Exploring the Ion Channel TRPV2 and Testicular Macrophages in Mouse Testis

The cation channel TRPV2 is known to be expressed by murine macrophages and is crucially involved in their functionality. Macrophages are frequent cells of the mouse testis, an immune-privileged and steroid-producing organ. TRPV2 expression by testicular macrophages and possible changes associated with age or inflammation have not been investigated yet. Therefore, we studied testes of young adult and old wild-type (WT) and AROM⁺ mice, i.e., transgenic mice overexpressing aromatase. In these animals, inflammatory changes are described in the testis, involving active macrophages, which increase with age. This is associated with impaired spermatogenesis and therefore AROM⁺ mice are a model for male infertility associated with sterile inflammation. In WT animals, testicular TRPV2 expression was mapped to interstitial CD206⁺ and peritubular MHC II⁺ macrophages, with higher levels in CD206⁺ cells. Expression levels of TRPV2 and most macrophage markers did not increase significantly in old mice, with the exception of CD206. As the number of TRPV2⁺ testicular macrophages was relatively small, their possible involvement in testicular functions and in aging in WT mice remains to be further studied. In AROM⁺ testis, TRPV2 was readily detected and levels increased significantly with age, together with macrophage markers and TNF-α. TRPV2 co-localized with F4/80 in macrophages and further studies showed that TRPV2 is mainly expressed by unusual CD206⁺MHC II⁺ macrophages, arising in the testis of these animals. Rescue experiments (aromatase inhibitor treatment and crossing with ERαKO mice) restored the testicular phenotype and also abolished the elevated expression of TRPV2, macrophage and inflammation markers. This suggests that TRPV2⁺ macrophages of the testis are part of an inflammatory cascade initiated by an altered sex hormone balance in AROM⁺ mice. The changes in testis are distinct from the described alterations in other organs of AROM⁺, such as prostate and spleen. When we monitored TRPV2 levels in another immune-privileged organ, namely the brain, we found that levels of TRPV2 were not elevated in AROM⁺ and remained stable during aging. In the adrenal, which similar to the testis produces steroids, we found slight, albeit not significant increases in TRPV2 in both AROM⁺ and WT mice, which were associated with age. Thus, the changes in the testis are specific for this organ.     

Hepatic Macrophage Abundance and Phenotype in Aging and Liver Iron Accumulation

Liver macrophages serve important roles in iron homeostasis through phagocytosis of effete erythrocytes and the export of iron into the circulation. Conversely, intracellular iron can alter macrophage phenotype. Aging increases hepatic macrophage number and nonparenchymal iron, yet it is unknown whether age-related iron accumulation alters macrophage number or phenotype. To evaluate macrophages in a physiological model of iron loading that mimicked biological aging, young (6 mo) Fischer 344 rats were given one injection of iron dextran (15 mg/kg), and macrophage number and phenotype were evaluated via immunohistochemistry. A separate group of old (24 mo) rats was treated with 200 mg/kg deferoxamine every 12 h for 4 days. Iron administration to young rats resulted in iron concentrations that matched the values and pattern of tissue iron deposition observed in aged animals; however, iron did not alter macrophage number or phenotype. Aging resulted in significantly greater numbers of M1 (CD68⁺) and M2 (CD163⁺) macrophages in the liver, but neither macrophage number nor phenotype were affected by deferoxamine. Double-staining experiments demonstrated that both M1 (iNOS⁺) and M2 (CD163⁺) macrophages contained hemosiderin, suggesting that macrophages of both phenotypes stored iron. These results also suggest that age-related conditions other than iron excess are responsible for the accumulation of hepatic macrophages with aging.     

Roles of Macrophages in Advanced Liver Fibrosis,Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c⁺/Ly6C⁻ and CD11c⁻/Ly6C⁺ cells. CD11c⁺ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c⁺ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C⁺ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c⁺/Ly6C⁻ cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c⁻/Ly6C⁺ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.     

