant findings which contribute to understanding the role of TLRs in modulating properties of MSC. First, TLR3 primed MSCs increase expression of GCS-F while TLR4 primed MSCs showed an increase in the expression of CXCL-1, CXCL-10 and CXCL-12. Second, TLR3-MSC treatment had an impact on the ratio of CD4 TNAI cells into TEFF, TMEM or TMEM-EM cells and increased TREGS marginally. Third, TLR3 MSCs were able to alter the proliferation profile of T cells. Fourth, CM failed to produce any relevant results in comparison to the equivalent number of MSCs used.

In our study, the pre-treatment of MSCs with TLR3 agonist (poly I:C) led to an increase in the anti-inflammatory protein and cytokine GCS-F and IL-10 while TLR4 agonist (LPS) led to an increase in the proinflammatory cytokines CXCL-1, CXCL-10 and CXCL-12. MSCs are known to cause direct immunomodulation of TEFF cells which are attracted towards them by the secretion CXCL-1, CXCL-10, CXCL-12 47,50.

Studies have shown that both TLR3-MSCs and TLR4-MSCs when directly co-cultured with T cells, act by suppressing the proliferation of T cells [20,23]. However, our findings from our co-culture assay revealed that only TLR3-MSCs had an effect on proliferative ability of T cells.

Interestingly, TLR3-MSC pre-treatment increased the percentage of TNAI cells while decreasing TEFF and TMEM subset proportions. TMEM cells indicate heightened T cell responsiveness, and they are known to interfere with the graft survival in the transplant patients by directly stimulating the TEFF cells [50,51]. Therefore, expansion of TNAI cell population with low TEFF/TMEM cell differentiation post TLR3/4 primed MSC treatment is of immense clinical importance.

The immunomodulatory capacity of MSCs is often evaluated by their ability to induce regulatory cells. We found that TLR3-MSC treatment led to slight expansion of TREGS . BREGs cells however showed a slight increase that was statistically insignificant in the current in vitro set-up.

Many reports have been published in favour of cell-free therapy [52,53]. Few studies have shown that conditioned medium derived from MSCs (CM-MSCs) can produce the same effects as the live MSCs [54,55]. However, we found that CM prepared from an equivalent number of cells failed to produce any relevant change in immune cell subsets. A comparative study (MSC Vs CM-MSCs) in mice model of acute kidney injury also demonstrated the incompetence of CM-MSCs in kidney repair [56]. Another study in a preclinical model of ventilation-induced lung injury indicated the ineffectiveness of CM-MSCs in tissue repair and restoration [57].

This study is first of its kind which has used an in vitro co-culture based assay to imitate an in vivo transplantation setting, to elucidate the effect of MSC priming on the immune cell profile. The current report is based on the results of an in vitro setup and additional studies using an in vivo model are required. TLRs have been known to influence the biology of MSCs, which in return affects their therapeutic potential. Our data showed that MSCs after priming with TLR-3 agonist have a higher capacity to induce a state of immunotolerance. Our findings, however, primarily rely on cell proliferation and phenotypic markers to characterize T and B cell populations that have differentiated in response to TLR3/4 primed MSCs. While immune cell proliferation and differentiation can serve as a surrogate for function, incorporating intracellular cytokine staining to confirm cell activation could be beneficial. Furthermore, employing multiomic techniques like single-cell sequencing could provide a more comprehensive evaluation of reduced heterogeneity in immune cell populations following stimulation with TLR-primed MSCs.

Further, the effect of microenvironment on MSCs has been reported; however, the underlying decision of MSCs to bind to a specific TLR with higher affinity still needs to be investigated. In this context, it would be interesting to explore the possibility of induction of a similar or better level of immunotolerance after engagement of other TLRs or pro or anti-inflammatory cytokines. Besides this, such studies would form a basis for a better understanding of the in vivo mechanism of MSC immunomodulation. The graphical in Fig. 5 depicts our experimental setup with changes in analysed parameters..Fig. 5 Schematic of the study depicting study design, parameters analysed and changes observed by various priming protocols. Dotted black lines depict no effects in our experimental setup while green dotted lines depict significant changes in parameters analysed.

