Immunomodulatory effect of IFN-γ licensed adipose-mesenchymal stromal cells in an in vitro model of inflammation generated by SARS-CoV-2 antigens

Abstract

In recent years, clinical studies have shown positive results of the application of Mesenchymal Stromal Cells (MSCs) in severe cases of COVID-19. However, the mechanisms of immunomodulation of IFN-γ licensed MSCs in SARS-CoV-2 infection are only partially understood. In this study, we first tested the effect of IFN-γ licensing in the MSC immunomodulatory profile. Then, we established an in vitro model of inflammation by exposing Calu-3 lung cells to SARS-CoV-2 nucleocapsid and spike (NS) antigens, and determined the toxicity of SARS-CoV-2 NS antigen and/or IFN-γ stimulation to Calu-3. The conditioned medium (iCM) generated by Calu-3 cells exposed to IFN-γ and SARS-CoV-2 NS antigens was used to stimulate T-cells, which were then co-cultured with IFN-γ-licensed MSCs. The exposure to IFN-γ and SARS-CoV-2 NS antigens compromised the viability of Calu-3 cells and induced the expression of the inflammatory mediators ICAM-1, CXCL-10, and IFN-β by these cells. Importantly, despite initially stimulating T-cell activation, IFN-γ-licensed MSCs dramatically reduced IL-6 and IL-10 levels secreted by T-cells exposed to NS antigens and iCM. Moreover, IFN-γ-licensed MSCs were able to significantly inhibit T-cell apoptosis induced by SARS-CoV-2 NS antigens. Taken together, our data show that, in addition to reducing the level of critical cytokines in COVID-19, IFN-γ-licensed MSCs protect T-cells from SARS-CoV-2 antigen-induced apoptosis. Such observations suggest that MSCs may contribute to COVID-19 management by preventing the lymphopenia and immunodeficiency observed in critical cases of the disease.

Keywords: COVID-19; IFN-γ; Mesenchymal stem cells; Nucleocapsid; SARS-CoV-2; Spike; T-cells.

Fig. 1

Experimental design. Calu-3 lung cells were exposed to IFN-γ and SARS-CoV-2 NS antigens. After 48 h, this inflamed conditioned medium (iCM) was collected and used in the proposed experiments. PBMCs were exposed to NS antigens in iCM medium and treated for 24 h with IFN-γ-licensed MSCs. After this period, the expression of activation markers was determined in T-cells. In parallel, PBMCs were exposed to NS antigens in iCM medium and after 72 h they were treated with IFN-γ-licensed MSCs. Following 72 h of treatment, the percentage of memory T-cells, the production of pro- and anti-inflammatory cytokines, the levels of T-cell apoptosis, and the expression of genes related to the immune response and cell death in both MSCs and PBMCs were determined.

Fig. 2

Characterization of MSCs. (A) Expression levels of CD90, CD73, CD44, CD105, and negative cocktail (NC) antibodies on MSCs. Representative flow cytometry histograms are shown on the left. (B) Proliferation of T-cells after their activation with PHA and co-culture with MSCs (10:1 ratio). (C) Proliferation of T-cells after their activation with PHA and co-culture with control MSCs or MSCs previously licensed with increasing doses of IFN-γ. (D-J) Gene expression analysis of IL-10, TGF-β, TSG-6, IDO, PDL-1, TMPRSS2, and ACE2 in MSCs or IFN-γ-licensed MSCs. The fold changes were determined by the 2^{− ΔΔCt} method, using the mean Ct value of MSCs as a reference. Results are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3

Characterization of the viability and inflammatory status of Calu-3 cells exposed to NS antigens. (A) ACE2 and TMPRSS2 expression in Calu-3 and A549 lung cell lines. The fold changes were determined by the 2^{− ΔΔCt} method, using the mean Ct value of A549 as a reference. (C) Cell viability determined by MTT of Calu-3 cells exposed to IFN-γ and NS antigens. (D) LDH release by Calu-3 exposed to IFN-γ and NS antigens. (E-L) Transcriptional levels of CASP-1, CASP-8, GSDMD, IL-1β, IL-6, CXCL10, ICAM-1, and IFN-β in Calu-3 cells exposed or not to IFN-γ and NS antigens. The fold changes were determined by the 2^{− ΔΔCt} method, using the mean Ct value of Calu-3 cells as a reference. (M) Caspase 1 activity in Calu-3 cells exposed or not to IFN-γ and NS antigens. Results are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 4

Assessment of activation markers and memory T-cells. (A-E) Expression of CD69, CD137, CD38, and CD25 on T-cells after the culture of PBMCs with NS antigens in iCM, and coculture or not with IFN-γ-licensed MSCs. Representative flow cytometry plots of T-cells positive for CD69, CD137, CD38, and CD25 are shown on the left. (F-J) Percentage of naive T-cells, TEM, TEMRA, and TCM after culture of PBMCs in RPMI medium, with NS antigens in iCM, and coculture with IFN-γ-licensed MSCs. Representative gating strategy and flow cytometry plots of CCR7 and CD45RA expression on T-cells are shown on the left. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 5

T-cell apoptosis and gene expression analysis. (A) Representative flow cytometry plots of annexin-V staining on CD4 and CD8 T-cells. (B, C) Percentage of annexin-V + CD4 and CD8 T-cells after the culture of PBMCs with NS antigens in iCM, and treatment or not with IFN-γ-licensed MSCs. (D-M) Transcriptional levels of IDO, IL-10, TSG-6, TGF-β, FGF2, ANGPT1, ANGPT2, VEGF, EGF, and HGF in IFN-γ licensed MSCs, IFN-γ-licensed MSCs that were cultured in iCM medium, and IFN-γ-licensed MSCs that were co-cultured with PBMCs in iCM medium. (N) Heatmap showing the expression of inflammatory, regenerative, and apoptosis-related genes in IFN-γ licensed MSCs, IFN-γ-licensed MSCs that were cultured in iCM medium, and IFN-γ-licensed MSCs that were co-cultured with PBMCs in iCM medium. All real-time PCRs reactions were performed in technical duplicate, and the relative fold change was obtained with the 2^−∆∆Ct method. Data are expressed as mean with SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 6

Cytokine quantification. Levels of (A) IFN-γ, (B) TNF-α, (C) IL-2, (D) IL-10, and (E) IL-6 secreted in the culture of PBMCs exposed to NS antigens in iCM, treated or not with IFN-γ-licensed MSCs. *p < 0.05; **p < 0.01; ****p < 0.0001.