Myeloid-derived suppressor cells in the therapy of autoimmune diseases.

Myeloid-derived suppressor cells (MDSCs) are well recognized as critical factors in the pathology of tumors. However, their roles in autoimmune diseases are still unclear, which hampers the development of efficient immunotherapies. The role of different MDSCs subsets in multiple sclerosis, inflammatory bowel diseases, rheumatoid arthritis, type 1 diabetes, and systemic lupus erythematosus displayed different mechanisms of immune suppression, and several studies pointed to MDSCs' capacity to induce T-helper (Th)17 cells and tissue damage. These results also suggested that MDSCs could be present in different functional states and utilize different mechanisms for controlling the activity of T and B cells. Therefore, various therapeutic strategies should be employed to restore homeostasis in autoimmune diseases. The therapies harnessing MDSCs could be designed either as cell therapy or rely on the expansion and activation of MDSCs in vivo, or their depletion. Cumulatively, MDSCs are inevitable players in autoimmunity, and rational approaches in developing therapies are required to avoid the adverse effects of MDSCs and harness their suppressive mechanisms to improve the overall efficacy of autoimmunity therapy.

The association of human leucocyte antigen (HLA) alleles with COVID-19 severity: A systematic review and meta-analysis.

Due to their pivotal role in orchestrating the immune response, HLA loci were recognized as candidates for genetic association studies related to the severity of COVID-19. Since the findings on the effects of HLA alleles on the outcome of SARS-CoV-2 infection remain inconclusive, we aimed to elucidate the potential involvement of genetic variability within HLA loci in the molecular genetics of COVID-19 by classifying the articles according to different disease severity/outcomes and by conducting a systematic review with meta-analysis. Potentially eligible studies were identified by searching PubMed, Scopus and Web of Science literature databases. A total of 28 studies with 13,073 participants were included in qualitative synthesis, while the results of 19 studies with 10,551 SARS-CoV-2-positive participants were pooled in the meta-analysis. According to the results of quantitative data synthesis, association with COVID-19 severity or with the lethal outcome was determined for the following alleles and allele families: HLA-A*01, HLA-A*03, HLA-A*11, HLA-A*23, HLA-A*31, HLA-A*68, HLA-A*68:02, HLA-B*07:02, HLA-B*14, HLA-B*15, HLA-B*40:02, HLA-B*51:01, HLA-B*53, HLA-B*54, HLA-B*54:01, HLA-C*04, HLA-C*04:01, HLA-C*06, HLA-C*07:02, HLA-DRB1*11, HLA-DRB1*15, HLA-DQB1*03 and HLA-DQB1*06 (assuming either allelic or dominant genetic model). We conclude that alleles of HLA-A, -B, -C, -DRB1 and -DQB1 loci may represent potential biomarkers of COVID-19 severity and/or mortality, which needs to be confirmed in a larger set of studies.

Pomegranate Peel Extract Differently Modulates Gene Expression in Gingiva-Derived Mesenchymal Stromal Cells under Physiological and Inflammatory Conditions.

Pomegranate has shown a favorable effect on gingivitis/periodontitis, but the mechanisms involved are poorly understood. The aim of this study was to test the effect of pomegranate peel extract (PoPEx) on gingiva-derived mesenchymal stromal cells (GMSCs) under physiological and inflammatory conditions. GMSC lines from healthy (H) and periodontitis (P) gingiva (n = 3 of each) were established. The lines were treated with two non-toxic concentrations of PoPEX (low-10; high-40 µg/mL), with or without additional lipopolysaccharide (LPS) stimulation. Twenty-four genes in GMSCs involved in different functions were examined using real-time polymerase chain reaction (RT-PCR). PoPEx (mostly at higher concentrations) inhibited the basal expression of IL-6, MCP-1, GRO-α, RANTES, IP-10, HIF-1α, SDF-1, and HGF but increased the expression of IL-8, TLR3, TGF-ß, TGF-ß/LAP ratio, IDO-1, and IGFB4 genes in H-GMSCs. PoPEx increased IL-6, RANTES, MMP3, and BMP2 but inhibited TLR2 and GRO-α gene expression in P-GMSCs. LPS upregulated genes for proinflammatory cytokines and chemokines, tissue regeneration/repair (MMP3, IGFBP4, HGF), and immunomodulation (IP-10, RANTES, IDO-1, TLR3, COX-2), more strongly in P-GMSCs. PoPEx also potentiated most genes' expression in LPS-stimulated P-GMSCs, including upregulation of osteoblastic genes (RUNX2, BMP2, COL1A1, and OPG), simultaneously inhibiting cell proliferation. In conclusion, the modulatory effects of PoPEx on gene expression in GMSCs are complex and dependent on applied concentrations, GMSC type, and LPS stimulation. Generally, the effect is more pronounced in inflammation-simulating conditions.

