TNF-Stimulated Gene-6 Is a Key Regulator in Switching Stemness and Biological Properties of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well established to have promising therapeutic properties. TNF-stimulated gene-6 (TSG-6), a potent tissue-protective and anti-inflammatory factor, has been demonstrated to be responsible for a significant part of the tissue-protecting properties mediated by MSCs. Nevertheless, current knowledge about the biological function of TSG-6 in MSCs is limited. Here, we demonstrated that TSG-6 is a crucial factor that influences many functional properties of MSCs. The transcriptomic sequencing analysis of wild-type (WT) and TSG-6-/- -MSCs shows that the loss of TSG-6 expression leads to the perturbation of several transcription factors, cytokines, and other key biological pathways. TSG-6-/- -MSCs appeared morphologically different with dissimilar cytoskeleton organization, significantly reduced size of extracellular vesicles, decreased cell proliferative rate, and loss of differentiation abilities compared with the WT cells. These cellular effects may be due to TSG-6-mediated changes in the extracellular matrix (ECM) environment. The supplementation of ECM with exogenous TSG-6, in fact, rescued cell proliferation and changes in morphology. Importantly, TSG-6-deficient MSCs displayed an increased capacity to release interleukin-6 conferring pro-inflammatory and pro-tumorigenic properties to the MSCs. Overall, our data provide strong evidence that TSG-6 is crucial for the maintenance of stemness and other biological properties of murine MSCs.

MFSD2A Promotes Endothelial Generation of Inflammation-Resolving Lipid Mediators and Reduces Colitis in Mice.

BACKGROUND & AIMS: Alterations in signaling pathways that regulate resolution of inflammation (resolving pathways) contribute to pathogenesis of ulcerative colitis (UC). The resolution process is regulated by lipid mediators, such as those derived from the ω-3 docosahexaenoic acid (DHA), whose esterified form is transported by the major facilitator superfamily domain containing 2A (MFSD2A) through the endothelium of brain, retina, and placenta. We investigated if and how MFSD2A regulates lipid metabolism of gut endothelial cells to promote resolution of intestinal inflammation. METHODS: We performed lipidomic and functional analyses of MFSD2A in mucosal biopsies and primary human intestinal microvascular endothelial cells (HIMECs) isolated from surgical specimens from patients with active, resolving UC and healthy individuals without UC (controls). MFSD2A was knocked down in HIMECs with small hairpin RNAs or overexpressed from a lentiviral vector. Human circulating endothelial progenitor cells that overexpress MFSD2A were transferred to CD1 nude mice with dextran sodium sulfate-induced colitis, with or without oral administration of DHA. RESULTS: Colonic biopsies from patients with UC had reduced levels of inflammation-resolving DHA-derived epoxy metabolites compared to healthy colon tissues or tissues with resolution of inflammation. Production of these metabolites by HIMECs required MFSD2A, which is required for DHA retention and metabolism in the gut vasculature. In mice with colitis, transplanted endothelial progenitor cells that overexpressed MFSD2A not only localized to the inflamed mucosa but also restored the ability of the endothelium to resolve intestinal inflammation, compared with mice with colitis that did not receive MFSD2A-overexpressing endothelial progenitors. CONCLUSIONS: Levels of DHA-derived epoxides are lower in colon tissues from patients with UC than healthy and resolving mucosa. Production of these metabolites by gut endothelium requires MFSD2A; endothelial progenitor cells that overexpress MFSD2A reduce colitis in mice. This pathway might be induced to resolve intestinal inflammation in patients with colitis.

Mesenchymal Stem Cells Reduce Colitis in Mice via Release of TSG6, Independently of Their Localization to the Intestine.

