Human Omental Mesothelial Cells Impart an Immunomodulatory Landscape Impeding B- and T-Cell Activation

Citation: Gauthier, B.R.; Rubio-Contreras, D.; Gómez-Rosado, J.C.; Capitán-Morales, L.C.; Hmadcha, A.; Soria, B.; Lachaud, C.C. Human Omental Mesothelial Cells Impart an Immunomodulatory Landscape Impeding Band T-Cell Activation. Int. J. Mol. Sci. 2022,23, 5924. https://doi.org/10.3390/ ijms23115924 Academic Editor: Joseph Barbi Received: 16 March 2022 Accepted: 23 May 2022 Published: 25 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Human Omental Mesothelial Cells Impart an Immunomodulatory Landscape Impeding Band T-Cell Activation Benoit R. Gauthier 1,2,* , Diana Rubio-Contreras 3,4, Juan Carlos Gómez-Rosado 5,6, Luis Cristobal Capitán-Morales 5,6, Abdelkrim Hmadcha 2,7 , Bernat Soria 2,8 and Christian Claude Lachaud 1,* 1Andalusian Center for Molecular Biology and Regenerative Medicine-CABIMER, Junta de Andalucía-University of Pablo de Olavide-University of Seville-CSIC, 41092 Seville, Spain 2Biomedical Research Network on Diabetes and Related Metabolic Diseases (CIBERDEM), Institute of Health Carlos III, 28029 Madrid, Spain; [email protected] (A.H.); [email protected] (B.S.) 3Instituto de Biomedicina de Sevilla (IBiS), Hospital Virgen del Rocío-CSIC-Universidad de Sevilla, and Departamento de Genética, Universidad de Sevilla, 41013 Seville, Spain; [email protected] 4Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41012 Seville, Spain 5Unidad de Gestión Clínica de Cirugía General y Digestiva, Hospital Universitario Virgen Macarena, Avda. Dr Fedriani s/n, 41009 Sevilla, Spain; dr[email protected] (J.C.G.-R.); [email protected] (L.C.C.-M.) 6Departamento de Cirugía, Universidad de Sevilla, Avda. Dr Fedriani s/n, 41009 Sevilla, Spain 7General Hospital, Alicante Institute for Health and Biomedical Research (ISABIAL), 03010 Alicante, Spain 8 Institute of Bioengineering and Health Research Institute (ISABIAL), Dr Balmis University Hospital (HGUA), Miguel Hernández University School of Medicine, 03010 Alicante, Spain *Correspondence: [email protected] (B.R.G.); [email protected] (C.C.L.); Tel.: +34-954-468-004 (B.R.G. & C.C.L.) Abstract: Mesothelial cells form the mesothelium, a simple epithelium lining the walls of serous cavities and the surface of visceral organs. Although mesothelial cells are phenotypically well characterized, their immunoregulatory properties remain largely unknown, with only two studies reporting their capacity to inhibit T cells through TGFβ and their consumption of L-arginine by arginase-1. Whether human mesothelial cells can suppress other immune cells and possess additional leukosuppressive mechanisms, remain to be addressed to better delineate their therapeutic potential for cell therapy. Herein, we generated secretomes from omental mesothelial cells (OMC) and assess their capacity to inhibit lymphocytes proliferation, suppress activated T and B cells, as well as to modify macrophage activation markers. The secretome from mesenchymal stromal cells (MSC) served as a control of immuno-suppression. Although OMC and MSC were phenotypically divergent, their cytokine secretion patterns as well as expression of inflammatory and immunomodulary genes were similar. As such, OMCand MSC-derived secretomes (OMC-S and MSC-S) both polarized RAW 264.7 macrophages towards a M2-like anti-inflammatory phenotype and suppressed mouse and human lymphocytes proliferation. OMC-S displayed a strong ability to suppress mouseand human-activated CD19 + /CD25 + B cells as compared to MSC-S. The lymphosuppressive activity of the OMC-S could be significantly counteracted either by SB-431542, an inhibitor of TGF β and activin signaling pathways, or with a monoclonal antibody against the TGF β 1, β 2, and β 3 isoforms. A strong blockade of the OMC-S-mediated lymphosuppressive activity was achieved using L-NMMA, a specific inhibitor of nitric oxide synthase (NOS). Taken together, our results suggest that OMC are potent immunomodulators. Keywords: mesothelial cells; adult stem cells; lymphocytes; macrophages; immunosuppression; immunomodulation Int. J. Mol. Sci. 2022,23, 5924. https://doi.org/10.3390/ijms23115924 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022,23, 5924 2 of 18 1. Introduction Mesothelial cells and their basement membrane form the mesothelium, a simple epithelium that lines the wall of serous cavities as well as visceral organs [ 1 , 2 ]. Although initially viewed as a support tissue, the mesothelium is now considered a highly dynamic tissue, whose functions are critical for the proper maintenance of body serous cavities [ 3 – 5 ]. A main function of mesothelial cells is to secrete large amounts of surfactant molecules, principally glycosaminoglycans that create a protective and viscous barrier that allows visceral organs to freely move inside serous cavities [ 2 ]. In addition to this mechanostructural function, several in vitro studies have shown that mesothelial cells can secrete either antior pro-inflammatory factors pending the environment [ 6 – 9 ]. In this context, co-injection of Freund adjuvant, a potent immunopotentiator, in combination with GMCSF inhibits expression of IL-10 in rat peritoneal mesothelial cells while gaining strong IL-6 expression [ 8 ]. Additionally, it was demonstrated that peritoneal mesothelial cells secrete a relevant quantity of colony-stimulating factor 1 (CSF1) which strongly induces macrophages’ proliferation and regulates their homeostatic maintenance [ 10 ]. Despite these