Sitagliptin Induces Tolerogenic Human Dendritic Cells.

Sitagliptin, an anti-diabetic drug, is a dipeptidyl peptidase (DPP)-4/CD26 inhibitor with additional anti-inflammatory and immunomodulatory properties. In this study, we investigated for the first time the effect of sitagliptin on the differentiation and functions of human dendritic cells generated from monocytes (MoDCs) for 4 days using the standard GM-CSF/IL-4 procedure. LPS/IFN-γ treatment for an additional 24 h was used for maturation induction of MoDCs. Sitagliptin was added at the highest non-cytotoxic concentration (500 µg/mL) either at the beginning (sita 0d protocol) or after MoDC differentiation (sita 4d protocol). Sitagliptin impaired differentiation and maturation of MoDCs as judged with the lower expression of CD40, CD83, CD86, NLRP3, and HLA-DR, retention of CD14 expression, and inhibited production of IL-ß, IL-12p70, IL-23, and IL-27. In contrast, the expression of CD26, tolerogenic DC markers (ILT4 and IDO1), and production of immunoregulatory cytokines (IL-10 and TGF-ß) were increased. Generally, the sita 0d protocol was more efficient. Sitagliptin-treated MoDCs were poorer allostimulators of T-cells in MoDC/T-cell co-culture and inhibited Th1 and Th17 but augmented Th2 and Treg responses. Tolerogenic properties of sitagliptin-treated MoDCs were additionally confirmed by an increased frequency of CD4+CD25+CD127- FoxP3+ Tregs and Tr1 cells (CD4+IL-10+FoxP3-) in MoDC/T-cell co-culture. The differentiation of IL-10+ and TGF-ß+ Tregs depended on the sitagliptin protocol used. A Western blot analysis showed that sitagliptin inhibited p65 expression of NF-kB and p38MAPK during the maturation of MoDCs. In conclusion, sitagliptin induces differentiation of tolerogenic DCs, and the effect is important when considering sitagliptin for treating autoimmune diseases and allotransplant rejection.

Ex vivo study of IL-6 expression and function in immune cell subsets from human periapical lesions.

AIM: Even though IL-6 is a key inflammatory cytokine in periapical lesions (PLs), its function in stable periapical disease is unknown. Therefore, the aim of this study was to investigate the following: first, the ex vivo production of IL-6 by clinically different PLs; next, subsets of immune cells expressing IL-6 in PLs according to their inflammatory status and finally, modulatory effect of IL-6 on T-cell cytokine production in cell cultures. METHODOLOGY: Inflammatory cells were isolated from a total of 95 human PLs. Detection of IL-6+ cells within the myeloid and lymphoid populations was performed by multicolour flow cytometry. ELISA and FlowCytomix Microbeads Assay were used to measure cytokine levels in culture supernatants. To study the role of IL-6 in PLs, mononuclear cells (MNC) from symptomatic (Sy) or asymptomatic (Asy) PLs were treated with IL-6 or Tocilizumab, an IL-6R blocking antibody. The differences between groups were tested by unpaired t-test, Mann-Whitney or Friedman tests. RESULTS: The levels of IL-6 in PL MNC culture supernatants were significantly higher compared with total PL cells and PL granulocytes (p < .001). MNC from Sy PLs produced significantly higher levels of IL-6 than MNC from Asy PLs (p < .001). Flow cytometry analysis showed that NKT cells, CD8+ T cells and M2 macrophages (MØ), were dominant IL-6+ cells, in contrast to CD4+ T cells. However, CD8+ and CD4+ T cells contributed the most to IL-6 production. IL-6hi producing MNC cultures had higher levels of Th1 (IFN-γ), Th17 (IL-17A), Tfh (IL-21) and RANKL, whereas Th2 (IL-4) and Tregs cytokines (IL-10, TGF-ß) were lower compared with IL-6low -producing cultures. Exogenous IL-6 stimulated 17A, IL-21 and RANKL, independently of PL activation status, but decreased IFN-γ, IL-4 and IL-33 levels in IL-6hi -producing cultures. Tocilizumab increased IL-10 and TGF-ß in IL-6low -producing cultures. All differences were p < .05. CONCLUSIONS: Most immune cells from Sy PLs expressed higher levels of IL-6 compared with Asy PLs, which correlated with IL-6 production in culture. Analysis of cytokines suggested a dominant pro-inflammatory T-cell response and osteodestructive function of IL-6 in PLs judging by Th17/Tfh cell activation, Tregs inhibition and increased RANKL/OPG ratio. Excessive IL-6 production decreased Th1/Th2 responses.