BACKGROUND & AIMS: Mesenchymal stem cells (MSCs) are pluripotent cells that can promote expansion of immune regulatory cells and might be developed for the treatment of immune disorders, including inflammatory bowel diseases. MSCs were reported to reduce colitis in mice; we investigated whether MSC localization to the intestine and production of paracrine factors, including tumor necrosis factor-induced protein 6 (TSG6), were required for these effects. METHODS: MSCs were isolated from bone marrow (BM-MSCs) of 4- to 6-week-old C57BL/6, C57BL/6-green fluorescent protein, or Balb/c Tsg6-/- male mice. Colitis was induced by ad libitum administration of dextran sulfate sodium for 10 days; after 5 days the mice were given intraperitoneal injections of BM-MSCs or saline (controls). Blood samples and intestinal tissues were collected 24, 48, 96, and 120 hours later; histologic and flow cytometry analyses were performed. RESULTS: Injection of BM-MSCs reduced colitis in mice, increasing body weight and reducing markers of intestinal inflammation, compared with control mice. However, fewer than 1% of MSCs reached the inflamed colon. Most of the BM-MSCs formed aggregates in the peritoneal cavity. The aggregates contained macrophages and B and T cells, and produced immune-regulatory molecules including FOXP3, interleukin (IL)10, transforming growth factor-ß, arginase type II, chemokine (C-C motif) ligand 22 (CCL22), heme oxygenase-1, and TSG6. Serum from mice given BM-MSCs, compared with mice given saline, had increased levels of TSG6. Injection of TSG6 reduced the severity of colitis in mice, along with the numbers of CD45+ cells, neutrophils and metalloproteinase activity in the mucosa, while increasing the percentage of Foxp3CD45+ cells. TSG6 injection also promoted the expansion of regulatory macrophages that expressed IL10 and inducible nitric oxide synthase, and reduced serum levels of interferon-γ, IL6, and tumor necrosis factor. Tsg6-/- MSCs did not suppress the mucosal inflammatory response in mice with colitis. CONCLUSIONS: BM-MSCs injected into mice with colitis do not localize to the intestine but instead form aggregates in the peritoneum where they produce immunoregulatory molecules, including TSG6, that reduce intestinal inflammation. TSG6 is sufficient to reduce intestinal inflammation in mice with colitis.

Inhibition of CCR7/CCL19 axis in lesional skin is a critical event for clinical remission induced by TNF blockade in patients with psoriasis.

Despite the evidence that tumor necrosis factor (TNF) inhibitors block TNF and the downstream inflammatory cascade, their primary mechanism of action in inhibiting the self-sustaining pathogenic cycle in psoriasis is not completely understood. This study has the aim to identify early critical events for the resolution of inflammation in skin lesions using anti-TNF therapy. We used a translational approach that correlates gene expression fold change in lesional skin with the Psoriasis Area and Severity Index score decrease induced by TNF blockade after 4 weeks of treatment. Data were validated by immunofluorescence microscopy on skin biopsy specimens. We found that the anti-TNF-modulated genes that mostly associated with the clinical amelioration were Ccr7, its ligand, Ccl19, and dendritic cell maturation genes. Decreased expression of T-cell activation genes and Vegf also associated with the clinical response. More important, the down-regulation of Ccr7 observed at 4 weeks significantly correlated with the clinical remission occurring at later time points. Immunofluorescence microscopy on skin biopsy specimens showed that reduction of CCR7(+) cells and chemokine ligand (CCL) 19 was paralleled by disaggregation of the dermal lymphoid-like tissue. These data show that an early critical event for the clinical remission of psoriasis in response to TNF inhibitors is the inhibition of the CCR7/CCL19 axis and support its role in psoriasis pathogenesis.

LLT1-CD161 Interaction in Cancer: Promises and Challenges.

The success of immune checkpoint therapy in cancer has changed our way of thinking, promoting the design of future cancer treatments that places the immune system at the center stage. The knowledge gained on immune regulation and tolerance helped the identification of promising new clinical immune targets. Among them, the lectin-like transcript 1 (LLT1) is the ligand of CD161 (NKR-P1A) receptor expressed on natural killer cells and T cells. LLT1/CD161 interaction modulates immune responses but the exact nature of the signals delivered is still partially resolved. Investigation on the role of LLT1/CD161 interaction has been hampered by the lack of functional homologues in animal models. Also, some studies have been misled by the use of non-specific reagents. Recent studies and meta-analyses of single cell data are bringing new insights into the function of LLT1 and CD161 in human pathology and notably in cancer. The advances made on the characterization of the tumor microenvironment prompt us to integrate LLT1/CD161 interaction into the equation. This review recapitulates the key findings on the expression profile of LLT1 and CD161, their regulation, the role of their interaction in cancer development, and the relevance of targeting LLT1/CD161 interaction.