Isolation of Decidual Macrophages and Hofbauer Cells from Term Placenta—Comparison of the Expression of CD163 and CD80

(1) Background: Placental immune cells are playing a very important role in a successful placentation and the prevention of pregnancy complications. Macrophages dominate in number and relevance in the maternal and the fetal part of the placenta. The evidence on the polarization state of fetal and maternal macrophages involved in both, healthy and pregnancy-associated diseases, is limited. There is no representative isolation method for the direct comparison of maternal and fetal macrophages so far. (2) Material and Methods: For the isolation of decidual macrophages and Hofbauer cells from term placenta, fresh tissue was mechanically dissected and digested with trypsin and collagenase A. Afterwards cell enrichment was increased by a Percoll gradient. CD68 is represented as pan-macrophage marker, the surface markers CD80 and CD163 were further investigated. (3) Results: The established method revealed a high cell yield and purity of the isolated macrophages and enabled the comparison between decidual macrophages and Hofbauer cells. No significant difference was observed in the percentage of single CD163⁺ cells in the distinct macrophage populations, by using FACS and immunofluorescence staining. A slight increase of CD80⁺ cells could be found in the decidual macrophages. Considering the percentage of CD80⁺CD163⁻ and CD80⁻CD163⁺ cells we could not find differences. Interestingly we found an increased number of double positive cells (CD80⁺CD163⁺) in the decidual macrophage population in comparison to Hofbauer cells. (4) Conclusion: In this study we demonstrate that our established isolation method enables the investigation of decidual macrophages and Hofbauer cells in the placenta. It represents a promising method for direct cell comparison, enzyme independently, and unaffected by magnetic beads, to understand the functional subsets of placental macrophages and to identify therapeutic targets of pregnancy associated diseases.     

C3 Deficiency Leads to Increased Angiogenesis and Elevated Pro-Angiogenic Leukocyte Recruitment in Ischemic Muscle Tissue

The complement system is a potent inflammatory trigger, activator, and chemoattractant for leukocytes, which play a crucial role in promoting angiogenesis. However, little information is available about the influence of the complement system on angiogenesis in ischemic muscle tissue. To address this topic and analyze the impact of the complement system on angiogenesis, we induced muscle ischemia in complement factor C3 deficient (C3−/−) and wildtype control mice by femoral artery ligation (FAL). At 24 h and 7 days after FAL, we isolated the ischemic gastrocnemius muscles and investigated them by means of (immuno-)histological analyses. C3−/− mice showed elevated ischemic damage 7 days after FAL, as evidenced by H&E staining. In addition, angiogenesis was increased in C3−/− mice, as demonstrated by increased capillary/muscle fiber ratio and increased proliferating endothelial cells (CD31⁺/BrdU⁺). Moreover, our results showed that the total number of leukocytes (CD45⁺) was increased in C3−/− mice, which was based on an increased number of neutrophils (MPO⁺), neutrophil extracellular trap formation (MPO⁺/CitH3⁺), and macrophages (CD68⁺) displaying a shift toward an anti-inflammatory and pro-angiogenic M2-like polarized phenotype (CD68⁺/MRC1⁺). In summary, we show that the deficiency of complement factor C3 increased neutrophil and M2-like polarized macrophage accumulation in ischemic muscle tissue, contributing to angiogenesis.     

