Targeted Deletion of Pparβ/δ in Myeloid Cells Enhances Their Tumor-Promoting Function

Abstract

Peroxisome proliferator activated receptors (PPARs) are transcription factors of the nuclear hormone transcription superfamily. Thee different PPAR subtypes have been described, PPARalpha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ), with distinct tissue-specific allocations. All PPARs have been shown to be expressed in immune cells and, due to their ability to regulate lipid metabolism, these transcription factors can affect immune cell differentiation and function. While the effects of PPARα and PPARγ on M2 macrophage polarization are well established, the role of PPARβ/δ in myeloid cells is less studied. In the context of autoimmunity, it was shown that targeted deletion of PPARβ/δ in the myeloid compartment altered the Th1/Th17 polarization of CD4+ cells, and exacerbated experimental autoimmune encephalomyelitis in mice. In tumor mouse models, it was reported that M-CSF secreted by tumor cells could activate PPARβ/δ in tumor infiltrating myeloid cells, resulting in upregulation of genes responsible for their tumor-promoting properties such as IL-10, ARG-1 and VEGF. To investigate how PPARβ/δ can affect the myeloid cell responses in tumor immunity, we combined genetic and pharmacologic approaches. We generated mice with conditional targeting of Pparβ/δ gene and crossed them with LysMCre ( Pparβ/δ f/fLysMCre) to delete Pparβ/δ in myeloid cells. We used the MC38 colon adenocarcinoma syngeneic mouse tumor model and after implantation of tumor cells in Pparβ/δ f/fLysMCre and Pparβ/δ f/f control mice, we monitored tumor growth. Surprisingly, in contrast to what has been previously reported, we observed significantly larger and heavier tumors in PPAR β / δ f/fLysMCre mice compared to control mice, indicating that selective deletion of Pparβ/δ in the myeloid compartment was sufficient to impair anti-tumor immune responses in vivo. We assessed the intratumoral immune infiltrates including CD11b +F4/80 + tumor-associated macrophages (TAMs), CD11b +Ly6C hiLy6G - monocytic (M-MDSC), and CD11b +Ly6C loLy6G + polymorphonuclear (PMN-MDSC) MDSCs by flow cytometry. Although no numerical differences were detected in these populations between the two experimental groups, all myeloid cells isolated from tumor bearing Ppar β / δ f/fLysMCre mice demonstrated a less activated and pro-inflammatory phenotype, as indicated by lower levels of IFN-g expression. Notably, CD38 and CD88, two markers that have been associated with enhanced suppressive properties of MDSCs, were significantly upregulated in M-MDSCs and PMN-MDSC from Ppar β / δ f/fLysMCre tumor bearing mice compared to control Pparβ/δ f/f tumor-bearing mice. Hence, we sought to investigate how Pparβ/δ affects the suppressor properties of MDSCs. By using magnetic beads, we isolated GR1 + MDSC from the spleens of tumor-bearing Ppar β / δ f/fLysMCre and control mice and assessed suppression function in co-cultures with OTI TCR-transgenic T cells stimulated with cognate antigen, by DNA synthesis. Suppression capacity of MDSC from Ppar β / d f/fLysMCre tumor-bearing mice was significantly enhanced compared to MDSC from control tumor bearing mice. These results indicate that conditional deletion of Pparβ/δ in the myeloid cells prevented myeloid cell activation and augmented MDSCs suppressor properties by cell intrinsic mechanisms resulting in acceleration of tumor growth.