Fig. 5

Funding

AR was awarded Inspire Faculty Award (IFA11- LSBM-11; http://www.dst.gov.in/) by Department of Science and Technology (DST), India, for execution of this project. UR was provided with a fellowship as a stipend for the period of this study by UGC-CSIR, India (Ref. No.: 23/12/2012(ii)EU-V; http://www.ugc.ac.in/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

CRediT authorship contribution statement

Urvashi Kaundal: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Aruna Rakha: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Aruna Rakha reports financial support was provided by 10.13039/100019473 Department of Scince and Technology (DST) under the Indian Ministry of Science and Technology .

Abbreviations

BREGS regulatory B cells (all subsets)

Breg regulatory B cell subset

B10 transitional B cells

BIM immature transitional B cells

CM-MSCs conditioned medium derived from mesenchymal stromal cells

MSC mesenchymal stromal cells

TEFF effector T cells

TMEM memory T cells

TMEM-EM effector memory T cells

TMEM-CM central memory T cells

TNAI naive T cells

TREGS regulatory T cells

TLR toll-like receptors

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Data availability

Data will be made available on request.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrep.2024.101809.
==== Refs
References

1 Shi Y. Hu G. Su J. Mesenchymal stem cells: a new strategy for immunosuppression and tissue repair Cell Res. 20 5 May 2010 510 518 10.1038/cr.2010.44 20368733
2 Aggarwal S. Pittenger M.F. Human mesenchymal stem cells modulate allogeneic immune cell responses Blood 105 4 Feb 15 2005 1815 1822 10.1182/blood-2004-04-1559 15494428
3 Deng W. Han Q. Liao L. You S. Deng H. Zhao R.C. Effects of allogeneic bone marrow-derived mesenchymal stem cells on T and B lymphocytes from BXSB mice DNA Cell Biol. 24 7 Jul 2005 458 463 10.1089/dna.2005.24.458 16008514
4 Rasmusson I. Ringden O. Sundberg B. Le Blanc K. Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms Exp. Cell Res. 305 1 Apr 15 2005 33 41 S0014-4827(04)00738-4 [pii]10.1016/j.yexcr.2004.12.013 [doi] 15777785
5 Zhao Z.-G. Xu W. Sun L. Immunomodulatory function of regulatory dendritic cells induced by mesenchymal stem cells Immunol. Invest. 41 2 2012/02/01 2012 183 198 10.3109/08820139.2011.607877
6 Rutz S. Janke M. Kassner N. Hohnstein T. Krueger M. Scheffold A. Notch regulates IL-10 production by T helper 1 cells Proc. Natl. Acad. Sci. U.S.A. 105 9 Mar 4 2008 3497 3502 10.1073/pnas.0712102105 18292228
7 Ma O.K. Chan K.H. Immunomodulation by mesenchymal stem cells: interplay between mesenchymal stem cells and regulatory lymphocytes World J. Stem Cell. 8 9 Sep 26 2016 268 278 10.4252/wjsc.v8.i9.268
8 Cho D.-I. Kim M.R. Jeong H-y Mesenchymal stem cells reciprocally regulate the M1/M2 balance in mouse bone marrow-derived macrophages Experimental & Molecular Medicine. 01/1007/01/received 08/20/revised 09/12/accepted 46 1 2014 e70 10.1038/emm.2013.135
9 Bartholomew A. Sturgeon C. Siatskas M. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo Exp. Hematol. 30 1 Jan 2002 42 48 11823036
10 Huang H. He J. Teng X. Combined intrathymic and intravenous injection of mesenchymal stem cells can prolong the survival of rat cardiac allograft associated with decrease in miR-155 expression J. Surg. Res. 185 2 Dec 2013 896 903 10.1016/j.jss.2013.06.015 23870834