A Heterotypic Tridimensional Model to Study the Interaction of Macrophages and Glioblastoma In Vitro

Background: Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor, and macrophages account for 30–40% of its composition. Most of these macrophages derive from bone marrow monocytes playing a crucial role in tumor progression. Unraveling the mechanisms of macrophages-GBM crosstalk in an appropriate model will contribute to the development of specific and more successful therapies. We investigated the interaction of U87MG human GBM cells with primary human CD14⁺ monocytes or the THP-1 cell line with the aim of establishing a physiologically relevant heterotypic culture model. Methods: primary monocytes and THP-1 cells were cultured in the presence of U87MG conditioned media or co-cultured together with previously formed GBM spheroids. Monocyte differentiation was determined by flow cytometry. Results: primary monocytes differentiate to M2 macrophages when incubated with U87MG conditioned media in 2-dimensional culture, as determined by the increased percentage of CD14⁺CD206⁺ and CD64⁺CD206⁺ populations in CD11b⁺ cells. Moreover, the mitochondrial protein p32/gC1qR is expressed in monocytes exposed to U87MG conditioned media. When primary CD14⁺ monocytes or THP-1 cells are added to previously formed GBM spheroids, both invade and establish within them. However, only primary monocytes differentiate and acquire a clear M2 phenotype characterized by the upregulation of CD206, CD163, and MERTK surface markers on the CD11b⁺CD14⁺ population and induce alterations in the sphericity of the cell cultures. Conclusion: our results present a new physiologically relevant model to study GBM/macrophage interactions in a human setting and suggest that both soluble GBM factors, as well as cell-contact dependent signals, are strong inducers of anti-inflammatory macrophages within the tumor niche.     

Changes in T-Cell Subpopulations and Cytokine Levels in Patients with Treatment-Resistant Depression—A Preliminary Study

Although there is some evidence for the involvement of cytokines and T cells in the pathophysiology of treatment-resistant depression (TRD), the nature of this relationship is not entirely clear. Therefore, we compared T-cell subpopulations and serum cytokine levels in TRD patients to find relationships between their immunological profiles, clinical presentation, and episode severity. Blood samples from TRD patients (n = 20) and healthy people (n = 13) were collected and analyzed by flow cytometry. We analyzed the percentages of helper and cytotoxic T cells according to the expression of selected activation markers, including CD28, CD69, CD25, CD95, and HLA-DR. The serum levels of inflammatory cytokines IL12p70, TNF-α, IL-10, IL-6, IL-1β, and IL-8 were also determined. TRD patients had a lower percentage of CD3⁺CD4⁺CD25⁺ and CD3⁺CD8⁺CD95⁺ cells than healthy people. They also had lower serum levels of IL-12p70 and TNF-α, whereas IL-8 levels were significantly higher. Receiver operating characteristic (ROC) analysis demonstrated that serum IL-8 values above 19.55 pg/mL were associated with a 10.26 likelihood ratio of developing TRD. No connections were found between the MADRS score and immunological parameters. These results show that TRD patients have reduced percentages of T cells expressing activation antigens (CD25 and CD95) and higher serum concentrations of proinflammatory and chemotactic IL-8. These changes may indicate reduced activity of the immune system and the important role of IL-8 in maintaining chronic inflammation in the course of depression.     

Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma

A predictive biomarker of immune checkpoint inhibitor (ICI)-based treatments in hepatocellular carcinoma (HCC) has not been clearly demonstrated. In this study, we focused on the infiltration and programmed death ligand 1 (PD-L1) expression of tumor-associated macrophages (TAMs) in the tumor microenvironment of HCC. Immunohistochemistry demonstrated that PD-L1 was preferentially expressed on CD68⁺ macrophages in the tumor microenvironment of HCC, suggestive of its expression in TAMs rather than in T cells or tumor cells (P < 0.05). A co-culture experiment using activated T cells and M2 macrophages confirmed a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8⁺ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4⁺ and CD8⁺ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8⁺ T cells in HCC.     