advances, little is known on the biological role of the different cytokines or inflammatory mediators secreted by mesothelial cells in either regulating the homeostasis of serous cavities or the activation state of serous fluid immune cells. In recent years, the development of novel mesothelial-cell-based therapies for the treatment of human diseases has come into the limelight. Indeed, mesothelial cells fulfill diverse criteria for cell types considered with “high therapeutic potential” in regenerative medicine, as in the case for mesenchymal stromal cells (MSC). A key advantage of mesothelial cells is accessibility from diverse sources and ease of expansion in vitro [11–13]. In this context, the greater omentum is a large peritoneal fold containing abundant adipose tissue and is considered an optimal source from which omental mesothelial cells (OMC) can be harvested in therapeutically relevant numbers and with minimal health concerns [ 11 , 12 , 14 ]. Mesothelial cells possess significant plasticity as indicated by their capacity to differentiate in vitro and acquire features of vascular smooth muscle cells, osteocytes, adipocytes, and chondrocytes [ 13 , 15 – 17 ]. Of particular interest, mesothelial cell transplantation in a rat myocardial infarction model was shown to promote local tissue regeneration [ 18 , 19 ]. Several studies have also demonstrated that autologous peritoneal grafts efficiently prevented post-operative peritoneal adhesions [ 14 , 20 , 21 ]. As such, current developments in mesothelial-cell-based therapies are principally focusing on tissue engineering of autologous mesothelial cell sheets for the regeneration of the damaged mesothelium [ 5 , 22 , 23 ]. Notwithstanding, as the mesothelium also share morpho-structural and biochemical similarities with other simple epitheliums, mesothelial cells have also been highlighted as useful surrogate cells to regenerate the corneal endothelium, vascular endothelium, or synovium [24–27]. Although mesothelial cells have been well defined phenotypically and are already engaged in the development of diverse therapeutic applications, very little is known about their immunoregulatory properties and whether they could be useful cells for the treatment of autoimmune disorders. In support of such function, OMC can efficiently suppress reactive T-cell responses through action of TGFβ 1 while human mesothelial-like cells derived from peritoneal fluid cells can efficiently suppress the proliferation of CD3-activated peripheral blood lymphocytes [ 28 , 29 ]. The lymphosuppressive activity of the mesothelial-like cells was attributed to high expression of arginase-1, which leads to L-arginine deprivation from the culture media, an amino acid essential for the survival and proliferation of lymphocytes [ 30 , 31 ]. More recently, mesothelial cells were shown to regulate the immune peritoneal homeostasis through secretion of diverse immune regulators such as IL-6, CSF1 further substantiating their immunoregulatory properties [9,10]. In order to further grasp the immunomodulatory function conveyed by human mesothelial cells, herein we focused on the capacity of the secretome released by human OMC to inhibit the activation and proliferation of lymphocytes, as well as to modify the Int. J. Mol. Sci. 2022,23, 5924 3 of 18 phenotype of macrophages. The latter was compared and contrasted to the MSC-derived secretome, a reference for immunosuppression [ 32 ]. Despite the fact that OMC and MSC are defined as two distinct mesodermal cell types, we find that their Th1/Th2/Th17 cytokine secretory profiles are similar. Accordingly, the OMCand MSC-derived secretomes were equally efficient in inhibiting the proliferation of both activated mouse and human lymphocytes. Nonetheless, the OMC-derived secretome suppressed mouse activated B cells more efficiently than the MSC-derived secretome. We also observed that the lymphosuppressive activity of OMC-derived secretome could be counteracted through inhibition of TGF β or by the addition of L-arginine [ 28 , 29 ]. Furthermore, we found that the immunosuppressive capacity of OMC-derived secretome was efficiently ablated by blocking endogenous nitric oxide synthases (NOS) in lymphocytes. Taken together, our results suggest that OMC are potent immunomodulators. 2. Results 2.1. Omental-Derived Mesothelial Cells Exhibit a Typical Phenotype Human mesothelial cells were isolated from the greater omentum of several donors and expanded in culture. Similar to mesothelial cells derived from the pleural cavity [ 33 ], OMC typically formed a cobblestone-like monolayer at confluency with no evidence of fibroblast contamination. By contrast, human adipose-tissue-derived MSC displayed a fibroblastic shape (Figure 1A). We next phenotypically characterized and compared to MSC these newly established cell lines, including their capacity to differentiate into several cell lineages. Both OMC and MSC lacked expression of the cell surface markers, HLA-DR/DP/DQ (MHC-II receptors) CD45 (hematopoietic) and CD31 (endothelial) whereas they expressed similar levels of the stromal cell markers CD90 and CD29 (Figure 1B). Consistent with their developmental origin, MSC expressed higher levels of the stroma/mesenchymal markers CD44, CD73, CD13, and CD105 as compared to OMC (Figure 1B). In contrast, cytokeratin (epithelial) and WT1 (mesothelial) were only expressed in OMC (Figure 1C). Interestingly, beta-catenin was highly expressed at the cell surface of OMC while only low levels could be discerned on MSC (Figure 1C). In contrast to previous reports using pericardial-fluid-derived mesothelial cells [ 16 ], OMC poorly differentiated into adipocytes as compared to MSC while readily acquired either an osteocyte or chondrocyte lineage (Figure 1D). Taken together, these results establish the bona fide mesothelial phenotype of OMC with stem/progenitor characteristics and differentiation potential. 