11 Xu D.M. Yu X.F. Zhang D. Mesenchymal stem cells differentially mediate regulatory T cells and conventional effector T cells to protect fully allogeneic islet grafts in mice Diabetologia 55 4 Apr 2012 1091 1102 10.1007/s00125-011-2433-9 22270222
12 Perico N. Casiraghi F. Introna M. Autologous mesenchymal stromal cells and kidney transplantation: a pilot study of safety and clinical feasibility Clin. J. Am. Soc. Nephrol. : CJASN 6 2 Feb 2011 412 422 10.2215/cjn.04950610 20930086
13 Reinders M.E. de Fijter J.W. Roelofs H. Autologous bone marrow-derived mesenchymal stromal cells for the treatment of allograft rejection after renal transplantation: results of a phase I study Stem Cells Translational Medicine 2 2 Feb 2013 107 111 doi:sctm.2012-0114 [pii] 10.5966/sctm.2012-0114 [doi] 23349326
14 Kaundal U. Ramachandran R. Arora A. B cells: a new player in mesenchymal stromal cell-mediated immune modulation in renal transplant patients Kidney Int. 96 1 2019 249 250 10.1016/j.kint.2019.03.028
15 Kaundal U. Ramachandran R. Arora A. Mesenchymal stromal cells mediate clinically unpromising but favourable immune responses in kidney transplant patients Stem Cell. Int. 2022 2154544 10.1155/2022/2154544 2022/02/15 2022
16 Perico N. Casiraghi F. Todeschini M. Long-term clinical and immunological profile of kidney transplant patients given mesenchymal stromal cell immunotherapy. Clinical trial Front. Immunol. 9 1359 2018-June-14 2018 10.3389/fimmu.2018.01359
17 Sheng H. Wang Y. Jin Y. A critical role of IFNgamma in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1 Cell Res. 18 8 Aug 2008 846 857 10.1038/cr.2008.80 18607390
18 Krampera M. Cosmi L. Angeli R. Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells Stem Cell. 24 2 Feb 2006 386 398 10.1634/stemcells.2005-0008
19 Waterman R.S. Tomchuck S.L. Henkle S.L. Betancourt A.M. A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an immunosuppressive MSC2 phenotype PLoS One 5 4 2010 e10088 10.1371/journal.pone.0010088
20 Rashedi I. Gomez-Aristizabal A. Wang X.H. Viswanathan S. Keating A. TLR3 or TLR4 activation enhances mesenchymal stromal cell-mediated treg induction via notch signaling Stem Cell. 35 1 Jan 2017 265 275 10.1002/stem.2485
21 Behm C. Blufstein A. Gahn J. Continuing effect of cytokines and toll-like receptor agonists on indoleamine-2,3-dioxygenase-1 in human periodontal ligament stem/stromal cells Cells 9 12 Dec 16 2020 10.3390/cells9122696
22 Kawasaki T. Kawai T. Toll-like receptor signaling pathways Front. Immunol. 5 2014 461 10.3389/fimmu.2014.00461 25309543
23 Opitz C.A. Litzenburger U.M. Lutz C. Toll-like receptor engagement enhances the immunosuppressive properties of human bone marrow-derived mesenchymal stem cells by inducing indoleamine-2,3-dioxygenase-1 via interferon-beta and protein kinase R Stem Cell. 27 4 Apr 2009 909 919 10.1002/stem.7
24 Kim D.S. Lee W.H. Lee M.W. Involvement of TLR3-dependent PGES expression in immunosuppression by human bone marrow mesenchymal Stem Cell. 14 2 Apr 2018 286 293 10.1007/s12015-017-9793-6
25 Qiu Y. Guo J. Mao R. TLR3 preconditioning enhances the therapeutic efficacy of umbilical cord mesenchymal stem cells in TNBS-induced colitis via the TLR3-Jagged-1-Notch-1 pathway Mucosal Immunol. 10 3 May 2017 727 742 10.1038/mi.2016.78 27649928
26 Fuenzalida P. Kurte M. Fernandez-O'ryan C. Toll-like receptor 3 pre-conditioning increases the therapeutic efficacy of umbilical cord mesenchymal stromal cells in a dextran sulfate sodium-induced colitis model Cytotherapy 18 5 May 2016 630 641 10.1016/j.jcyt.2016.02.002 27059200