Mycobacterium tuberculosis H37Rv Strain Increases the Frequency of CD3+TCR+ Macrophages and Affects Their Phenotype,but Not Their Migration Ability

In mycobacterial infections, the number of cells from two newly discovered subpopulations of CD3⁺ myeloid cells are increased at the infection site; one type expresses the T cell receptor (CD3⁺TCRαβ⁺) and the other does not (CD3⁺TCRαβ⁻). The role of Mycobacterium tuberculosis (Mtb) virulence in generating these subpopulations and the ability of these cells to migrate remains unclear. In this study, monocyte-derived macrophages (MDMs) infected in vitro with either a virulent (H37Rv) or an avirulent (H37Ra) Mtb strain were phenotypically characterized based on three MDM phenotypes (CD3⁻, CD3⁺TCRαβ⁺, and CD3⁺TCRαβ⁻); then, their migration ability upon Mtb infection was evaluated. We found no differences in the frequency of CD3⁺ MDMs at 24 h of infection with either Mtb strain. However, H37Rv infection increased the frequency of CD3⁺TCRαβ⁺ MDMs at a multiplicity of infection of 1 and altered the expression of CD1b, CD1c, and TNF on the surface of cells from both the CD3⁺ MDM subpopulations; it also modified the expression of CCR2, CXCR1, and CCR7, thus affecting CCL2 and IL-8 levels. Moreover, H37Rv infection decreased the migration ability of the CD3⁻ MDMs, but not CD3⁺ MDMs. These results confirm that the CD3⁺ macrophage subpopulations express chemokine receptors that respond to chemoattractants, facilitating cell migration. Together, these data suggest that CD3⁺ MDMs are a functional subpopulation involved in the immune response against Mtb.     

CD26 Deficiency Controls Macrophage Polarization Markers and Signal Transducers during Colitis Development and Resolution

Ulcerative colitis (UC) is a multifactorial condition characterized by a destructive immune response that failed to be attenuated by common regulatory mechanisms which reduce inflammation and promote mucosa healing. The inhibition of CD26, a multifunctional glycoprotein that controls the immune response via its dipeptidyl peptidase (DP) 4 enzyme activity, was proven to have beneficial effects in various autoimmune inflammatory diseases. The polarization of macrophages into either pro-inflammatory M1 or anti-inflammatory M2 subclass is a key intersection that mediates the immune-inflammatory process in UC. Hence, we hypothesized that the deficiency of CD26 affects that process in the dextran sulfate sodium (DSS)-induced model of UC. We found that mRNA expression of M2 markers arginase 1 and Fizz were increased, while the expression of M1 marker inducible NO synthase was downregulated in CD26^−/− mice. Decreased STAT1 mRNA, as well as upregulated pSTAT6 and pSTAT3, additionally support the demonstrated activation of M2 macrophages under CD26 deficiency. Finally, we investigated DP8 and DP9, proteins with DP4-like activity, and found that CD26 deficiency is not a key factor for the noted upregulation of their expression in UC. In conclusion, we demonstrate that CD26 deficiency regulates macrophage polarization toward the anti-inflammatory M2 phenotype, which is driven by STAT6/STAT3 signaling pathways. This process is additionally enhanced by the reduction of M1 differentiation via the suppression of proinflammatory STAT1. Therefore, further studies should investigate the clinical potential of CD26 inhibitors in the treatment of UC.     

The Absence of Extracellular Cold-Inducible RNA-Binding Protein (eCIRP) Promotes Pro-Angiogenic Microenvironmental Conditions and Angiogenesis in Muscle Tissue Ischemia

Extracellular Cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released from cells upon hypoxia and cold-stress. The overall absence of extra- and intracellular CIRP is associated with increased angiogenesis, most likely induced through influencing leukocyte accumulation. The aim of the present study was to specifically characterize the role of eCIRP in ischemia-induced angiogenesis together with the associated leukocyte recruitment. For analyzing eCIRPs impact, we induced muscle ischemia via femoral artery ligation (FAL) in mice in the presence or absence of an anti-CIRP antibody and isolated the gastrocnemius muscle for immunohistological analyses. Upon eCIRP-depletion, mice showed increased capillary/muscle fiber ratio and numbers of proliferating endothelial cells (CD31⁺/CD45⁻/BrdU⁺). This was accompanied by a reduction of total leukocyte count (CD45⁺), neutrophils (MPO⁺), neutrophil extracellular traps (NETs) (MPO⁺CitH3⁺), apoptotic area (ascertained via TUNEL assay), and pro-inflammatory M1-like polarized macrophages (CD68⁺/MRC1⁻) in ischemic muscle tissue. Conversely, the number of regenerative M2-like polarized macrophages (CD68⁺/MRC1⁺) was elevated. Altogether, we observed that eCIRP depletion similarly affected angiogenesis and leukocyte recruitment as described for the overall absence of CIRP. Thus, we propose that eCIRP is mainly responsible for modulating angiogenesis via promoting pro-angiogenic microenvironmental conditions in muscle ischemia.     

