Activation of Cannabinoid Receptor 2 Prevents Colitis-Associated Colon Cancer through Myeloid Cell De-activation Upstream of IL-22 Production.

Intestinal disequilibrium leads to inflammatory bowel disease (IBD), and chronic inflammation predisposes to oncogenesis. Antigen-presenting dendritic cells (DCs) and macrophages can tip the equilibrium toward tolerance or pathology. Here we show that delta-9-tetrahydrocannabinol (THC) attenuates colitis-associated colon cancer and colitis induced by anti-CD40. Working through cannabinoid receptor 2 (CB2), THC increases CD103 expression on DCs and macrophages and upregulates TGF-ß1 to increase T regulatory cells (Tregs). THC-induced Tregs are necessary to remedy systemic IFNγ and TNFα caused by anti-CD40, but CB2-mediated suppression of APCs by THC quenches pathogenic release of IL-22 and IL-17A in the colon. By examining tissues from multiple sites, we confirmed that THC affects DCs, especially in mucosal barrier sites in the colon and lungs, to reduce DC CD86. Using models of colitis and systemic inflammation we show that THC, through CB2, is a potent suppressor of aberrant immune responses by provoking coordination between APCs and Tregs.

Arginase-1 is neither constitutively expressed in nor required for myeloid-derived suppressor cell-mediated inhibition of T-cell proliferation.

Although previous reports suggest that tumor-induced myeloid-derived suppressor cells (MDSC) inhibit T cells by L-arginine depletion through arginase-1 activity, we herein show that arginase-1 is neither inherently expressed in MDSC nor required for MDSC-mediated inhibition. Employing Percoll density gradients, large expansions of MDSC in the bone marrow of tumor-bearing mice were isolated and demonstrated potent inhibition in T-cell proliferation activated by TCR-ligation, Concanavalin A, PMA plus ionomycin, or IL-2. Despite demonstrating characteristic immunosuppressive capacity, these MDSC exhibit no arginase-1 expression and/or exert their inhibitory effects independent of arginase-1 activity. However, arginase-1 expression in MDSC can be induced by exposure to TCR-activated T cells or their culture medium, but not T cells activated by other means or growing tumor cells. Further investigation reveals multiple cytokines secreted by TCR-activated T cells as orchestrating two signaling-relay axes, IL-6-to-IL-4 and GM-CSF/IL-4-to-IL-10, leading to arginase-1 expression in MDSC. Specifically, IL-6 signaling increases IL-4R, enabling IL-4 to induce arginase-1 expression; similarly, GM-CSF in concert with IL-4 induces IL-10R, allowing IL-10-mediated induction. Surprisingly, our study indicates that induction of arginase-1 expression is not conducive to the critical MDSC-mediated inhibition toward T cells, which is rather dependent on direct cell contacts undiminished by PD-L1 blockade or SIRPα deficiency.

Tumor conditions induce bone marrow expansion of granulocytic, but not monocytic, immunosuppressive leukocytes with increased CXCR2 expression in mice.

Myeloid-derived suppressor cells (MDSCs) promote tumor growth through, in part, inhibiting T-cell immunity. However, mechanisms underlying MDSC expansion and guidance of MDSCs toward the tumor microenvironment remain unclear. Employing Percoll density gradients, we separate bone marrow (BM) leukocytes from tumor-bearing mice into four density-increasing bands with myeloid leukocytes enriched in bands III and IV. Band III comprises monocytes and low-density granulocytes, both confirmed to be M-MDSCs and G-MDSCs, respectively, by displaying potent inhibition of T-cell proliferation. However, monocytes act as M-MDSCs not only under tumor conditions but also the healthy condition. In contrast, band IV contains non-inhibitory, mature granulocytes. Only band III G-MDSCs display significant expansion in mice bearing B16 melanoma, Lewis lung carcinoma, or MC38 colon carcinoma. The expanded G-MDSCs also show increased CXCR2 expression, which guides egress out of BM, and produce arginase-1 and ROS upon encountering antigen-activated T cells. Adoptive transfer assays demonstrate that both G-MDSCs and mature granulocytes infiltrate tumors, but only the former displays sustention and accumulation. Intratumoral administrations of granulocytes further demonstrate that G-MDSCs promote tumor growth, whereas mature granulocytes exert minimal effects, or execute powerful anti-tumor effects providing the presence of PMN activation mechanisms in the tumor microenvironment.

Cd47-Sirpα interaction and IL-10 constrain inflammation-induced macrophage phagocytosis of healthy self-cells.

Rapid clearance of adoptively transferred Cd47-null (Cd47(-/-)) cells in congeneic WT mice suggests a critical self-recognition mechanism, in which CD47 is the ubiquitous marker of self, and its interaction with macrophage signal regulatory protein α (SIRPα) triggers inhibitory signaling through SIRPα cytoplasmic immunoreceptor tyrosine-based inhibition motifs and tyrosine phosphatase SHP-1/2. However, instead of displaying self-destruction phenotypes, Cd47(-/-) mice manifest no, or only mild, macrophage phagocytosis toward self-cells except under the nonobese diabetic background. Studying our recently established Sirpα-KO (Sirpα(-/-)) mice, as well as Cd47(-/-) mice, we reveal additional activation and inhibitory mechanisms besides the CD47-SIRPα axis dominantly controlling macrophage behavior. Sirpα(-/-) mice and Cd47(-/-) mice, although being normally healthy, develop severe anemia and splenomegaly under chronic colitis, peritonitis, cytokine treatments, and CFA-/LPS-induced inflammation, owing to splenic macrophages phagocytizing self-red blood cells. Ex vivo phagocytosis assays confirmed general inactivity of macrophages from Sirpα(-/-) or Cd47(-/-) mice toward healthy self-cells, whereas they aggressively attack toward bacteria, zymosan, apoptotic, and immune complex-bound cells; however, treating these macrophages with IL-17, LPS, IL-6, IL-1ß, and TNFα, but not IFNγ, dramatically initiates potent phagocytosis toward self-cells, for which only the Cd47-Sirpα interaction restrains. Even for macrophages from WT mice, phagocytosis toward Cd47(-/-) cells does not occur without phagocytic activation. Mechanistic studies suggest a PKC-Syk-mediated signaling pathway, to which IL-10 conversely inhibits, is required for activating macrophage self-targeting, followed by phagocytosis independent of calreticulin Moreover, we identified spleen red pulp to be one specific tissue that provides stimuli constantly activating macrophage phagocytosis albeit lacking in Cd47(-/-) or Sirpα(-/-) mice.