A Systematic Study of the Effect of Different Molecular Weights of Hyaluronic Acid on Mesenchymal Stromal Cell-Mediated Immunomodulation

Abstract

Introduction: Osteoarthritis (OA) is associated with chronic inflammation, and mesenchymal stromal cells (MSCs) have been shown to provide pain relief and reparative effects in clinical investigations. MSCs are often delivered with hyaluronic acid (HA), although the combined mechanism of action is not fully understood; we thus investigated the immunomodulatory effects of combining MSCs with different molecular weights (MW) of HA.

Methods: HAs with MWs of 1.6 MDa (hHA), 150 kDa or 7.5 kDa, were added to MSCs alone or MSC-immune cell co-cultures. Gene expression analyses, flow cytometry and cytokine measurements were assessed to determine the effect of HAs on the MSC interactions with immune cells.

Results: MSCs in the presence of HAs, in both normal and lymphocyte-conditioned medium, showed negligible changes in gene expression. While addition of hHA resulted in increased proliferation of activated lymphocytes, both in the presence and absence of MSCs, the overall combined effect was a more regulated, homeostatic one; this was supported by higher ratios of secreted IL10/IFNγ and IL10/IL2, in lymphocyte cultures, than with lower MW HAs or no HA, both in the presence and absence of MSCs. In addition, examination of monocyte-derived macrophages showed an increased M2 macrophage frequency (CD14+CD163+CD206+) in the presence of hHA, both with and without MSCs.

Conclusions: hHA produces a less pro-inflammatory environment than lower MW HAs. Moreover, combining hHA with MSCs has an additive effect on the MSC-mediated immunomodulation, suggestive of a more potent combination treatment modality for OA.

Fig 1. Overall experimental design.

Legend of cell types shown on top. A) General experimental setup for MSC-immune cell interactions. A) Experimental setup to analyze the effects of HAs on the gene expression of MSCs. B) Experimental setup to analyze the effects of HAs on the capacity of MSCs to inhibit PBLs and Th cell proliferation, and to modulate Th cell activation (left); (right), the experimental setup to test the effects of HAs on the induction of Tregs by MSCs. C) Experimental setup to assay the effects of HAs on the immunomodulatory role of MSCs on MDMs.

Fig 2. Effect of different MWs of HA on the MSC-mediated inhibition of PBL proliferation.

(A) and (C) representative histograms of carboxyfluorescein succinimidyl ester (CFSE) fluorescence. Lines: Black solid line, hHA; black dashed line, 150 kDa HA; black dotted line, 7.5 kDa HA; and gray solid line, no HA. Gray shaded histogram represents resting PBMC control. (B) and (D) PBL proliferation from the whole PBMC population. (A) and (B), normal conditions. (C) and (D), IFNγ-supplemented conditions. Solid filled bars stand for hHA; hatched bars, 150 kDa HA; dotted bars, 7.5 kDa HA and empty bars, no HA. Black color, normal conditions and gray color with red frame, IFNγ-supplemented conditions. All data shown is from one representative MSC donor; additional data from three other donors and at different PBMC:MSC ratios are shown in S4 Fig. Error bars represent 95% CI. Statistical differences in the presence of MSCs account for MSCs from 4 donors, each tested in triplicate. Statistical differences in the absence of MSCs account for PBMCs from one donor tested in quadruplicate. * p<0.05, ** p<0.01 and ***<0.001.

Fig 3. Effect of different MWs of HA on the interaction of MSCs and Th cells.

Proliferation of Th cells, in the presence or absence of MSCs, without (A), and with IFNγ supplementation (B). (C) IL10, IFNγ and IL2 secreted levels in medium from activated Th cells with or without MSCs, and with or without IFNγ supplementation. (D) Ratios of secreted IL10/IFNγ, IL10/IL2. Solid filled bars stand for hHA; hatched bars, 150 kDa HA; dotted bars, 7.5 kDa HA and empty bars, no HA. Black color, normal conditions and gray color with red frame, IFNγ-supplemented conditions. All data shown is from one representative MSC donor; additional data from three other donors shown in S7 Fig. Statistical differences in the presence of MSCs account for MSCs from 4 donors, each tested in triplicate. Statistical differences in the absence of MSCs account for Th cells from one donor tested in quadruplicate. Error bars represent 95% CI. * p<0.05, ** p<0.01 and ***<0.001.

Fig 4. Effect of different MWs of HA on the MSC-mediated induction of Tregs.

Frequency of Tregs (CD25+CD127-) within CD4+ T cells is shown (A, B). A) Effect of MSC donor-to-donor variability and different MWs of HA on Treg frequency. B) Effect of IFNγ-supplemented conditions on the Treg ratio from CD4+ T cells when MSCs and/or HAs are present. (A) and (B) show data from two separate experiments; normal conditions are shown in (B) for comparison purposes with IFNγ- supplemented conditions. Solid filled bars stand for hHA; hatched bars, 150 kDa HA; dotted bars, 7.5 kDa HA and empty bars, no HA. Black color, normal conditions and gray color with red frame, IFNγ-supplemented conditions. Each bar indicates the mean of triplicates replicates from MSCs or Th cell from one donor. Error bars represent 95% CI. * p<0.05, ** p<0.01 and ***<0.001.

Fig 5. Effect of different MWs of HA on MSC-mediated induction of M2 MDMs.

(A) and (B) Frequency of M2 MDMs within the whole MDM population. (C) mean fluorescence intensity (MFI) levels of CD163 from whole MDM population. All data shown is from one representative experiment; additional data from 3 MSC donors is shown in S9 Fig. (D) and (E) Levels of secreted IL10 and IL1RA as measured by ELISA. Solid filled bars stand for hHA; hatched bars, 150 kDa HA; dotted bars, 7.5 kDa HA and empty bars, no HA. Black color, normal conditions and gray color with red frame, IFNγ-supplemented conditions. Statistical differences account for data from three independent experiments; each with MSCs and monocytes from different donors in allogeneic co-cultures. Each bar indicates the mean of triplicates. Error bars represent 95% CI. * p<0.05, ** p<0.01 and ***<0.001.