Iron Released after Cryo-Thermal Therapy Induced M1 Macrophage Polarization,Promoting the Differentiation of CD4+ T Cells into CTLs

Macrophages play critical roles in both innate and adaptive immunity and are known for their high plasticity in response to various external signals. Macrophages are involved in regulating systematic iron homeostasis and they sequester iron by phagocytotic activity, which triggers M1 macrophage polarization and typically exerts antitumor effects. We previously developed a novel cryo-thermal therapy that can induce the mass release of tumor antigens and damage-associated molecular patterns (DAMPs), promoting M1 macrophage polarization. However, that study did not examine whether iron released after cryo-thermal therapy induced M1 macrophage polarization; this question still needed to be addressed. We hypothesized that cryo-thermal therapy would cause the release of a large quantity of iron to augment M1 macrophage polarization due to the disruption of tumor cells and blood vessels, which would further enhance antitumor immunity. In this study, we investigated iron released in primary tumors, the level of iron in splenic macrophages after cryo-thermal therapy and the effect of iron on macrophage polarization and CD4⁺ T cell differentiation in metastatic 4T1 murine mammary carcinoma. We found that a large amount of iron was released after cryo-thermal therapy and could be taken up by splenic macrophages, which further promoted M1 macrophage polarization by inhibiting ERK phosphorylation. Moreover, iron promoted DC maturation, which was possibly mediated by iron-induced M1 macrophages. In addition, iron-induced M1 macrophages and mature DCs promoted the differentiation of CD4⁺ T cells into the CD4 cytolytic T lymphocytes (CTL) subset and inhibited differentiation into Th2 and Th17 cells. This study explains the role of iron in cryo-thermal therapy-induced antitumor immunity from a new perspective.     

Chromatin Regulator SRG3 Overexpression Protects against LPS/D-GalN-Induced Sepsis by Increasing IL10-Producing Macrophages and Decreasing IFNγ-Producing NK Cells in the Liver

We previously showed that ubiquitous overexpression of the chromatin remodeling factor SWItch3-related gene (SRG3) promotes M2 macrophage differentiation, resulting in anti-inflammatory responses in the experimental autoimmune encephalomyelitis model of multiple sclerosis. Since hepatic macrophages are responsible for sepsis-induced liver injury, we investigated herein the capacity of transgenic SRG3 overexpression (SRG3^β-actin mice) to modulate sepsis in mice exposed to lipopolysaccharide (LPS) plus d-galactosamine (d-GalN). Our results demonstrated that ubiquitous SRG3 overexpression significantly protects mice from LPS/d-GalN-induced lethality mediated by hepatic M1 macrophages. These protective effects of SRG3 overexpression correlated with the phenotypic conversion of hepatic macrophages from an M1 toward an M2 phenotype. Furthermore, SRG3^β-actin mice had decreased numbers and activation of natural killer (NK) cells but not natural killer T (NKT) cells in the liver during sepsis, indicating that SRG3 overexpression might contribute to cross-talk between NK cells and macrophages in the liver. Finally, we demonstrated that NKT cell-deficient CD1d KO/SRG3^β-actin mice are protected from LPS/d-GalN-induced sepsis, indicating that NKT cells are dispensable for SRG3-mediated sepsis suppression. Taken together, our findings provide strong evidence that SRG3 overexpression may serve as a therapeutic approach to control overwhelming inflammatory diseases such as sepsis.     