27 Tolstova T. Dotsenko E. Kozhin P. The effect of TLR3 priming conditions on MSC immunosuppressive properties Stem Cell Res. Ther. 14 1 Nov 29 2023 344 10.1186/s13287-023-03579-y 38031182
28 Yao Y. Zhang F. Wang L. Lipopolysaccharide preconditioning enhances the efficacy of mesenchymal stem cells transplantation in a rat model of acute myocardial infarction J. Biomed. Sci. 16 Aug 20 2009 74 10.1186/1423-0127-16-74 19691857
29 Liotta F. Angeli R. Cosmi L. Toll-like receptors 3 and 4 are expressed by human bone marrow-derived mesenchymal stem cells and can inhibit their T-cell modulatory activity by impairing Notch signaling Stem Cell. 26 1 Jan 2008 279 289 10.1634/stemcells.2007-0454
30 Huang S. Wang D. Gu F. No significant effects of Poly(I:C) on human umbilical cord-derived mesenchymal stem cells in the treatment of B6.MRL-Fas(lpr) mice Current research in translational medicine 64 2 Apr-Jun 2016 55 60 10.1016/j.retram.2016.03.002 27316386
31 Pezzanite L.M. Chow L. Engiles J.B. Targeted transcriptomic analysis of synovial tissues from horses with septic arthritis treated with immune-activated mesenchymal stromal cells reveals induction of T-cell response pathways J. Am. Vet. Med. Assoc. 262 S1 Jun 1 2024 S73 s82 10.2460/javma.23.10.0561 38295517
32 Cassatella M.A. Mosna F. Micheletti A. Toll-like receptor-3-activated human mesenchymal stromal cells significantly prolong the survival and function of neutrophils Stem Cell. 29 6 Jun 2011 1001 1011 10.1002/stem.651
33 Niu G.C. Liu L. Zheng L. Zhang H. Shih D.Q. Zhang X. Mesenchymal stem cell transplantation improves chronic colitis-associated complications through inhibiting the activity of toll-like receptor-4 in mice BMC Gastroenterol. 18 1 Aug 13 2018 127 10.1186/s12876-018-0850-7 30103680
34 Brewster B.D. Rouch J.D. Wang M. Meldrum D.R. Toll-like receptor 4 ablation improves stem cell survival after hypoxic injury J. Surg. Res. 177 2 Oct 2012 330 333 10.1016/j.jss.2012.04.042 22703984
35 Dominici M. Le Blanc K. Mueller I. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement Cytotherapy 8 4 2006 315 317 10.1080/14653240600855905 16923606
36 Mudrabettu C. Kumar V. Rakha A. Safety and efficacy of autologous mesenchymal stromal cells transplantation in patients undergoing living donor kidney transplantation: a pilot study Nephrology 20 1 2015 25 33 10.1111/nep.12338 25230334
37 Hwang S.M. See C.-J. Choi J. The application of an in situ karyotyping technique for mesenchymal stromal cells: a validation and comparison study with classical G-banding Exp. Mol. Med. 45 12 2013 e68 10.1038/emm.2013.133 e68 24357832
38 Knutsen T. Spurbeck J.L. Barch M.J. The AGT Cytogenetics Laboratory Manual 1991
39 Cassatella M.A. Mosna F. Micheletti A. Toll-like receptor-3-activated human mesenchymal stromal cells significantly prolong the survival and function of neutrophils Stem Cell. 29 6 2011 1001 1011 10.1002/stem.651
40 Kim D.S. Jang I.K. Lee M.W. Enhanced immunosuppressive properties of human mesenchymal stem cells primed by interferon-γ EBioMedicine 28 Feb 2018 261 273 10.1016/j.ebiom.2018.01.002 29366627
41 Fuss I.J. Kanof M.E. Smith P.D. Zola H. Isolation of whole mononuclear cells from peripheral blood and cord blood Curr. Protoc. Im. 85 1 2009 7.1.1 7.1.8 10.1002/0471142735.im0701s85