2.2. Omental Mesothelial Cells Display a Mixed Proand Anti-Inflammatory Signature In order to better grasp the immunomodulatory function potentially conveyed by OMC, we profiled the Th1/Th2/Th17 cytokines secreted by OMC and compared it to that of MSC (Figure 2A,B). Although globally the cytokine secretion profiles were similar, some important differences were discerned between OMCand MSC-derived secretomes (OMC-S and MSC-S, respectively) (Figure 2A,B). As such, OMC-S and MSC-S secreted similarly high levels of interleukin-6 (IL-6), a pleiotropic cytokine with context-dependent proand antiinflammatory properties [ 34 ]. Both cell types also secreted similarly high levels of TGF β 3 (Figure 2B). In contrast, secretion of MIP-3 α (CCL20), Sgp130, IL-1sRI, TNFα , IL-21, and IL-21R was significantly higher in OMC as compared to MSC whereas CD40, CD40L, IL17R, IL-12p70, and IL-12p40 secretion was higher in MSC (Figure 2B). We next assessed transcript levels of several key inflammatory and immunomodulary genes (Figure 2C). OMC expressed significantly higher levels of IL-15 (T cells activation), IL-1 β ,IFN γ , and TNF α (pro-inflammatory cytokines), iNOS (inflammatory and immunosuppression), and ARG1,LGALS9, and IL-10 (anti-inflammatory). Of particular interest, the expression of the potent anti-inflammatory IL-10 cytokine was 100-fold higher in OMC as compared to MSC. Int. J. Mol. Sci. 2022,23, 5924 4 of 18 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 of 18 type of macrophages. The latter was compared and contrasted to the MSC-derived secretome, a reference for immunosuppression [32]. Despite the fact that OMC and MSC are defined as two distinct mesodermal cell types, we find that their Th1/Th2/Th17 cytokine secretory profiles are similar. Accordingly, the OMCand MSC-derived secretomes were equally efficient in inhibiting the proliferation of both activated mouse and human lymphocytes. Nonetheless, the OMC-derived secretome suppressed mouse activated B cells more efficiently than the MSC-derived secretome. We also observed that the lymphosuppressive activity of OMC-derived secretome could be counteracted through inhibition of TGFβ or by the addition of L-arginine [28,29]. Furthermore, we found that the immunosuppressive capacity of OMC-derived secretome was efficiently ablated by blocking endogenous nitric oxide synthases (NOS) in lymphocytes. Taken together, our results suggest that OMC are potent immunomodulators. 2. Results 2.1. Omental-Derived Mesothelial Cells Exhibit a Typical Phenotype Human mesothelial cells were isolated from the greater omentum of several donors and expanded in culture. Similar to mesothelial cells derived from the pleural cavity [33], OMC typically formed a cobblestone-like monolayer at confluency with no evidence of fibroblast contamination. By contrast, human adipose-tissue-derived MSC displayed a fibroblastic shape (Figure 1A). We next phenotypically characterized and compared to MSC these newly established cell lines, including their capacity to differentiate into several cell lineages. Both OMC and MSC lacked expression of the cell surface markers, HLADR/DP/DQ (MHC-II receptors) CD45 (hematopoietic) and CD31 (endothelial) whereas they expressed similar levels of the stromal cell markers CD90 and CD29 (Figure 1B). Consistent with their developmental origin, MSC expressed higher levels of the stroma/mesenchymal markers CD44, CD73, CD13, and CD105 as compared to OMC (Figure 1B). In contrast, cytokeratin (epithelial) and WT1 (mesothelial) were only expressed in OMC (Figure 1C). Interestingly, beta-catenin was highly expressed at the cell surface of OMC while only low levels could be discerned on MSC (Figure 1C). In contrast to previous reports using pericardial-fluid-derived mesothelial cells [16], OMC poorly differentiated into adipocytes as compared to MSC while readily acquired either an osteocyte or chondrocyte lineage (Figure 1D). Taken together, these results establish the bona fide mesothelial phenotype of OMC with stem/progenitor characteristics and differentiation potential. Figure 1. Phenotypic characterization of human omental mesothelial cells (OMC) and human mesenchymal stem cells (MSC). ( A ) Phase contrast pictures of confluent OMC and MSC cultures displaying typical cobblestone-type and fibroblastic morphologies, respectively. Scale bar is 100 µm . ( B ) Flow cytometric analysis of OMC and MSC for expression of hematopoietic (CD45), endothelial (CD31), HLA class II (HLA-DR/DP/DQ), and stromal/mesenchymal (CD90, CD29, CD44, CD73, CD73, CD13, CD105, and CD166) cell markers. Histograms for OMC and MSC are red and blue, respectively. M1 bar marks positivity delimited from isotype histograms (not shown). ( C ) Immunofluorescence analysis of OMC and MSC showing highly expressed epithelial (pan-cytokeratin or pan-CK) and mesothelial (Wilm’s tumor protein 1 or WT1) cell markers in OMC, but not in MSC, while the cell–cell junction protein β -catenin was expressed in both cell types. ( D ) Multilineage differentiation assay of OMC and MSC revealed limited adipogenic differentiation of OMC (Oilred O staining) compared to MSC. In contrast, OMC displayed more similar osteogenic and chondrogenic differentiation compared to MSC. (A–C) OMC used are ALIC1 cells. 