Autofluorescence identifies highly phagocytic tissue-resident macrophages in mouse and human skin and cutaneous squamous cell carcinoma.

Macrophages from human and mouse skin share phenotypic and functional features, but remain to be characterized in pathological skin conditions. Skin-resident macrophages are known to derive from embryonic precursors or from adult hematopoiesis. In this report, we investigated the origins, phenotypes and functions of macrophage subsets in mouse and human skin and in cutaneous squamous cell carcinoma (cSCC) using the spectral flow cytometry technology that enables cell autofluorescence to be considered as a full-fledged parameter. Autofluorescence identifies macrophage subsets expressing the CD206 mannose receptor in human peri-tumoral skin and cSCC. In mouse, all AF+ macrophages express the CD206 marker, a subset of which also displaying the TIM-4 marker. While TIM-4-CD206+ AF+ macrophages can differentiate from bone-marrow monocytes and infiltrate skin and tumor, TIM-4 identifies exclusively a skin-resident AF+ macrophage subset that can derive from prenatal hematopoiesis which is absent in tumor core. In mouse and human, AF+ macrophages from perilesional skin and cSCC are highly phagocytic cells contrary to their AF- counterpart, thus identifying autofluorescence as a bona fide marker for phagocytosis. Our data bring to light autofluorescence as a functional marker characterizing subsets of phagocytic macrophages in skin and cSCC. Autofluorescence can thus be considered as an attractive marker of function of macrophage subsets in pathological context.

High Dimensional Imaging Mass Cytometry Panel to Visualize the Tumor Immune Microenvironment Contexture.

The integrative analysis of tumor immune microenvironment (TiME) components, their interactions and their microanatomical distribution is mandatory to better understand tumor progression. Imaging Mass Cytometry (IMC) is a high dimensional tissue imaging system which allows the comprehensive and multiparametric in situ exploration of tumor microenvironments at a single cell level. We describe here the design of a 39-antibody IMC panel for the staining of formalin-fixed paraffin-embedded human tumor sections. We also provide an optimized staining procedure and details of the experimental workflow. This panel deciphers the nature of immune cells, their functions and their interactions with tumor cells and cancer-associated fibroblasts as well as with other TiME structural components known to be associated with tumor progression like nerve fibers and tumor extracellular matrix proteins. This panel represents a valuable innovative and powerful tool for fundamental and clinical studies that could be used for the identification of prognostic biomarkers and mechanisms of resistance to current immunotherapies.

Impact of Tenascin-C on Radiotherapy in a Novel Syngeneic Oral Squamous Cell Carcinoma Model With Spontaneous Dissemination to the Lymph Nodes.

Radiotherapy, the most frequent treatment of oral squamous cell carcinomas (OSCC) besides surgery is employed to kill tumor cells but, radiotherapy may also promote tumor relapse where the immune-suppressive tumor microenvironment (TME) could be instrumental. We established a novel syngeneic grafting model from a carcinogen-induced tongue tumor, OSCC13, to address the impact of radiotherapy on OSCC. This model revealed similarities with human OSCC, recapitulating carcinogen-induced mutations found in smoking associated human tongue tumors, abundant tumor infiltrating leukocytes (TIL) and, spontaneous tumor cell dissemination to the local lymph nodes. Cultured OSCC13 cells and OSCC13-derived tongue tumors were sensitive to irradiation. At the chosen dose of 2 Gy mimicking treatment of human OSCC patients not all tumor cells were killed allowing to investigate effects on the TME. By investigating expression of the extracellular matrix molecule tenascin-C (TNC), an indicator of an immune suppressive TME, we observed high local TNC expression and TIL infiltration in the irradiated tumors. In a TNC knockout host the TME appeared less immune suppressive with a tendency towards more tumor regression than in WT conditions. Altogether, our novel syngeneic tongue OSCC grafting model, sharing important features with the human OSCC disease could be relevant for future anti-cancer targeting of OSCC by radiotherapy and other therapeutic approaches.