42 Rakha A. Todeschini M. Casiraghi F. Assessment of anti-donor T cell proliferation and cytotoxic T lymphocyte-mediated lympholysis in living donor kidney transplant patients Methods Mol. Biol. 1213 2014 355 364 10.1007/978-1-4939-1453-1_29 25173397
43 Juvet S.C. Zhang L. Double negative regulatory T cells in transplantation and autoimmunity: recent progress and future directions J. Mol. Cell Biol. 4 1 Feb 2012 48 58 10.1093/jmcb/mjr043 22294241
44 Hendrikx T.K. van Gurp E.A. Sewgobind V.D. Generation of donor-specific regulatory T-cell function in kidney transplant patients Transplantation 87 3 Feb 15 2009 376 383 10.1097/TP.0b013e3181901b69 19202442
45 Reinders M.E.J. van Kooten C. Rabelink T.J. de Fijter J.W. Mesenchymal stromal cell therapy for solid organ transplantation Transplantation 102 1 Jan 2018 35 43 10.1097/tp.0000000000001879 28704335
46 Kaundal U. Bagai U. Rakha A. Immunomodulatory plasticity of mesenchymal stem cells: a potential key to successful solid organ transplantation. journal article J. Transl. Med. 16 1 February 15 2018 31 10.1186/s12967-018-1403-0 29448956
47 Ren G. Zhang L. Zhao X. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide Cell Stem Cell 2 2 Feb 7 2008 141 150 10.1016/j.stem.2007.11.014 18371435
48 Kyurkchiev D. Bochev I. Ivanova-Todorova E. Secretion of immunoregulatory cytokines by mesenchymal stem cells World J. Stem Cell. 6 5 Nov 26 2014 552 570 10.4252/wjsc.v6.i5.552
49 Chow L. Johnson V. Impastato R. Coy J. Strumpf A. Dow S. Antibacterial activity of human mesenchymal stem cells mediated directly by constitutively secreted factors and indirectly by activation of innate immune effector cells Stem Cells Translational Medicine 9 2 2019 235 249 10.1002/sctm.19-0092 31702119
50 Benichou G. Gonzalez B. Marino J. Ayasoufi K. Valujskikh A. Role of memory T cells in allograft rejection and tolerance Front. Immunol. 8 2017 170 10.3389/fimmu.2017.00170 28293238
51 Su C.A. Fairchild R.L. Memory T cells in transplantation Current Transplantation Reports 1 3 2014/09/01 2014 137 146 10.1007/s40472-014-0018-5
52 Vackovcá I. Kubinová S. Stem cell conditioned medium for cell-free therapies Cesk. Fysiol. 65 1 Oct 2016 25 31 29489089
53 Gama K.B. Santos D.S. Evangelista A.F. Conditioned medium of bone marrow-derived mesenchymal stromal cells as a therapeutic approach to neuropathic pain: a preclinical evaluation Stem Cell. Int. 2018 1 2018 8179013 10.1155/2018/8179013
54 Kay A.G. Long G. Tyler G. Mesenchymal stem cell-conditioned medium reduces disease severity and immune responses in inflammatory arthritis Sci. Rep. 7 1 Dec 21 2017 18019 10.1038/s41598-017-18144-w
55 Dahbour S. Jamali F. Alhattab D. Mesenchymal stem cells and conditioned media in the treatment of multiple sclerosis patients: clinical, ophthalmological and radiological assessments of safety and efficacy CNS Neurosci. Ther. 23 11 Nov 2017 866 874 10.1111/cns.12759 28961381
56 Xing L. Cui R. Peng L. Mesenchymal stem cells, not conditioned medium, contribute to kidney repair after ischemia-reperfusion injury Stem Cell Res. Ther. 5 4 Aug 21 2014 101 10.1186/scrt489 25145540
57 Hayes M. Curley G.F. Masterson C. Devaney J. O'Toole D. Laffey J.G. Mesenchymal stromal cells are more effective than the MSC secretome in diminishing injury and enhancing recovery following ventilator-induced lung injury Intensive Care Med Exp 3 1 Dec 2015 29 10.1186/s40635-015-0065-y 26472334
