Herein, we demonstrate a role of AMP-activated protein kinase (AMPK) as a potent counterregulator of inflammatory signaling pathways in macrophages. Stimulation of macrophages with anti-inflammatory cytokines (i.e., IL-10 and TGFβ) resulted in the rapid phosphorylation/activation of AMPK, whereas stimulation of macrophages with a proinflammatory stimulus (LPS) resulted in AMPK dephosphorylation/inactivation. Inhibition of AMPKα expression by RNA interference dramatically increased the mRNA levels of LPS-induced TNF-α, IL-6, and cyclooxygenase-2. Likewise, expression of a dominant negative AMPKα1 in macrophages enhanced TNF-α and IL-6 protein synthesis in response to LPS stimulation, while diminishing the production of IL-10. In contrast, transfection of macrophages with a constitutively active form of AMPKα1 resulted in decreased LPS-induced TNF-α and IL-6 production, and heightened production of IL-10. In addition, we found that AMPK negatively regulated LPS-induced IκB-α degradation and positively regulated Akt activation, accompanied by inhibition of glycogen synthase kinase β and activation of CREB. Thus, AMPK directs signaling pathways in macrophages in a manner that suppresses proinflammatory responses and promotes macrophage polarization to an anti-inflammatory functional phenotype.

The immune system plays an important role in regulating tumor growth and metastasis. For example, classical monocytes promote tumorigenesis and cancer metastasis; however, how nonclassical “patrolling” monocytes interact with tumors is unknown. Here we show that patrolling monocytes are enriched in the microvasculature of the lung and reduce tumor metastasis to lung in multiple mouse metastatic tumor models. Nr4a1-deficient mice, which specifically lack patrolling monocytes, showed increased lung metastasis in vivo. Transfer of Nr4a1-proficient patrolling monocytes into Nr4a1-deficient mice prevented tumor invasion in lung. Patrolling monocytes established early interactions with metastasizing tumor cells, scavenged tumor material from the lung vasculature and promoted natural killer cell recruitment and activation. Thus, patrolling monocytes contribute to cancer immunosurveillance and may be targets for cancer immunotherapy.

No abstract

High concentrations of adenosine in tumor microenvironments inhibit anti-tumor cytotoxic lymphocyte responses. Although T cells express inhibitory adenosine A2A receptors (A2ARs) that suppress their activation and inhibit immune killing of tumors, a role for myeloid-cell A2ARs in suppressing the immune response to tumors has yet to be investigated. In this study we show that the growth of transplanted syngeneic B16F10 melanoma or Lewis lung carcinoma cells is slowed in Adora2af/f–LysMCre+/− mice, which selectively lack myeloid A2ARs. Reduced melanoma growth is associated with significant increases in MHCII and IL-12 expression in tumor-associated macrophages and with > 90% reductions in IL-10 expression in tumor-associated macrophages, dendritic cells and Ly6C+ or Ly6G+ myeloid-derived suppressor cells. Myeloid deletion of A2ARs significantly increases CD44 expression on tumor-associated T cells and NK cells. Depletion of CD8+ T cells or NK cells in tumor-bearing mice indicates that both cell types initially contribute to slowing melanoma growth in mice lacking myeloid A2A receptors, but tumor suppression mediated by CD8+ T cells is more persistent. Myeloid-selective A2AR deletion significantly reduces lung metastasis of melanomas that express luciferase (for in vivo tracking) and ovalbumin (as a model antigen). Reduced metastasis is associated with increased numbers and activation of NK cells and antigen specific CD8+ T cells in lung infiltrates. Overall the findings indicate that myeloid cell A2ARs have direct myelosupressive effects that indirectly contribute to the suppression of T cells and NK cells in primary and metastatic tumor microenvironments. The results indicate that tumor-associated myeloid cells, including macrophages, DCs and MDSCs all express immunosuppressive A2ARs that are potential targets of adenosine receptor blockers to enhance immune killing of tumors.

The accumulation of high levels of adenosine in tumors activates A2A and A2B receptors on immune cells and inhibits their ability to suppress tumor growth. Deletion of A2AARs has been reported to activate anti-tumor T cells, stimulates DC function and inhibits angiogenesis. Here we evaluated the effects of intermittent intratumor injection of a non-selective adenosine receptor antagonist, aminophylline (AMO, theophylline ethylenediamine) and, for the first time, a selective A2BAR antagonist, ATL801. AMO and ATL801 slowed the growth of MB49 bladder and 4T1 breast tumors in syngeneic mice, and reduced by 85% metastasizes of breast cancer cells from mammary fat to lung. Based on experiments with A2AAR−/− or A2BAR−/− mice, the effect of AMO injection was unexpectedly attributed to A2BAR and not to A2AAR blockade. AMO and ATL801 significantly increased tumor levels of IFNγ and the interferon-inducible chemokine CXCL10, which is a ligand for CXCR3. This was associated with an increase in activated tumor-infiltrating CXCR3+ T cells and a decrease in endothelial cell precursors within tumors. Tumor growth inhibition by AMO or ATL801 was eliminated in CXCR3−/− mice and in RAG1−/− mice that lack mature T cells. In RAG1−/− mice A2BAR deletion enhanced CD86 expression on CD11b- DCs. Bone marrow chimera experiments demonstrated that CXCR3 and A2BAR expression on bone marrow cells are required for the anti-tumor effects of AMO. The data suggest that blockade of A2BARs enhances DC activation and CXCR3-dependent anti-tumor responses.