Mesenchymal Stromal Cells from Healthy and Inflamed Human Gingiva Respond Differently to Porphyromonas gingivalis.

Gingiva-Derived Mesenchymal Stromal Cells (GMSCs) have been shown to play an important role in periodontitis. However, how P. gingivalis, one of the key etiological agents of the disease, affects healthy (H)- and periodontitis (P)-GMSCs is unknown. To address this problem, we established 10 H-GMSC and 12 P-GMSC lines. No significant differences in morphology, differentiation into chondroblasts and adipocytes, expression of characteristic MSCS markers, including pericyte antigens NG2 and PDGFR, were observed between H- and P-GMSC lines. However, proliferation, cell size and osteogenic potential were higher in P-GMSCs, in contrast to their lower ability to suppress mononuclear cell proliferation. P. gingivalis up-regulated the mRNA expression of IL-6, IL-8, MCP-1, GRO-α, RANTES, TLR-2, HIF-1α, OPG, MMP-3, SDF-1, HGF and IP-10 in P-GMSCs, whereas only IL-6, MCP-1 and GRO-α were up-regulated in H-GMSCs. The expression of MCP-1, RANTES, IP-10 and HGF was significantly higher in P-GMSCs compared to H-GMSCs, but IDO1 was lower. No significant changes in the expression of TLR-3, TLR-4, TGF-ß, LAP, IGFBP4 and TIMP-1 were observed in both types of GMSCs. In conclusion, our results suggest that P-GMSCs retain their pro-inflammatory properties in culture, exhibit lower immunosuppressive potential than their healthy counterparts, and impaired regeneration-associated gene induction in culture. All these functions are potentiated significantly by P. gingivalis treatment.

Immunomodulatory Activity of Punicalagin, Punicalin, and Ellagic Acid Differs from the Effect of Pomegranate Peel Extract.

BACKGROUND: Our recent study has shown that pomegranate peel extract (PEx) showed significant immunomodulatory activity, which might be caused by ellagitannins. The aim of this work was to test the hypothesis that ellagitannin components act synergistically in the modulation of cytokine production. METHODS: Human peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with phytohemagglutinin and treated with different concentrations of PEx or punicalagin (PG), punicalin (PN), and ellagic acid (EA), alone or with their combinations. Cytotoxicity, cell proliferation, and cytokine production were determined. RESULTS: Non-cytotoxic concentrations of all compounds significantly inhibited cell proliferation. IC50 values (µg/mL) were: EA (7.56), PG (38.52), PEx (49.05), and PN (69.95). PEx and all ellagitannins inhibited the levels of TNF-α, IL-6, and IL-8, dose-dependently, and their combinations acted synergistically. PEx and all ellagitannins inhibited Th1 and Th17 responses, whereas the lower concentrations of PEx stimulated the production of IL-10, a Treg cytokine, as did lower concentrations of EA. However, neither component of ellagitannins increased Th2 response, as was observed with PEx. CONCLUSIONS: The combination of PG, PN, and EA potentiated the anti-inflammatory response without any significant synergistic down-modulatory effect on T-cell cytokines. The increased production of IL-10 observed with PEx could be attributable to EA, but the examined ellagitannins are not associated with the stimulatory effect of PEx on Th2 response.

Differences in cytocompatibility, dynamics of the oxide layers' formation, and nickel release between superelastic and thermo-activated nickel-titanium archwires.