Adipose Tissue-Derived CCL5 Enhances Local Pro-Inflammatory Monocytic MDSCs Accumulation and Inflammation via CCR5 Receptor in High-Fat Diet-Fed Mice

The C-C chemokine motif ligand 5 (CCL5) and its receptors have recently been thought to be substantially involved in the development of obesity-associated adipose tissue inflammation and insulin resistance. However, the respective contributions of tissue-derived and myeloid-derived CCL5 to the etiology of obesity-induced adipose tissue inflammation and insulin resistance, and the involvement of monocytic myeloid-derived suppressor cells (MDSCs), remain unclear. This study used CCL5-knockout mice combined with bone marrow transplantation (BMT) and mice with local injections of shCCL5/shCCR5 or CCL5/CCR5 lentivirus into bilateral epididymal white adipose tissue (eWAT). CCL5 gene deletion significantly ameliorated HFD-induced inflammatory reactions in eWAT and protected against the development of obesity and insulin resistance. In addition, tissue (non-hematopoietic) deletion of CCL5 using the BMT method not only ameliorated adipose tissue inflammation by suppressing pro-inflammatory M-MDSC (CD11b⁺Ly6G⁻Ly6C^hi) accumulation and skewing local M1 macrophage polarization, but also recruited reparative M-MDSCs (CD11b⁺Ly6G⁻Ly6C^low) and M2 macrophages to the eWAT of HFD-induced obese mice, as shown by flow cytometry. Furthermore, modulation of tissue-derived CCL5/CCR5 expression by local injection of shCCL5/shCCR5 or CCL5/CCR5 lentivirus substantially impacted the distribution of pro-inflammatory and reparative M-MDSCs as well as macrophage polarization in bilateral eWAT. These findings suggest that an obesity-induced increase in adipose tissue CCL5-mediated signaling is crucial in the recruitment of tissue M-MDSCs and their trans-differentiation to tissue pro-inflammatory macrophages, resulting in adipose tissue inflammation and insulin resistance.     

DFMO Improves Survival and Increases Immune Cell Infiltration in Association with MYC Downregulation in the Pancreatic Tumor Microenvironment

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor five-year survival rate of less than 10%. Immune suppression along with chemoresistance are obstacles for PDAC therapeutic treatment. Innate immune cells, such as tumor-associated macrophages, are recruited to the inflammatory environment of PDAC and adversely suppress cytotoxic T lymphocytes. KRAS and MYC are important oncogenes associated with immune suppression and pose a challenge to successful therapies. Here, we targeted KRAS, through inhibition of downstream c-RAF with GW5074, and MYC expression via difluoromethylornithine (DFMO). DFMO alone and with GW5074 reduced in vitro PDAC cell viability. Both DFMO and GW5074 showed efficacy in reducing in vivo PDAC growth in an immunocompromised model. Results in immunocompetent syngeneic tumor-bearing mice showed that DFMO and combination treatment markedly decreased tumor size, but only DFMO increased survival in mice. To further investigate, immunohistochemical staining showed DFMO diminished MYC expression and increased tumor infiltration of macrophages, CD86⁺ cells, CD4⁺ and CD8⁺ T lymphocytes. GW5074 was not as effective in modulating the tumor infiltration of total CD3⁺ lymphocytes or tumor progression and maintained MYC expression. Collectively, this study highlights that in contrast to GW5074, the inhibition of MYC through DFMO may be an effective treatment modality to modulate PDAC immunosuppression.     

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

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 TGF-β 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, OMC- and 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 mouse- and 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 TGFβ and activin signaling pathways, or with a monoclonal antibody against the TGFβ1, β2, and β3 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.