Probes for INS

Cannabinoid CB2 receptors (CB2R) are importantly involved in drug reward and addiction. However, the cellular mechanisms underlying CB2R action remain unclear. We have previously reported that cocaine self-administration upregulates CB2R expression in midbrain dopamine (DA) neurons. In the present study, we investigated whether cocaine or heroin also alters CB2R expression in striatal medium-spiny neurons that express dopamine D1 or D2 receptors (D1-MSNs, D2-MSNs) and microglia. Due to the concern of CB2R antibody specificity, we developed three mouse CB2-specific probes to detect CB2R mRNA using quantitative RT-PCR and RNAscope in situ hybridization (ISH) assays. We found that a single injection of cocaine failed to alter, while repeated cocaine injections or self-administration dose-dependently upregulated CB2R gene expression in both brain (cortex and striatum) and periphery (spleen). In contrast, repeated administration of heroin produced a dose-dependent reduction in striatal CB2 mRNA expression. RNAscope ISH assays detected CB2R mRNA in striatal D1- and D2-MSNs, not in microglia. We then used transgenic CX3CR1eGFP/+ microglia reporter mice and D1- or D2-Cre-RiboTag mice to purify striatal microglia or ribosome-associated mRNAs from CX3CR1eGFP/+, D1-MSNs, or D2-MSNs, respectively. We found that CB2R upregulation occurred mainly in D1-MSNs, not in D2-MSNs or microglia, in the nucleus accumbens rather than the dorsal striatum. These findings indicate that repeated cocaine exposure may upregulate CB2R expression in both brain and spleen, with regional and cell type-specific profiles. In the striatum, CB2R upregulation occurs mainly in D1-MSNs in the nucleus accumbens. Given the important role of D1-MSNs in brain reward function, the present findings provide new insight into mechanisms by which brain CB2Rs modulate cocaine action.

Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal disease of unknown aetiology. In France, it ranks among the most frequent interstitial pathologies and affects 6 out of 8 people per 100,000 each year. IPF is characterized by dysregulated healing mechanisms that leads to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. Nintedanib and Pirfenidone are the only currently available treatments even though they are only able to slow down the disease without being curative. In this context, inhibiting HSPB5, a low molecular weight heat shock protein known to be involved in the development of fibrosis, could constitute a potential therapeutic target. Our aim consist to explore how NCI-41356 (a chemical inhibitor of HSPB5) can limit the development of pulmonary fibrosis. Methods In vivo, fibrosis was assessed in mice injected intratracheally (i.t.) with Bleomycin (BLM) and treated with NaCl or NCI-41356 (3 times i.t. or 3 times a week i.v.). Fibrosis was evaluated by collagen quantification (Sircol, Sirius Red staining), Immunofluorescence, TGF-β gene expression (RNAscope). In vitro, TGF-β1 signaling was evaluated in epithelial cells treated by TGF-β1 with or without NCI-41356 (Western Blot, Immunofluorescence, Proximity ligation assay). Results In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-β1 and several pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 explaining NCI-41356 anti-fibrotic properties. Conclusion In this study, we determined that inhibition of HSPB5/SMAD4 could limit IPF in mice. NCI-41356 modulates SMAD4 nuclear translocation thus limiting TGF-β1 signaling and synthesis of collagen and pro-fibrotic markers. Further investigations with human fibrotic lung tissues are needed to determine if these results can be transposed in human.

Epithelial-mesenchymal transition is a hallmark of uterine carcinosarcoma (UCS). Here, we used shotgun proteomics analysis to identify biomarkers associated with blebbistatin-mediated epithelial-mesenchymal transition in UCS, and found up-regulation of nucleobindin-2 (NUCB2) in endometrial carcinoma (Em Ca) cells. Expression of N-cadherin, Snail, Slug, and ZEB1 was reduced in NUCB2 knockout Em Ca cells, whereas ZEB1, Twist1, and vimentin were up-regulated in NUCB2-overexpressing Em Ca cells. NUCB2 knockout reduced cell proliferation and migration, whereas NUCB2 overexpression had the opposite effect. Treatment of Em Ca cells with transforming growth factor (TGF)-β1 dramatically altered morphology toward a fibroblastic appearance; concomitantly, expression of NUCB2 and ZEB1 increased. The NUCB2 promoter was also activated by transfection of Smad2. In UCS tissues, NUCB2 expression was significantly higher in sarcomatous compared with carcinomatous components; this was consistent with increased TGF-β1 mRNA expression in stromal and sarcomatous components compared with carcinomatous components. In addition, NUCB2 score correlated positively with ZEB1 and vimentin scores, whereas ZEB1 score correlated positively with Slug and vimentin scores and inversely with the E-cadherin score. We therefore suggest that TGF-β-dependent up-regulation of NUCB2 and ZEB1 contributes to the phenotypic characteristics of sarcomatous components in UCS.

Pulmonary hypertension (PH) can occur as a complication of schistosomiasis. In humans, schistosomiasis-PH persists despite antihelminthic therapy and parasite eradication. We hypothesized that persistent disease arises as a consequence of exposure repetition.Following intraperitoneal sensitization, mice were experimentally exposed to Schistosoma eggs by intravenous injection, either once or three times repeatedly. The phenotype was characterized by right heart catheterization and tissue analysis.Following intraperitoneal sensitization, a single intravenous Schistosoma egg exposure resulted in a PH phenotype that peaked at 7-14 days, followed by spontaneous resolution. Three sequential exposures resulted in a persistent PH phenotype. Inflammatory cytokines were not significantly different between mice exposed to one or three egg doses, but there was an increase in perivascular fibrosis in those who received three egg doses. Significant perivascular fibrosis was also observed in autopsy specimens from patients who died of this condition.Repeatedly exposing mice to schistosomiasis causes a persistent PH phenotype, accompanied by perivascular fibrosis. Perivascular fibrosis may contribute to the persistent schistosomiasis-PH observed in humans with this disease.

Collagens in the extracellular matrix (ECM) provide a physical barrier to tumor immune infiltration, while also acting as a ligand for immune inhibitory receptors. Transforming growth factor-β (TGF-β) is a key contributor to shaping the ECM by stimulating the production and remodeling of collagens. TGF-β-activation signatures and collagen-rich environments have both been associated with T-cell exclusion and lack of responses to immunotherapy. Here we describe the effect of targeting collagens that signal through the inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) in combination with blockade of TGF-β and programmed cell death ligand 1 (PD-L1). This approach remodeled the tumor collagenous matrix, enhanced tumor infiltration and activation of CD8+ T cells, and repolarized suppressive macrophage populations resulting in high cure rates and long-term tumor-specific protection across murine models of colon and mammary carcinoma. The results highlight the advantage of direct targeting of ECM components in combination with immune checkpoint blockade therapy.