Superelastic (SE) and thermo-activated (TA) nickel-titanium (NiTi) archwires are used in everyday orthodontic practice, based on their acceptable biocompatibility and well-defined shape memory properties. However, the differences in their surface microstructure and cytotoxicity have not been clearly defined, and the standard cytotoxicity tests are too robust to detect small differences in the cytotoxicity of these alloys, all of which can lead to unexpected adverse reactions in some patients. Therefore, we tested the hypothesis that the differences in manufacture and microstructure of commercially available SE and TA archwires may influence their biocompatibility. The archwires were studied as-received and after conditioning for 24 h or 35 days in a cell culture medium under static conditions. All of the tested archwires, including their conditioned medium (CM), were non-cytotoxic for L929 cells, but Rematitan SE (both as received and conditioned) induced the apoptosis of rat thymocytes in a direct contact. In contrast, TruFlex SE and Equire TA increased the proliferation of thymocytes. The cytotoxic effect of Rematitan SE correlated with the higher release of Ni ions in CM, higher concentration of surface Ni and an increased oxygen layer thickness after the conditioning. In conclusion, the apoptosis assay on rat thymocytes, in contrast to the less sensitive standard assay on L929 cells, revealed that Rematitan SE was less cytocompatible compared to other archwires and the effect was most probably associated with a higher exposition of the cells to Ni on the surface of the archwire, due to the formation of unstable oxide layer.

Tumor necrosis factor-α promotes survival and phenotypic maturation of poly(I:C)-treated dendritic cells but impairs their Th1 and Th17 polarizing capability.

BACKGROUND AIMS: Toll-like receptor (TLR)-3 synthetic agonist polyinosinic-polycytidylic acid (poly(I:C)) is a promising agent for dendritic cell (DC)-based anti-tumor vaccines because of its ability to induce a strong maturation of DCs, but such an effect is followed by stimulation of DC apoptosis. Tumor necrosis factor (TNF)-α may promote the survival of poly(I:C)-stimulated DCs, but it is not known in detail how this combination affects the maturation and polarization capacity of monocyte-derived (Mo)DCs. METHODS: Immature MoDCs, generated from human monocytes, were treated with different concentrations of poly(I:C) combined with TNF-α, and the effect on survival, phenotype, production of cytokines, allostimulatory and Th polarization capacity was assessed after 24 and 48 h. RESULTS: We showed that TNF-α inhibited the dose-dependent pro-apoptotic effect of poly(I:C). However, TNF-α also decreased poly(I:C)-induced production of interleukin (IL)-12 and IL-23 by MoDCs, which correlated with their diminished capacity to stimulate cellular proliferation, interferon-γ and IL-17 production by allogeneic CD4(+)T cells in co-culture. Such an effect was more pronounced after 24 h and could not be restored by CD40 ligation. In the presence of CD40L, TNF-α even stimulated IL-10 production and immunoglobulin-like transcript 3 expression by poly(I:C)-matured DCs, which correlated with their increased capacity to induce IL-10 production by CD4(+)T cells. CONCLUSION: Even though TNF-α could promote the survival of poly(I:C)-matured MoDCs, it also suppresses key anti-tumor functions of these cells, which could have important implications when considering this, already suggested, protocol for the DC-based anti-tumor therapy.

Fast dendritic cells matured with Poly (I:C) may acquire tolerogenic properties.

BACKGROUND AIMS: Because of the labor-intensive and time-consuming conventional protocols for the generation of dendritic cells (DCs) as the most promising tools for anti-cancer therapy that enable the induction of a T-helper (Th)1-mediated anti-tumor immune response, the use of short-term protocols has been proposed. However, data on the applicability of such protocols in cancer immunotherapy are quite limited. METHODS: We compared the phenotypic and functional capability of fast DCs (fDCs) differentiated for 24 h and then matured for 48 h with Poly (I:C), a strong Th1-promoting agent, with donor-matched conventional DCs (cDCs) differentiated for 5 days and matured likewise. RESULTS: Of 12 donors tested, we identified seven whose monocytes failed to develop into immunogenic DCs through the use of fDC protocol, on the basis of incomplete downregulation of CD14, low expression of CD1a and macrophage-like morphology. Such fDCs have significantly lower expression of CD83, CD86, CCR7 and CD40, weaker allo-stimulatory Th1- and Th17-polarizing capacity caused by poor production of interleukin (IL)-12p70 and IL-23 and high production of IL-10, and prominent Th2-polarizing capacity, compared with donor-matched cDCs. Furthermore, such fDCs had tolerogenic properties as judged by higher expression of indolamine dioxigenase-3, IDO-1 and IL-1ß and induction of a higher percentage of CD4(+)CD25(+)FoxP3(+) T cells. These findings correlated with increased transforming growth factor (TGF)-ß production by fDC-primed CD3(+)T cells and their stronger anti-proliferative capacity. CONCLUSIONS: We emphasize that although fDCs could probably be applied as an alternative to cDCs for cancer therapy, the fDC protocol should not be applied to donors whose DCs acquire tolerogenic capabilities.