2.3. The Secretome of OMC Induces M2 Polarization and Inhibits Mouse Lymphocyte Proliferation and Agglutination In order to determine whether the OMC secretome conveys either proor antiinflammatory properties, mouse Raw 264.7 cells were exposed to condition media obtained from OMC (OMC-S) cultures. Raw 264.7 cells cultured in the presence of OMC-S displayed reduced expression of the M1 pro-inflammatory markers CD54 and of the activator marker CD25 [ 35 ]. In contrast, expression of the M2 anti-inflammatory marker CD206 was increased as compared to cells that we cultured in fresh media (FM) (Figure 3A,B). Int. J. Mol. Sci. 2022,23, 5924 5 of 18 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 18 Figure 1. Phenotypic characterization of human omental mesothelial cells (OMC) and human mesenchymal stem cells (MSC). (A) Phase contrast pictures of confluent OMC and MSC cultures displaying typical cobblestone-type and fibroblastic morphologies, respectively. Scale bar is 100 µm. (B) Flow cytometric analysis of OMC and MSC for expression of hematopoietic (CD45), endothelial (CD31), HLA class II (HLA-DR/DP/DQ), and stromal/mesenchymal (CD90, CD29, CD44, CD73, CD73, CD13, CD105, and CD166) cell markers. Histograms for OMC and MSC are red and blue, respectively. M1 bar marks positivity delimited from isotype histograms (not shown). (C) Immunofluorescence analysis of OMC and MSC showing highly expressed epithelial (pan-cytokeratin or pan-CK) and mesothelial (Wilm’s tumor protein 1 or WT1) cell markers in OMC, but not in MSC, while the cell–cell junction protein β-catenin was expressed in both cell types. (D) Multilineage differentiation assay of OMC and MSC revealed limited adipogenic differentiation of OMC (Oilred O staining) compared to MSC. In contrast, OMC displayed more similar osteogenic and chondrogenic differentiation compared to MSC. (A–C) OMC used are ALIC1 cells. 2.2. Omental Mesothelial Cells Display a Mixed Proand Anti-Inflammatory Signature In order to better grasp the immunomodulatory function potentially conveyed by OMC, we profiled the Th1/Th2/Th17 cytokines secreted by OMC and compared it to that of MSC (Figure 2A,B). Figure 2. OMC and MSC display similarities in their expression patterns of antiand proinflammatory markers. ( A ) Representative membrane antibody arrays incubated within fresh media, OMCor MSC-derived secretome (OMC-S and MSC-S, respectively). Array map (upper left) is shown for cytokine localization and positive (POS) and negative (NEG) controls. (B) Quantification of cytokines expressed in OMC-S and MSC-S. Results are shown as mean ± s.e.m of signal densities from (n= 3) independent batches of ALIC1 OMC-S and MSC-S. ( C ) Quantitative PCR analysis of the expression of proand anti-inflammatory and immunoregulation genes in OMC and MSC. Results are shown as mean fold change ± s.e.m in mRNA expression relative to MSC (values set as 1), from ( n= 4 ) distinct batches of ALIC1 OMC and MSC cultures. ( B , C ) Statistical significance was determined by Student’s t-test. * is for p≤0.05; ** is for p≤0.01; *** is for p≤0.001. MSC-S induced similar changes in the expression pattern of these markers ( Figure 3A,B) . Interestingly, markers of macrophage activation, MHC-II and CD86, but not CD80 were also upregulated by both OMC-S and MSC-S (Figure 3A,B). Overall, the OMC immunomodulatory secretory profile appears to convey an antiinflammatory M2 phenotype to Raw 264.7 cells. To further substantiate this premise, we assessed the capacity of OMC to inhibit lymphocyte proliferation. To this end, lymphocytes isolated from lymph nodes of C57BL6 and FVB mice were fluorescently labeled using Int. J. Mol. Sci. 2022,23, 5924 6 of 18 CFSE and subjected to a two-way mixed lymphocyte reaction (MLR) culture containing phytohemagglutinin (PHA) to induce their agglutination and promote a strong proliferation induced by double polyclonal activation (Figure 4A). Remarkably, OMC-S produced a clear dose-dependent reduction in lymphocytes agglutination and the percentages of CFSE low lymphocytes, indicative of blunted proliferation (Figure 4B,C). Similar effects were observed with MSC-S (Figure 4B,C). Consistent with their reduced agglutination, further experiments indicated that the expression of CD54/ICAM-1 which is associated with cell–cell adhesion, was robustly inhibited by OMC-S (Figure S1). Taken together, these results suggest that OMC convey anti-, rather than pro-, inflammatory properties. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 18 Figure 2. OMC and MSC display similarities in their expression patterns of antiand pro-inflammatory markers. (A) Representative membrane antibody arrays incubated within fresh media, OMCor MSC-derived secretome (OMC-S and MSC-S, respectively). Array map (upper left) is shown for cytokine localization and positive (POS) and negative (NEG) controls. (B) Quantification of cytokines expressed in OMC-S and MSC-S. Results are shown as mean ± s.e.m of signal densities from (n = 3) independent batches of ALIC1 OMC-S and MSC-S. (C) Quantitative PCR analysis of the expression of proand anti-inflammatory and immunoregulation genes in OMC and MSC. Results are shown as mean fold change ± s.e.m in mRNA expression relative to MSC (values set as 1), from (n = 4) distinct batches of ALIC1 OMC and MSC cultures. (B,C) Statistical significance was