Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro.

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16Ink4a or p21Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces.

Functional changes of immune cells: signal of immune tolerance of the ectopic lesions in endometriosis?

RESEARCH QUESTION: What is the potential role of immune cells and their inflammatory cytokines in the pathogenesis, development and establishment of endometriosis? DESIGN: Peritoneal fluid from 59 women (43 with endometriosis and 16 controls) who had undergone laparoscopic surgery was analysed. Changes in the population of innate and adaptive immune cells, cytokines, chemokines and growth factor expression were measured by flow cytometry, Luminex Technology and enzyme-linked immunosorbent assay. RESULTS: No differences were found in the frequencies of the innate and adaptive immune cells between women with and without endometriosis. In the peritoneal fluid of women with endometriosis, IL-1ß, IL-1RN, IL-2, IL-4, IL-8, IL-10, IL-12 (p70), IL-17α, FGF2, G-CSF, MCP-1, MIP-1α and TNF-α were significantly increased compared with controls. A correlation between IL-2, MCP-1, MIP-1α, TNF-α and the severity of endometriosis was observed. The concentration of neopterin, a possible biomarker for this disease, was increased in women with endometriosis compared with controls. CONCLUSIONS: The functional activity of immune cells seemed to be reduced despite their numbers remaining unchanged. The data indicate that a shift of TH cytokine profile occurs, which increases the TH1-TH2 ratio. This is driven by the increased levels of the cytokines (TNF-α and IL-2) in women with severe endometriosis.

IL-4 induces M2 macrophages to produce sustained analgesia via opioids.

IL-4 is a pleiotropic antiinflammatory cytokine, which can be neuroprotective after nervous system injury. The beneficial actions of IL-4 are thought to result from the blunting of action of inflammatory mediators, such as proinflammatory cytokines. Here, we demonstrate that IL-4 induces M2 macrophages to continuously produce opioid peptides and ameliorate pain. IL-4 application at injured nerves in mice shifted F4/80+ macrophages from the proinflammatory M1 to the antiinflammatory M2 phenotype, which synthesized opioid peptides (Met-enkephalin, ß-endorphin, and dynorphin A 1-17). These effects were accompanied by a long-lasting attenuation of neuropathy-induced mechanical hypersensitivity, beyond the IL-4 treatment. This IL-4-induced analgesia was decreased by opioid peptide antibodies and opioid receptor (δ, µ, κ) antagonists applied at injured nerves, which confirms the involvement of the local opioid system. The participation of M2 macrophages was supported by analgesia in recipient mice injected at injured nerves with F4/80+ macrophages from IL-4-treated donors. Together, IL-4-induced M2 macrophages at injured nerves produced opioid peptides, which activated peripheral opioid receptors to diminish pain. Fostering the opioid-mediated actions of intrinsic M2 macrophages may be a strategy to tackle pathological pain.

Lipid droplet-dependent fatty acid metabolism controls the immune suppressive phenotype of tumor-associated macrophages.

Tumor-associated macrophages (TAMs) promote tumor growth and metastasis by suppressing tumor immune surveillance. Herein, we provide evidence that the immunosuppressive phenotype of TAMs is controlled by long-chain fatty acid metabolism, specifically unsaturated fatty acids, here exemplified by oleate. Consequently, en-route enriched lipid droplets were identified as essential organelles, which represent effective targets for chemical inhibitors to block in vitro polarization of TAMs and tumor growth in vivo. In line, analysis of human tumors revealed that myeloid cells infiltrating colon cancer but not gastric cancer tissue indeed accumulate lipid droplets. Mechanistically, our data indicate that oleate-induced polarization of myeloid cells depends on the mammalian target of the rapamycin pathway. Thus, our findings reveal an alternative therapeutic strategy by targeting the pro-tumoral myeloid cells on a metabolic level.

Microbial Colonization in Adulthood Shapes the Intestinal Macrophage Compartment.

