Tumor-derived GCSF Alters Tumor and Systemic Immune System Cell Subset Composition and Signaling.

While immunotherapies such as immune checkpoint blockade and adoptive T-cell therapy improve survival for a subset of human malignancies, many patients fail to respond. Phagocytes including dendritic cells (DC), monocytes, and macrophages (MF) orchestrate innate and adaptive immune responses against tumors. However, tumor-derived factors may limit immunotherapy effectiveness by altering phagocyte signal transduction, development, and activity. Using Cytometry by Time-of-Flight, we found that tumor-derived GCSF altered myeloid cell distribution both locally and systemically. We distinguished a large number of GCSF-induced immune cell subset and signal transduction pathway perturbations in tumor-bearing mice, including a prominent increase in immature neutrophil/myeloid-derived suppressor cell (Neut/MDSC) subsets and tumor-resident PD-L1+ Neut/MDSCs. GCSF expression was also linked to distinct tumor-associated MF populations, decreased conventional DCs, and splenomegaly characterized by increased splenic progenitors with diminished DC differentiation potential. GCSF-dependent dysregulation of DC development was recapitulated in bone marrow cultures in vitro, using medium derived from GCSF-expressing tumor cell cultures. Importantly, tumor-derived GCSF impaired T-cell adoptive cell therapy effectiveness and was associated with increased tumor volume and diminished survival of mice with mammary cancer. Treatment with neutralizing anti-GCSF antibodies reduced colonic and circulatory Neut/MDSCs, normalized colonic immune cell composition and diminished tumor burden in a spontaneous model of mouse colon cancer. Analysis of human colorectal cancer patient gene expression data revealed a significant correlation between survival and low GCSF and Neut/MDSC gene expression. Our data suggest that normalizing GCSF bioactivity may improve immunotherapy in cancers associated with GCSF overexpression. Significance: Tumor-derived GCSF leads to systemic immune population changes. GCSF blockade restores immune populations, improves immunotherapy, and reduces tumor size, paralleling human colorectal cancer data. GCSF inhibition may synergize with current immunotherapies to treat GCSF-secreting tumors.

Deep Phenotyping by Mass Cytometry and Single-Cell RNA-Sequencing Reveals LYN-Regulated Signaling Profiles Underlying Monocyte Subset Heterogeneity and Lifespan.

RATIONALE: Monocytes are key effectors of the mononuclear phagocyte system, playing critical roles in regulating tissue homeostasis and coordinating inflammatory reactions, including those involved in chronic inflammatory diseases such as atherosclerosis. Monocytes have traditionally been divided into 2 major subsets termed conventional monocytes and patrolling monocytes (pMo) but recent systems immunology approaches have identified marked heterogeneity within these cells, and much of what regulates monocyte population homeostasis remains unknown. We and others have previously identified LYN tyrosine kinase as a key negative regulator of myeloid cell biology; however, LYN's role in regulating specific monocyte subset homeostasis has not been investigated. OBJECTIVE: We sought to comprehensively profile monocytes to elucidate the underlying heterogeneity within monocytes and dissect how Lyn deficiency affects monocyte subset composition, signaling, and gene expression. We further tested the biological significance of these findings in a model of atherosclerosis. METHODS AND RESULTS: Mass cytometric analysis of monocyte subsets and signaling pathway activation patterns in conventional monocytes and pMos revealed distinct baseline signaling profiles and far greater heterogeneity than previously described. Lyn deficiency led to a selective expansion of pMos and alterations in specific signaling pathways within these cells, revealing a critical role for LYN in pMo physiology. LYN's role in regulating pMos was cell-intrinsic and correlated with an increased circulating half-life of Lyn-deficient pMos. Furthermore, single-cell RNA sequencing revealed marked perturbations in the gene expression profiles of Lyn-/- monocytes with upregulation of genes involved in pMo development, survival, and function. Lyn deficiency also led to a significant increase in aorta-associated pMos and protected Ldlr-/- mice from high-fat diet-induced atherosclerosis. CONCLUSIONS: Together our data identify LYN as a key regulator of pMo development and a potential therapeutic target in inflammatory diseases regulated by pMos.

Preventive and therapeutic oral administration of the pentacyclic triterpene α,ß-amyrin ameliorates dextran sulfate sodium-induced colitis in mice: the relevance of cannabinoid system.

The pentacyclic triterpene α,ß-amyrin has been previously reported as an effective compound in the treatment of several inflammatory conditions. Recent evidence indicates that α,ß-amyrin displayed its effects through interaction with the cannabinoid pathway. We assessed the anti-inflammatory effects of the α,ß-amyrin in the dextran sulfate sodium (DSS)-induced colitis in mice and investigated whether its effects were associated with the interaction with the cannabinoid system. Our results showed that the oral preventive or therapeutic treatment with α,ß-amyrin significantly reduced disease activity, body weight loss, colonic damage, as well as colonic myeloperoxidase and N-acetylglucosaminidase activities. Moreover, α,ß-amyrin decreases the colonic pro-inflammatory mediators tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and keratinocyte-derived chemokine (CXCL1/KC), while up-regulating the IL-4 levels. Additionally, we also observed that the α,ß-amyrin caused a significant reduction of the adhesion molecules mRNA expression for intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), platelet cell adhesion molecule 1 (PCAM-1), ß(2)-integrin and protein expression for proliferation marker Ki67, the macrophage molecule CD68 and for adhesion molecule P-selectin. Interestingly, our results also showed that the cannabinoid receptor 1 (CB(1)), but not CB(2), pharmacological blockade significantly reversed the beneficial effects of α,ß-amyrin in DSS-induced colitis. Besides, our data demonstrated that mRNA expression for both the endocannabinoid hydrolase monoglyceride lipase 1 (MGL1) and fatty acid amide hydrolase (FAAH) were significantly reduced in the colon of α,ß-amyrin-treated mice. Altogether, these results suggest that the α,ß-amyrin might possess potential therapeutic interest for the treatment of IBD, and also provide new insights for the underlying mechanisms.