Unveiling role of sphingosine-1-phosphate receptor 2 as a brake of epithelial stem cell proliferation and a tumor suppressor in colorectal cancer.

BACKGROUND: Sphingosine-1-phosphate receptor 2 (S1PR2) mediates pleiotropic functions encompassing cell proliferation, survival, and migration, which become collectively de-regulated in cancer. Information on whether S1PR2 participates in colorectal carcinogenesis/cancer is scanty, and we set out to fill the gap. METHODS: We screened expression changes of S1PR2 in human CRC and matched normal mucosa specimens [N = 76]. We compared CRC arising in inflammation-driven and genetically engineered models in wild-type (S1PR2+/+) and S1PR2 deficient (S1PR2-/-) mice. We reconstituted S1PR2 expression in RKO cells and assessed their growth in xenografts. Functionally, we mimicked the ablation of S1PR2 in normal mucosa by treating S1PR2+/+ organoids with JTE013 and characterized intestinal epithelial stem cells isolated from S1PR2-/-Lgr5-EGFP- mice. RESULTS: S1PR2 expression was lost in 33% of CRC; in 55%, it was significantly decreased, only 12% retaining expression comparable to normal mucosa. Both colitis-induced and genetic Apc+/min mouse models of CRC showed a higher incidence in size and number of carcinomas and/or high-grade adenomas, with increased cell proliferation in S1PR2-/- mice compared to S1PR2+/+ controls. Loss of S1PR2 impaired mucosal regeneration, ultimately promoting the expansion of intestinal stem cells. Whereas its overexpression attenuated cell cycle progression, it reduced the phosphorylation of AKT and augmented the levels of PTEN. CONCLUSIONS: In normal colonic crypts, S1PR2 gains expression along with intestinal epithelial cells differentiation, but not in intestinal stem cells, and contrasts intestinal tumorigenesis by promoting epithelial differentiation, preventing the expansion of stem cells and braking their malignant transformation. Targeting of S1PR2 may be of therapeutic benefit for CRC expressing high Lgr5.

Tenascin-C Orchestrates an Immune-Suppressive Tumor Microenvironment in Oral Squamous Cell Carcinoma.

Inherent immune suppression represents a major challenge in the treatment of human cancer. The extracellular matrix molecule tenascin-C promotes cancer by multiple mechanisms, yet the roles of tenascin-C in tumor immunity are incompletely understood. Using a 4NQO-induced oral squamous cell carcinoma (OSCC) model with abundant and absent tenascin-C, we demonstrated that tenascin-C enforced an immune-suppressive lymphoid stroma via CCL21/CCR7 signaling, leading to increased metastatic tumors. Through TLR4, tenascin-C increased expression of CCR7 in CD11c+ myeloid cells. By inducing CCL21 in lymphatic endothelial cells via integrin α9ß1 and binding to CCL21, tenascin-C immobilized CD11c+ cells in the stroma. Inversion of the lymph node-to-tumor CCL21 gradient, recruitment of T regulatory cells, high expression of anti-inflammatory cytokines, and matrisomal components were hallmarks of the tenascin-C-instructed lymphoid stroma. Ablation of tenascin-C or CCR7 blockade inhibited the lymphoid immune-suppressive stromal properties, reducing tumor growth, progression, and metastasis. Thus, targeting CCR7 could be relevant in human head and neck tumors, as high tenascin-C expression and an immune-suppressive stroma correlate to poor patient survival.

Hyaluronan Accelerates Intestinal Mucosal Healing through Interaction with TSG-6.