determined by Student’s t-test. * is for p ≤ 0.05; ** is for p ≤ 0.01; *** is for p ≤ 0.001. Although globally the cytokine secretion profiles were similar, some important differences were discerned between OMCand MSC-derived secretomes (OMC-S and MSCS, respectively) (Figure 2A,B). As such, OMC-S and MSC-S secreted similarly high levels of interleukin-6 (IL-6), a pleiotropic cytokine with context-dependent proand anti-inflammatory properties [34]. Both cell types also secreted similarly high levels of TGFβ3 (Figure 2B). In contrast, secretion of MIP-3α (CCL20), Sgp130, IL-1sRI, TNF-α, IL-21, and IL-21R was significantly higher in OMC as compared to MSC whereas CD40, CD40L, IL17R, IL-12p70, and IL-12p40 secretion was higher in MSC (Figure 2B). We next assessed transcript levels of several key inflammatory and immunomodulary genes (Figure 2C). OMC expressed significantly higher levels of IL-15 (T cells activation), IL-1β, IFNγ, and TNFα (pro-inflammatory cytokines), iNOS (inflammatory and immunosuppression), and ARG1, LGALS9, and IL-10 (anti-inflammatory). Of particular interest, the expression of the potent anti-inflammatory IL-10 cytokine was 100-fold higher in OMC as compared to MSC. 2.3. The Secretome of OMC Induces M2 Polarization and Inhibits Mouse Lymphocyte Proliferation and Agglutination In order to determine whether the OMC secretome conveys either proor anti-inflammatory properties, mouse Raw 264.7 cells were exposed to condition media obtained from OMC (OMC-S) cultures. Raw 264.7 cells cultured in the presence of OMC-S displayed reduced expression of the M1 pro-inflammatory markers CD54 and of the activator marker CD25 [35]. In contrast, expression of the M2 anti-inflammatory marker CD206 was increased as compared to cells that we cultured in fresh media (FM) (Figure 3A,B). Figure 3. OMC-derived secretome favors an anti-inflammatory M2 phenotype. (A,B) RAW264.7 cells were cultured for 6 days in fresh media (FM) and either OMCor MSC-derived secretome (OMC-S and MSC-S, respectively). Expression levels of CD11b, MHC-II, CD54 (M1 associated marker), CD80, CD86, CD206 (M2 associated marker), and CD25 were assessed by flow cytometry. (A) Representative histograms and (B) quantification of mean fluorescence intensity (MFI) are depicted. Isotype histograms are not shown. Results are shown as mean ± s.e.m of (n = 3) independent batches of OMC-S (from ALIC1, SEV1, and SEV3 OMC lines) and MSC-S production. Statistical Figure 3. OMC-derived secretome favors an anti-inflammatory M2 phenotype. ( A , B ) RAW264.7 cells were cultured for 6 days in fresh media (FM) and either OMCor MSC-derived secretome (OMC-S and MSC-S, respectively). Expression levels of CD11b, MHC-II, CD54 (M1 associated marker), CD80, CD86, CD206 (M2 associated marker), and CD25 were assessed by flow cytometry. (A) Representative histograms and ( B ) quantification of mean fluorescence intensity (MFI) are depicted. Isotype histograms are not shown. Results are shown as mean ± s.e.m of (n= 3) independent batches of OMC-S (from ALIC1, SEV1, and SEV3 OMC lines) and MSC-S production. Statistical differences between conditions were calculated using Student’s ttest; * is for p ≤ 0.05; ** is for p ≤ 0.01; NS, not significant. 2.4. Omental Mesothelial Cell Secretome Inhibits the Activation of Mouse T and B Cells To further delineate the lymphocyte subpopulations targeted by OMC-S, we analyzed the ability of OMC-S to inhibit Tand B-cell activation in mixed T and B lymph node cell populations activated with PHA. Interestingly, the reduction of PHA-induced agglutination of lymphocytes provoked by OMC-S was also accompanied by a significant reduction of CD4 + /CD25 + and CD8 + /CD25 + activated T cells as well as CD19 + /CD25 + B cells (Figure 5A). While activation of CD4 + and CD8 + T cells was as equally reduced by MSC-S as OMC-S, inhibition of CD19 + B cell activation was significantly higher in 100% OMC-S as compared to 100% MSC-S (Figure 5A). In further support of these results, OMC-S was also found to significantly inhibit mouse peritoneal fluid CD19 + B cells, as evidenced by their significant increase in B cells displaying loss of CD19 expression (Figure 5B). 2.5. Activation and Proliferation of Human Lymphocytes and B Cells Are Blunted by OMC-S We next sought to translate our murine data to human immune cells. To this end, peripheral blood mononuclear cells (PBMC)-enriched lymphocytes from two distinct donors were subjected to a two-way mixed lymphocyte reaction (MLR) culture containing PHA to robustly induce proliferation (Figure 6A). Consistent with our mouse data, both the PHA-induced agglutination and proliferation of human lymphocytes were dose-dependently reduced by OMC-S (Figure 6A). We also determined whether OMC-S could lower the activation of PBMNC-enriched lymphocytes after CD3/CD28-mediated polyclonal activation and PHA stimulation. As expected, Int. J. Mol. Sci. 2022,23, 5924 7 of 18 polyclonally (TCR and PHA) activated lymphocytes cultured in fresh media (FM) proliferated and generated large free-floating clusters composed mainly of CD3 + and CD4 + T cells and a minor CD19 + B cells subpopulation (Figure S2). The vast majority of CD4 + T cells within FM/TCR/PHA cultures were also CD25 + (Figure 6B). Although not significant, OMC-S impeded expansion of the CD4 + /CD25 + T cells subpopulation while MSC-S significantly blunted its proliferation (Figure 6B). In contrast, activation of the CD19 + /CD25 + B cell subpopulation was significantly reduced by OMC-S and to a lesser extent by MSC-S (Figure 6C). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 18 differences between conditions were calculated using Student’s t test; * is for p ≤ 0.05; ** is for p ≤ 0.01; NS, not significant. MSC-S induced similar changes in the expression pattern of these markers (Figure 3A,B). Interestingly, markers of macrophage activation, MHC-II and CD86, but not CD80 were also upregulated by both OMC-S and MSC-S (Figure 3A,B). Overall, the OMC immunomodulatory secretory profile appears to convey an antiinflammatory M2 phenotype to Raw 264.7 cells. To further substantiate this premise, we assessed the capacity of OMC to inhibit lymphocyte proliferation. To this end, lymphocytes isolated from lymph nodes of C57BL6 and FVB mice were fluorescently labeled using CFSE and subjected to a two-way mixed lymphocyte reaction (MLR) culture containing phytohemagglutinin (PHA) to induce their agglutination and promote a strong proliferation induced by double polyclonal activation (Figure 4A). Remarkably, OMC-S produced a clear dose-dependent reduction in lymphocytes agglutination and the percentages of CFSElow lymphocytes, indicative of blunted proliferation (Figure 4B,C). Similar effects were observed with MSC-S (Figure 4B,C). Consistent with their reduced agglutination, further experiments indicated that the expression of CD54/ICAM-1 which is associated with cell–cell adhesion, was robustly inhibited by OMC-S (Figure S1). Taken together, these results suggest that OMC convey anti-, rather than pro-, inflammatory properties. Figure 4. The OMC-derived secretome inhibits mouse lymphocytes proliferation. (A) Left upper panel depicts representative images of lymph nodes (LN) lymphocytes cultured in the absence or presence of 25 µg/mL phytohemagglutinin (PHA) and MLR. Lower images depict histograms of carboxyfluorescein succinimidyl ester (CFSE) expression after 72 h of culture, showing loss of CFSE expression in the MLR + PHA condition (dashed black line shows LN lymphocytes just after initial CFSE labeling). Right panel, upper images, show images of MLR + PHA cultures performed with increasing amounts of OMCor MSC-derived secretome (OMC-S or MSC-S, respectively). Lower images, corresponding CFSE histogram expression. Dashed blue line is for CFSE expression level of Figure 4. The OMC-derived secretome inhibits mouse lymphocytes proliferation. ( A ) Left upper panel depicts representative images of lymph nodes (LN) lymphocytes cultured in the absence or presence of 25 µ g/mL phytohemagglutinin (PHA) and MLR. Lower images depict histograms of carboxyfluorescein succinimidyl ester (CFSE) expression after 72 h of culture, showing loss of CFSE expression in the MLR + PHA condition (dashed black line shows LN lymphocytes just after initial CFSE labeling). Right panel, upper images, show images of MLR + PHA cultures performed with increasing amounts of OMCor MSC-derived secretome (OMC-S or MSC-S, respectively). Lower images, corresponding CFSE histogram expression. Dashed blue line is for CFSE expression level of the MLR + PHA control condition in 100% fresh media. ( B ) Summary quantification of CFSE low proliferating lymphocytes in MLR + PHA cultures performed in 100% fresh media or increased proportions of OMC-S or MSC-S. Results shown are mean ± s.e.m percentages of cells with CFSE loss (CFSE low ) after 72 h of culture, from (n= 3) independent OMC-S batches from ALIC1 and SEV1 OMC lines. Statistical differences against control (100% fresh LM) were calculated with ANOVA. ** is for p≤0.01; *** is for p≤0.001. 2.6. The Lymphosuppressive Activity of OMC-Derived Secretome Is Conveyed via TGF β as Well as Nitric Oxide Synthase Activity We next sought to identify potential molecular pathways mediating the lymphosuppressive activity of OMC-S (Figure 7). We initially focused on the TGFβ signaling cascade as TFGβ 3 secretion was elevated in both OMC-S and MSC-S (Figure 2B). Inhibition of Int. J. Mol. Sci. 2022,23, 5924 8 of 18 TGFβ via the use of either the selective inhibitor, SB-431542, or a monoclonal anti-TGFβ antibody, blunted the immunosuppressive activity of OMC-S as assessed by reduced agglutinated particle sizes (Figure 7A,B). We next assessed the effect of arginine supplementation on OMC-S-mediated inhibition of cell aggregation. Indeed, ARG1, a key gene involved in inflammation resolution and for which the encoded enzyme converts arginine to ornithine thereby depleting arginine pools, was significantly increased in OMC as compared to MSC (Figure 2C). Accordingly, addition of L-arginine blunted the OMC-S-mediated inhibition of cell agglutination (Figure 7A,B). Interestingly, addition of L-NMMA, a specific inhibitor of the nitric oxide synthase (NOS), strongly abrogated the immunosuppressive effect of OMC-S (Figure 7A,B). Taken together, these results indicate that OMC-S exerts its lymphosuppressive activity through several independent signaling pathways, including the likely overstimulation of NOS activity in lymphocytes, leading to supraphysiological toxic nitric oxide levels that limit cell activity. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 18 the MLR + PHA control condition in 100% fresh media. (B) Summary quantification of CFSElow proliferating lymphocytes in MLR + PHA cultures performed in 100% fresh media or increased proportions of OMC-S or MSC-S. Results shown are mean ± s.e.m percentages of cells with CFSE loss (CFSElow) after 72 h of culture, from (n = 3) independent OMC-S batches from ALIC1 and SEV1 OMC lines. Statistical differences against control (100% fresh LM) were calculated with ANOVA. ** is for p ≤ 0.01; *** is for p ≤ 0.001. 