Probes for CCL2

Patients with primary solid malignancies frequently exhibit signs of systemic inflammation. Notably, elevated levels of neutrophils and their associated soluble mediators are regularly observed in cancer patients, and correlate with reduced survival and increased metastasis formation. Recently, we demonstrated a mechanistic link between mammary tumor-induced IL17-producing γδ T cells, systemic expansion of immunosuppressive neutrophils and metastasis formation in a genetically engineered mouse model for invasive breast cancer. How tumors orchestrate this systemic inflammatory cascade to facilitate dissemination remains unclear. Here we show that activation of this cascade relies on CCL2-mediated induction of IL1β in tumor-associated macrophages. In line with these findings, expression of CCL2 positively correlates with IL1Β and macrophage markers in human breast tumors. We demonstrate that blockade of CCL2 in mammary tumor-bearing mice results in reduced IL17 production by γδ T cells, decreased neutrophil expansion and enhanced CD8+ T cell activity. These results highlight a new role for CCL2 in facilitating the breast cancer-induced pro-metastatic systemic inflammatory γδ T cell – IL17 – neutrophil axis.

Inflammatory disorders of the CNS are frequently accompanied by synaptic loss, which is thought to involve phagocytic microglia and complement components. However, the mechanisms accounting for aberrant synaptic connectivity in the context of CD8+ T cell-driven neuronal damage are poorly understood. Here, we profiled the neuronal translatome in a murine model of encephalitis caused by CD8+ T cells targeting antigenic neurons. Neuronal STAT1 signaling and downstream CCL2 expression were essential for apposition of phagocytes, ensuing synaptic loss and neurological disease. Analogous observations were made in the brains of Rasmussen’s encephalitis patients. In this devastating CD8+T cell-driven autoimmune disease, neuronal STAT1 phosphorylation and CCL2 expression co-clustered with infiltrating CD8+ T cells as well as phagocytes. Taken together, our findings uncover an active role of neurons in coordinating phagocyte-mediated synaptic loss and highlight neuronal STAT1 and CCL2 as critical steps in this process that are amenable to pharmacological interventions.

Tumor-immune cell interactions shape the immune cell phenotype, with microRNAs (miRs) being crucial components of this crosstalk. How they are transferred and how they affect their target landscape, especially in tumor-associated macrophages (TAMs), is largely unknown. Here we report that breast cancer cells have a high constitutive expression of miR-375, which is released as a non-exosome entity during apoptosis. Deep sequencing of the miRome pointed to enhanced accumulation of miR-375 in TAMs, facilitated by the uptake of tumor-derived miR-375 via CD36. In macrophages, miR-375 directly targets TNS3 and PXN to enhance macrophage migration and infiltration into tumor spheroids and in tumors of a xenograft mouse model. In tumor cells, miR-375 regulates CCL2 expression to increase recruitment of macrophages. Our study provides evidence for miR transfer from tumor cells to TAMs and identifies miR-375 as a crucial regulator of phagocyte infiltration and the subsequent development of a tumor-promoting microenvironment.

Acute infection, if not kept in check, can lead to systemic inflammatory responses in the brain. Here, we show that within 2 hr of systemic inflammation, PDGFRβ mural cells of blood vessels rapidly secrete chemokine CCL2, which in turn increases total neuronal excitabilityby promoting excitatory synaptic transmission in glutamatergic neurons of multiple brain regions. By single-cell RNA sequencing, we identified Col1a1 and Rgs5 subgroups of PDGFRβ cells as the main source of CCL2. Lipopolysaccharide (LPS)- or Poly(I:C)-treated pericyte culture medium induced similar effects in a CCL2-dependent manner. Importantly, in Pdgfrb-Cre;Ccl2fl/fl mice, LPS-induced increase in excitatory synaptic transmission was significantly attenuated. These results demonstrate in vivo that PDGFRβ cells function as initial sensors of external insults by secreting CCL2, which relays the signal to the central nervous system. Through their gateway position in the brain, PDGFRβ cells are ideally positioned to respond rapidly to environmental changes and to coordinate responses.

The contributions of the innate immune system to the development of pancreatic cancer are still ill defined. Inflammatory macrophages can initiate metaplasia of pancreatic acinar cells to a duct-like phenotype (acinar-to-ductal metaplasia [ADM]), which then gives rise to pancreatic intraepithelial neoplasia (PanIN) when oncogenic KRas is present. However, it remains unclear when and how this inflammatory macrophage population is replaced by tumor-promoting macrophages. Here, we demonstrate the presence of interleukin-13 (IL-13), which can convert inflammatory into Ym1+ alternatively activated macrophages, at ADM/PanIN lesions. We further show that Ym1+ macrophages release factors, such as IL-1ra and CCL2, to drive pancreatic fibrogenesis and tumorigenesis. Treatment of mice expressing oncogenic KRas under an acinar cell-specific promoter with a neutralizing antibody for IL-13 significantly decreased the accumulation of alternatively activated macrophages at these lesions, resulting in decreased fibrosis and lesion growth.

Bovine γδ T cells are amongst the first cells to accumulate at the site of Mycobacterium bovis infection; however, their role in the developing lesion remains unclear. We utilized transcriptomics analysis, in situ hybridization, and a macrophage/γδ T cell co-culture system to elucidate the role of γδ T cells in local immunity to M. bovis infection. Transcriptomics analysis revealed that γδ T cells upregulated expression of several novel, immune-associated genes in response to stimulation with M. bovis antigen. BCG-infected macrophage/γδ T cell co-cultures confirmed the results of our RNAseq analysis, and revealed that γδ T cells from M. bovis-infected animals had a significant impact on bacterial viability. Analysis of γδ T cells within late-stage M. bovis granulomas revealed significant expression of IFN-γ and CCL2, but not IL-10, IL-22, or IL-17. Our results suggest γδ T cells influence local immunity to M. bovis through cytokine secretion and direct effects on bacterial burden.

As tumour protein 53 (p53) isoforms have tumour promoting, migration and inflammatory properties, this study investigated whether p53 isoforms contributed to glioblastoma progression. The expression levels of full-length TP53α (TAp53α) and six TP53 isoforms were quantitated by RT-qPCR in 89 glioblastomas and correlated with TP53 mutation status, tumour-associated macrophage content and various immune cell markers. Elevated levels of Δ133p53β mRNA characterised glioblastomas with increased CD163-positive macrophages and wild-type TP53. In situ based analyses found Δ133p53β expression localised to malignant cells in areas with increased hypoxia, and in cells with the monocyte chemoattractant protein C-C motif chemokine ligand 2 (CCL2) expressed. Tumours with increased Δ133p53β had increased numbers of cell positive for macrophage colony stimulating factor 1 receptor (CSF1R) and programmed death ligand 1 (PDL1). In addition, cells expressing a murine 'mimic' of Δ133p53 (Δ122p53) were resistant to temozolomide treatment and oxidative stress. Our findings suggest elevated Δ133p53β is an alternative pathway to TP53 mutation in glioblastoma that aids tumour progression by promoting an immunosuppressive and chemoresistant environment. Adding Δ133p53β to a TP53 signature along with TP53 mutation status will better predict treatment resistance in glioblastoma.