Hyaluronan (HA) has proven to be beneficial in the treatment of several diseases. Recently, it has been shown that the local application of HA (IBD98E) improves endoscopic and clinical outcomes in subjects with active distal ulcerative colitis (UC). However, the mechanisms by which this polysaccharide exerts its beneficial effects are unclear. Here, we demonstrated that HA treatment in vitro and in vivo improved mucosal healing by accelerating intestinal epithelial regeneration. Indeed, mice treated with HA showed a faster recovery from colitis and reduced endoscopic signs of mucosal inflammation compared to those receiving saline. Furthermore, histological analysis revealed less ulcerated mucosa in mice treated with HA, characterized by re-epithelialized areas. TSG-6, the secreted product of TNF-stimulated gene-6, is an HA-binding protein shown previously to have tissue-protective properties and promote wound healing. Mucosal levels of TSG-6 increased in UC patients compared to the healthy controls and also after wounding in mice. TSG-6 deletion prevented the beneficial properties of HA in mucosal wound repair, suggesting that the interaction of HA with TSG-6 is crucial for intestinal epithelial regeneration. Overall these results are consistent with HA having a therapeutic effect via the promotion of mucosal healing in patients with ulcerative colitis.

Reciprocal influence of B cells and tumor macro and microenvironments in the ApcMin/+ model of colorectal cancer.

One of the most fascinating aspects of the immune system is its dynamism, meant as the ability to change and readapt according to the organism needs. Following an insult, we assist to the spontaneous organization of different immune cells which cooperate, locally and at distance, to build up an appropriate response. Throughout tumor progression, adaptations within the systemic tumor environment, or macroenvironment, result in the promotion of tumor growth, tumor invasion and metastasis to distal organs, but also to dramatic changes in the activity and composition of the immune system. In this work, we show the changes of the B-cell arm of the immune system following tumor progression in the ApcMin/+ model of colorectal cancer. Tumor macroenvironment leads to an increased proportion of total and IL-10-competent B cells in draining LNs while activates a differentiation route that leads to the expansion of IgA+ lymphocytes in the spleen and peritoneum. Importantly, serum IgA levels were significantly higher in ApcMin/+ than Wt mice. The peculiar involvement of IgA response in the adenomatous transformation had correlates in the gut-mucosal compartment where IgA-positive elements increased from normal mucosa to areas of low grade dysplasia while decreasing upon overt carcinomatous transformation. Altogether, our findings provide a snapshot of the tumor education of B lymphocytes in the ApcMin/+ model of colorectal cancer. Understanding how tumor macroenvironment affects the differentiation, function and distribution of B lymphocytes is pivotal to the generation of specific therapies, targeted to switching B cells to an anti-, rather than pro-, tumoral phenotype.

Reduction of CD68+ macrophages and decreased IL-17 expression in intestinal mucosa of patients with inflammatory bowel disease strongly correlate with endoscopic response and mucosal healing following infliximab therapy.

BACKGROUND: Antibodies against tumor necrosis factor represent an effective therapy for patients with inflammatory bowel disease. Despite their successful results, the exact mechanism by which infliximab suppresses intestinal inflammation is still a matter of debate. In this study, we used a translational approach to identify the key mechanisms associated with resolution of mucosal inflammation induced by infliximab. METHODS: A total of 16 patients with active inflammatory bowel disease (9 with Crohn's disease and 7 with ulcerative colitis) and 16 controls were enrolled in the study. Patients received infliximab infusions at 0, 2, and 6 weeks. At enrollment and at week 6, patients underwent flexible sigmoidoscopy, and biopsies were taken from the sigmoid colon. RNA was extracted, and mucosal expression of 96 immune-related genes was evaluated by qRT-PCR and confirmed by immunofluorescence microscopy on tissue. Correlation between infliximab-induced gene expression modulation and endoscopic response to therapy was calculated. Lamina propria mononuclear cell apoptosis induced by infliximab was evaluated on tissue sections by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS: We found that infliximab-induced downregulation of macrophage and Th17 pathway genes was significantly associated with both endoscopic response to the therapy and achievement of mucosal healing. Importantly, the observed reduction of lamina propria CD68 macrophages was associated with an increased rate of macrophage apoptosis. CONCLUSIONS: The 2 mechanisms associated with infliximab-induced resolution of intestinal inflammation are the reduction of lamina propria infiltrating CD68 macrophages and the downregulation of interleukin 17A. Moreover, the data suggest that infliximab-induced macrophage apoptosis may represent a key mechanism for the therapeutic success of anti-tumor necrosis factor antibodies.