Investigating Chemokine-Matrix Networks in Breast Cancer: Tenascin-C Sets the Tone for CCL2.

Bidirectional dialogue between cellular and non-cellular components of the tumor microenvironment (TME) drives cancer survival. In the extracellular space, combinations of matrix molecules and soluble mediators provide external cues that dictate the behavior of TME resident cells. Often studied in isolation, integrated cues from complex tissue microenvironments likely function more cohesively. Here, we study the interplay between the matrix molecule tenascin-C (TNC) and chemokine CCL2, both elevated in and associated with the progression of breast cancer and playing key roles in myeloid immune responses. We uncover a correlation between TNC/CCL2 tissue levels in HER2+ breast cancer and examine the physical and functional interactions of these molecules in a murine disease model with tunable TNC levels and in in vitro cellular and cell-free models. TNC supported sustained CCL2 synthesis, with chemokine binding to TNC via two distinct domains. TNC dominated the behavior of tumor-resident myeloid cells; CCL2 did not impact macrophage survival/activation whilst TNC facilitated an immune suppressive macrophage phenotype that was not dependent on or altered by CCL2 co-expression. Together, these data map new binding partners within the TME and demonstrate that whilst the matrix exerts transcriptional control over the chemokine, each plays a distinct role in subverting anti-tumoral immunity.

TGF-ß is elevated in hyperuricemic individuals and mediates urate-induced hyperinflammatory phenotype in human mononuclear cells.

BACKGROUND: Soluble urate leads to a pro-inflammatory phenotype in human monocytes characterized by increased production of IL-1ß and downregulation of IL-1 receptor antagonist, the mechanism of which remains to be fully elucidated. Previous transcriptomic data identified differential expression of genes in the transforming growth factor (TGF)-ß pathway in monocytes exposed to urate in vitro. In this study, we explore the role of TGF-ß in urate-induced hyperinflammation in peripheral blood mononuclear cells (PBMCs). METHODS: TGF-ß mRNA in unstimulated PBMCs and protein levels in plasma were measured in individuals with normouricemia, hyperuricemia and gout. For in vitro validation, PBMCs of healthy volunteers were isolated and treated with a dose ranging concentration of urate for assessment of mRNA and pSMAD2. Urate and TGF-ß priming experiments were performed with three inhibitors of TGF-ß signalling: SB-505124, 5Z-7-oxozeaenol and a blocking antibody against TGF-ß receptor II. RESULTS: TGF-ß mRNA levels were elevated in gout patients compared to healthy controls. TGF-ß-LAP levels in serum were significantly higher in individuals with hyperuricemia compared to controls. In both cases, TGF-ß correlated positively to serum urate levels. In vitro, urate exposure of PBMCs did not directly induce TGF-ß but did enhance SMAD2 phosphorylation. The urate-induced pro-inflammatory phenotype of monocytes was partly reversed by blocking TGF-ß. CONCLUSIONS: TGF-ß is elevated in individuals with hyperuricemia and correlated to serum urate concentrations. In addition, the urate-induced pro-inflammatory phenotype in human monocytes is mediated by TGF-ß signalling. Future studies are warranted to explore the intracellular pathways involved and to assess the clinical significance of urate-TGF-ß relation.

TBK1 and IKKε act like an OFF switch to limit NLRP3 inflammasome pathway activation.

NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome activation is beneficial during infection and vaccination but, when uncontrolled, is detrimental and contributes to inflammation-driven pathologies. Hence, discovering endogenous mechanisms that regulate NLRP3 activation is important for disease interventions. Activation of NLRP3 is regulated at the transcriptional level and by posttranslational modifications. Here, we describe a posttranslational phospho-switch that licenses NLRP3 activation in macrophages. The ON switch is controlled by the protein phosphatase 2A (PP2A) downstream of a variety of NLRP3 activators in vitro and in lipopolysaccharide-induced peritonitis in vivo. The OFF switch is regulated by two closely related kinases, TANK-binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε). Pharmacological inhibition of TBK1 and IKKε, as well as simultaneous deletion of TBK1 and IKKε, but not of either kinase alone, increases NLRP3 activation. In addition, TBK1/IKKε inhibitors counteract the effects of PP2A inhibition on inflammasome activity. We find that, mechanistically, TBK1 interacts with NLRP3 and controls the pathway activity at a site distinct from NLRP3-serine 3, previously reported to be under PP2A control. Mutagenesis of NLRP3 confirms serine 3 as an important phospho-switch site but, surprisingly, reveals that this is not the sole site regulated by either TBK1/IKKε or PP2A, because all retain the control over the NLRP3 pathway even when serine 3 is mutated. Altogether, a model emerges whereby TLR-activated TBK1 and IKKε act like a "parking brake" for NLRP3 activation at the time of priming, while PP2A helps remove this parking brake in the presence of NLRP3 activating signals, such as bacterial pore-forming toxins or endogenous danger signals.