BACKGROUND AND AIMS: Contact with distinct microbiota early in life has been shown to educate the mucosal immune system, hence providing protection against immune-mediated diseases. However, the impact of early versus late colonization with regard to the development of the intestinal macrophage compartment has not been studied so far. METHODS: Germ-free mice were colonized with specific-pathogen-free [SPF] microbiota at the age of 5 weeks. The ileal and colonic macrophage compartment were analysed by immunohistochemistry, flow cytometry, and RNA sequencing 1 and 5 weeks after colonization and in age-matched SPF mice, which had had contact with microbiota since birth. To evaluate the functional differences, dextran sulfate sodium [DSS]-induced colitis was induced, and barrier function analyses were undertaken. RESULTS: Germ-free mice were characterized by an atrophied intestinal wall and a profoundly reduced number of ileal macrophages. Strikingly, morphological restoration of the intestine occurred within the first week after colonization. In contrast, ileal macrophages required 5 weeks for complete restoration, whereas colonic macrophages were numerically unaffected. However, following DSS exposure, the presence of microbiota was a prerequisite for colonic macrophage infiltration. One week after colonization, mild colonic inflammation was observed, paralleled by a reduced inflammatory response after DSS treatment, in comparison with SPF mice. This attenuated inflammation was paralleled by a lack of TNFα production of LPS-stimulated colonic macrophages from SPF and colonized mice, suggesting desensitization of colonized mice by the colonization itself. CONCLUSIONS: This study provides the first data indicating that after colonization of adult mice, the numeric, phenotypic, and functional restoration of the macrophage compartment requires the presence of intestinal microbiota and is time dependent.

HDAC inhibitors promote intestinal epithelial regeneration via autocrine TGFß1 signalling in inflammation.

Intact epithelial barrier function is pivotal for maintaining intestinal homeostasis. Current therapeutic developments aim at restoring the epithelial barrier in inflammatory bowel disease. Histone deacetylase (HDAC) inhibitors are known to modulate immune responses and to ameliorate experimental colitis. However, their direct impact on epithelial barrier function and intestinal wound healing is unknown. In human and murine colonic epithelial cell lines, the presence of the HDAC inhibitors Givinostat and Vorinostat not only improved transepithelial electrical resistance under inflammatory conditions but also attenuated the passage of macromolecules across the epithelial monolayer. Givinostat treatment mediated an accelerated wound closure in scratch assays. In vivo, Givinostat treatment resulted in improved barrier recovery and epithelial wound healing in dextran sodium sulphate-stressed mice. Mechanistically, these regenerative effects could be linked to an increased secretion of transforming growth factor beta1 and interleukin 8, paralleled by differential expression of the tight junction proteins claudin-1, claudin-2 and occludin. Our data reveal a novel tissue regenerative property of the pan-HDAC inhibitors Givinostat and Vorinostat in intestinal inflammation, which may have beneficial implications by repurposing HDAC inhibitors for therapeutic strategies for inflammatory bowel disease.

Oleate but not stearate induces the regulatory phenotype of myeloid suppressor cells.

Tumor infiltrating myeloid cells play contradictory roles in the tumor development. Dendritic cells and classical activated macrophages support anti-tumor immune activity via antigen presentation and induction of pro-inflammatory immune responses. Myeloid suppressor cells (MSCs), for instance myeloid derived suppressor cells (MDSCs) or tumor associated macrophages play a critical role in tumor growth. Here, treatment with sodium oleate, an unsaturated fatty acid, induced a regulatory phenotype in the myeloid suppressor cell line MSC-2 and resulted in an increased suppression of activated T cells, paralleled by increased intracellular lipid droplets formation. Furthermore, sodium oleate potentiated nitric oxide (NO) production in MSC-2, thereby increasing their suppressive capacity. In primary polarized bone marrow cells, sodium oleate (C18:1) and linoleate (C18:2), but not stearate (C18:0) were identified as potent FFA to induce a regulatory phenotype. This effect was abrogated in MSC-2 as well as primary cells by specific inhibition of droplets formation while the inhibition of de novo FFA synthesis proved ineffective, suggesting a critical role for exogenous FFA in the functional induction of MSCs. Taken together our data introduce a new unsaturated fatty acid-dependent pathway shaping the functional phenotype of MSCs, facilitating the tumor escape from the immune system.