2.4. Omental Mesothelial Cell Secretome Inhibits the Activation of Mouse T and B Cells To further delineate the lymphocyte subpopulations targeted by OMC-S, we analyzed the ability of OMC-S to inhibit Tand B-cell activation in mixed T and B lymph node cell populations activated with PHA. Interestingly, the reduction of PHA-induced agglutination of lymphocytes provoked by OMC-S was also accompanied by a significant reduction of CD4+/CD25+ and CD8+/CD25+ activated T cells as well as CD19+/CD25+ B cells (Figure 5A). While activation of CD4+ and CD8+ T cells was as equally reduced by MSC-S as OMC-S, inhibition of CD19+ B cell activation was significantly higher in 100% OMC-S as compared to 100% MSC-S (Figure 5A). In further support of these results, OMC-S was also found to significantly inhibit mouse peritoneal fluid CD19+ B cells, as evidenced by their significant increase in B cells displaying loss of CD19 expression (Figure 5B). Figure 5. OMC-derived secretome inhibits the activation of lymph node T and B cells and peritoneal fluid B cells. (A) Quantification (% of total cells) of CD4+/CD25+ and CD8+/CD25+ activated T cells and CD19+/CD25+ activated B cells within mouse lymph nodes (LN) cells cultured for 24 h in 100% fresh media supplemented with 25 µg/mL phytohemagglutinin (PHA) or increased proportions (10–100%) of OMC-S (ALIC1, SEV1, and SEV4) or MSC-S supplemented with PHA. Results are mean ± s.e.m from n = 4 independent experiments. Statistical differences between control (100% fresh media + PHA) and OMC-S and MSC-S conditions were calculated using ANOVA. Difference between 100% OMC-S and MSC-S for CD19+/CD25+ B cells was calculated with Student’s t test. (B) OMC-derived secretome inhibits peritoneal fluid B cells. Upper, shows representative coexpression of CD19 and CD25 in non-adherent mouse peritoneal fluid cells (PFC) cultured for 72 h in either fresh media (FM), OMC-S, or MSC-S. Two rectangles delimiting CD19low (grey rectangle) and CD19high (dark rectangle) expressing B cells are shown. Lower graph shows flow cytometric quantification of CD19−, CD19+, CD19low, and CD19high populations within non-adherent PFC cultured for 72 h in either fresh media or OMC-S (ALIC1, SEV1, and SEV4) and MSC-S. Results are shown as mean ± s.e.m from n = 3 independent experiments. Statistical differences for CD19low and CD19high between different culture conditions groups were calculated using Student’s t test. (A,B) * for p ≤ 0.05; ** for p ≤ 0.01. Figure 5. OMC-derived secretome inhibits the activation of lymph node T and B cells and peritoneal fluid B cells. ( A ) Quantification (% of total cells) of CD4 + /CD25 + and CD8 + /CD25 + activated T cells and CD19 + /CD25 + activated B cells within mouse lymph nodes (LN) cells cultured for 24 h in 100% fresh media supplemented with 25 µ g/mL phytohemagglutinin (PHA) or increased proportions ( 10–100% ) of OMC-S (ALIC1, SEV1, and SEV4) or MSC-S supplemented with PHA. Results are mean ±s.e.m from n= 4 independent experiments. Statistical differences between control (100% fresh media + PHA) and OMC-S and MSC-S conditions were calculated using ANOVA. Difference between 100% OMC-S and MSC-S for CD19 + /CD25 + B cells was calculated with Student’s ttest. (B) OMC-derived secretome inhibits peritoneal fluid B cells. Upper, shows representative coexpression of CD19 and CD25 in non-adherent mouse peritoneal fluid cells (PFC) cultured for 72 h in either fresh media (FM), OMC-S, or MSC-S. Two rectangles delimiting CD19 low (grey rectangle) and CD19 high (dark rectangle) expressing B cells are shown. Lower graph shows flow cytometric quantification of CD19 − , CD19 + , CD19 low , and CD19 high populations within non-adherent PFC cultured for 72 h in either fresh media or OMC-S (ALIC1, SEV1, and SEV4) and MSC-S. Results are shown as mean ± s.e.m from n= 3 independent experiments. Statistical differences for CD19 low and CD19 high between different culture conditions groups were calculated using Student’s ttest. (A,B) * for p≤0.05; ** for p≤0.01. Int. J. Mol. Sci. 2022,23, 5924 9 of 18 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 18 2.5. Activation and Proliferation of Human Lymphocytes and B Cells Are Blunted by OMC-S We next sought to translate our murine data to human immune cells. To this end, peripheral blood mononuclear cells (PBMC)-enriched lymphocytes from two distinct donors were subjected to a two-way mixed lymphocyte reaction (MLR) culture containing PHA to robustly induce proliferation (Figure 6A). Figure 6. OMC-derived secretome inhibits the proliferation of human lymphocytes and the activation of B cells. (A) Representative images (upper panel) of a mixed lymphocyte reaction (MLR) of PBMC-enriched lymphocytes labeled with CFSE and cultured in either 100% fresh media (FM) supplemented with 25 µg/mL phytohemagglutinin (PHA) or increased amounts of OMC-S and MSC-S supplemented with PHA. Scale bar is 500 µm. Lower histograms depict corresponding CFSE expression levels (red histograms) of disaggregated lymphocyte cultures. The yellow dashed line marks cells freshly labeled with CFSE. For reference, a black dashed line corresponding to CFSE expression histogram in 100% FM is shown in OMC-S and MSC-S histograms. Results are shown as mean percentages ± s.e.m of CFSElow cells calculated from n = 2 independent batches of OMC-S (ALIC1, SEV1) and MSC-S. (B,C) OMC-S efficiently