Adoptive transfer of M2 macrophages reduces neuropathic pain via opioid peptides.

BACKGROUND: During the inflammation which occurs following nerve damage, macrophages are recruited to the site of injury. Phenotypic diversity is a hallmark of the macrophage lineage and includes pro-inflammatory M1 and anti-inflammatory M2 populations. Our aim in this study was to investigate the ability of polarized M0, M1, and M2 macrophages to secrete opioid peptides and to examine their relative contribution to the modulation of neuropathic pain. METHODS: Mouse bone marrow-derived cells were cultured as unstimulated M0 macrophages or were stimulated into an M1 phenotype using lipopolysaccharide and interferon-γ or into an M2 phenotype using interleukin-4. The macrophage phenotypes were verified using flow cytometry for surface marker analysis and cytokine bead array for cytokine profile assessment. Opioid peptide levels were measured by radioimmunoassay and enzyme immunoassay. As a model of neuropathic pain, a chronic constriction injury (CCI) of the sciatic nerve was employed. Polarized M0, M1, and M2 macrophages (5 × 105 cells) were injected perineurally twice, on days 14 and 15 following CCI or sham surgery. Mechanical and heat sensitivity were measured using the von Frey and Hargreaves tests, respectively. To track the injected macrophages, we also transferred fluorescently stained polarized cells and analyzed the surface marker profile of endogenous and injected cells in the nerves ex vivo. RESULTS: Compared to M0 and M1 cells, M2 macrophages contained and released higher amounts of opioid peptides, including Met-enkephalin, dynorphin A (1-17), and ß-endorphin. M2 cells transferred perineurally at the nerve injury site reduced mechanical, but not heat hypersensitivity following the second injection. The analgesic effect was reversed by the perineurally applied opioid receptor antagonist naloxone methiodide. M2 cells did not affect sensitivity following sham surgery. Neither M0 nor M1 cells altered mechanical and heat sensitivity in CCI or sham-operated animals. Tracing the fluorescently labeled M0, M1, and M2 cells ex vivo showed that they remained in the nerve and preserved their phenotype. CONCLUSIONS: Perineural transplantation of M2 macrophages resulted in opioid-mediated amelioration of neuropathy-induced mechanical hypersensitivity, while M1 macrophages did not exacerbate pain. Therefore, rather than focusing on macrophage-induced pain generation, promoting opioid-mediated M2 actions may be more relevant for pain control.

A converse 4-1BB and CD40 ligand expression pattern delineates activated regulatory T cells (Treg) and conventional T cells enabling direct isolation of alloantigen-reactive natural Foxp3+ Treg.

Natural regulatory T cells (nTreg) play a central role in the induction and maintenance of immunological tolerance. Experimental transplant models and recent clinical trials demonstrate that nTreg can control alloreactivity. To upgrade Treg-based cell therapies to a selective suppression of undesired immune reactions, only the transfer of Ag-specific nTreg represents the appropriate therapeutic option. However, Ag-specific nTreg are present at extremely low frequencies in the periphery, and so far appropriate surface markers for their precise identification are missing. In this study, we demonstrate that activated nTreg and activated conventional T cells differ in their 4-1BB and CD40 ligand (CD40L) expression signatures, allowing a clear dissection from each other. Based on the expression of 4-1BB and absence of CD40L expression, human alloantigen-reactive Foxp3(+) nTreg can be directly isolated from MLR cultures with high purity. Alloantigen-reactive 4-1BB(+)CD40L(-) nTreg were characterized by a completely demethylated Treg-specific demethylated region and showed alloantigen-specific suppressive properties superior to polyclonal Treg. Importantly, isolated 4-1BB(+)CD40L(-) nTreg maintain the nTreg phenotype and alloantigen-reactivity after in vitro expansion. Our results offer the possibility to simultaneously analyze Ag-specific nTreg and conventional T cells, and to establish cellular therapies with Ag-specific nTreg aiming at a specific inhibition of unwanted immunity.