suppress B cells activation. Human PBMC-enriched lymphocytes subjected to double polyclonal stimulation (anti-CD3/CD28; PHA) were expanded for 6 days and finally incubated for 2 days in FM, OMC-S, or MSC-S. Cells were analyzed by flow cytometry. (B) Analysis of CD4 and CD25 coexpression. Upper panel shows representative dot plots of CD4/CD25 expression. Lower graph, quantification of mean percentages of total CD4+ Figure 6. OMC-derived secretome inhibits the proliferation of human lymphocytes and the activation of B cells. ( A ) Representative images (upper panel) of a mixed lymphocyte reaction (MLR) of PBMC-enriched lymphocytes labeled with CFSE and cultured in either 100% fresh media (FM) supplemented with 25 µ g/mL phytohemagglutinin (PHA) or increased amounts of OMC-S and MSC-S supplemented with PHA. Scale bar is 500 µ m. Lower histograms depict corresponding CFSE expression levels (red histograms) of disaggregated lymphocyte cultures. The yellow dashed line marks cells freshly labeled with CFSE. For reference, a black dashed line corresponding to CFSE expression histogram in 100% FM is shown in OMC-S and MSC-S histograms. Results are shown as mean percentages ± s.e.m of CFSE low cells calculated from n= 2 independent batches of OMC-S (ALIC1, SEV1) and MSC-S. ( B , C ) OMC-S efficiently suppress B cells activation. Human PBMC-enriched lymphocytes subjected to double polyclonal stimulation (anti-CD3/CD28; PHA) were expanded for 6 days and finally incubated for 2 days in FM, OMC-S, or MSC-S. Cells were analyzed by flow cytometry. ( B ) Analysis of CD4 and CD25 coexpression. Upper panel shows representative dot plots of CD4/CD25 expression. Lower graph, quantification of mean percentages of total CD4 + T cells and CD4 + /CD25 + -activated T cells. ( C ) Flow cytometry analysis of CD19 and CD25 coexpression. Upper panel shows representative dot plots of CD19/CD25 expression. Lower graph, quantification of mean percentages of total CD19 + B cells and CD19 + /CD25 + activated B cells. ( B , C ) Results are mean ± s.e.m results of n= 3 distinct batches of OMC-S (ALIC1, SEV1, and SEV3) and MSC-S. Statistical differences were calculated using ANOVA. * is for p ≤ 0.05; ** for p ≤ 0.01; *** p≤0.01. Int. J. Mol. Sci. 2022,23, 5924 16 of 18 25 µ g/mL phytohemagglutinin (PHA) (Sigma-Aldrich; L1668). The capacity of diverse inhibitors to counteract the lymphosuppression activity of OMC was tested and inhibitors used were as following: TGFβ RI Kinase Inhibitor VI, SB431542, (Ref: 616461); NGMethyl-L-arginine acetate salt or L-NMMA (Ref: M7033), L-Arginine (A5006), all from Sigma-Aldrich. Monoclonal antibody against TGF-beta I, II, III isoforms (Azide and BSA Free) was purchased from Novus Biological (Clone: 1D11.16.8; Ref: NBP2-47736). Cellular aggregation in the form of small to large spheroids was quantified with ImageJ (National Institutes of Health). Calculation of aggregates surface ( µ m 2 ) was automated by converting images to binary images and processed to find hedges, prior to quantifying the mean particle size. 4.17. Statistical Analysis Results are expressed as mean ± s.e.m (bar graphs). Statistical analyses were completed with GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). Statistical differences were estimated by ANOVA or Student’s ttest, whichever was appropriate. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms23115924/s1. Author Contributions: D.R.-C., J.C.G.-R., L.C.C.-M. and A.H.: collection and assembly of data, data analysis and interpretation, final approval of manuscript. B.S.: collection and/or assembly of data, data analysis and interpretation, conception and design, final approval of manuscript. B.R.G.: data analysis and interpretation, manuscript editing and final approval. C.C.L.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, financial support. All authors have read and agreed to the published version of the manuscript. Funding: This research is/was supported by the Consejería de Salud, Fundación Pública Andaluza Progreso y Salud, Junta de Andalucía (PI-0007-2016 to C.C.L.). Institutional Review Board Statement: Protocol for omental tissue collection was approved by the Ethical Committee of Investigation CEI de los hospitales universitarios Vírgen Macarena-Virgen del Rocío, Seville, Andalusia, Spain (Registration Number 08/2018). The animal study protocol was approved by the Animal Care Committee at CABIMER and performed in accordance with the Spanish law on animal use RD 53/2013. Informed Consent Statement: Written informed consent was obtained from the donors for collection of omental adipose tissue and establishment of OMC primary lines. Data Availability Statement: Data are available from the corresponding author on request. Acknowledgments: We thank María Isabel García Sánchez, head of the Biobanco Hospitalario Virgen Macarena for helpful assistance with the Ethical Committee of Investigation (CEI). Conflicts of Interest: The authors declare no conflict of interest. References 1. Mutsaers, S.E. The mesothelial cell. Int. J. Biochem. Cell Biol. 2004,36, 9–16. [CrossRef] 2. Mutsaers, S.E.; Wilkosz, S. Structure and function of mesothelial cells. Cancer Treat. Res. 2007,134, 1–19. 3. Mutsaers, S.E.; Prele, C.M.; Lansley, S.M.; Herrick, S.E. The origin of regenerating mesothelium: A historical perspective. Int. J. Artif. Organs 2007,30, 484–494. [CrossRef] 4. Mutsaers, S.E. Mesothelial cells: Their structure, function and role in serosal repair. 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