Tumor-associated macrophage heterogeneity is driven by tissue territories in breast cancer.

Tissue-resident macrophages adapt to local signals within tissues to acquire specific functions. Neoplasia transforms the tissue, raising the question as to how the environmental perturbations contribute to tumor-associated macrophage (TAM) identity and functions. Combining single-cell RNA sequencing (scRNA-seq) with spatial localization of distinct TAM subsets by imaging, we discover that TAM transcriptomic programs follow two main differentiation paths according to their localization in the stroma or in the neoplastic epithelium of the mammary duct. Furthermore, this diversity is exclusively detected in a spontaneous tumor model and tracks the different tissue territories as well as the type of tumor lesion. These TAM subsets harbor distinct capacity to activate CD8+ T cells and phagocyte tumor cells, supporting that specific tumor regions, rather than defined activation states, are the major drivers of TAM plasticity and heterogeneity. The distinctions created here provide a framework to design cancer treatment targeting specific TAM niches.

Imaging resident and recruited macrophage contribution to Wallerian degeneration.

Wallerian degeneration (WD) is a process of autonomous distal degeneration of axons upon injury. Macrophages (MPs) of the peripheral nervous system (PNS) are the main cellular agent controlling this process. Some evidence suggests that resident PNS-MPs along with MPs of hematogenous origin may be involved, but whether these two subsets exert distinct functions is unknown. Combining MP-designed fluorescent reporter mice and coherent anti-Stokes Raman scattering (CARS) imaging of the sciatic nerve, we deciphered the spatiotemporal choreography of resident and recently recruited MPs after injury and unveiled distinct functions of these subsets, with recruited MPs being responsible for efficient myelin stripping and clearance and resident MPs being involved in axonal regrowth. This work provides clues to tackle selectively cellular processes involved in neurodegenerative diseases.

Revising CX3CR1 Expression on Murine Classical and Non-classical Monocytes.

In mice, monocytes (Mo) are conventionally described as CX3CR1low classical Mo (CMo) and CX3CR1high non-classical Mo (NCMo) based on the expression of EGFP in Cx3cr1+/EGFP mice and by analogy with human CX3CR1 expression. Although this terminology is widely used, it may not reflect the expression of CX3CR1 on Mo subsets. Using an unsupervised multiparametric analysis of blood Mo in steady state and after sterile peritonitis, we observed that CX3CR1 expression did not discriminate the CMo from the NCMo subsets. Our results highlight that despite being a reliable reporter to discriminate Mo subpopulations, EGFP level in Cx3cr1+/EGFP mice does not reflect CX3CR1 expression measured by a fluorescently-labeled CX3CL1 chemokine and a CX3CR1 specific antibody. In conclusion, authors should be cautious not to identify murine classical and non-classical Mo as CX3CR1low and CX3CR1high but rather use alternative markers such as the combination of Ly6C and CD43.

Sepsis Triggers a Late Expansion of Functionally Impaired Tissue-Vascular Inflammatory Monocytes During Clinical Recovery.

Sepsis is characterized by a systemic inflammation that can cause an immune dysfunction, for which the underlying mechanisms are unclear. We investigated the impact of cecal ligature and puncture (CLP)-mediated polymicrobial sepsis on monocyte (Mo) mobilization and functions. Our results show that CLP led to two consecutive phases of Mo deployment. The first one occurred within the first 3 days after the induction of the peritonitis, while the second phase was of a larger amplitude and extended up to a month after apparent clinical recovery. The latter was associated with the expansion of Mo in the tissue reservoirs (bone marrow and spleen), their release in the blood and their accumulation in the vasculature of peripheral non-lymphoid tissues. It occurred even after antibiotic treatment but relied on inflammatory-dependent pathways and inversely correlated with increased susceptibility and severity to a secondary infection. The intravascular lung Mo displayed limited activation capacity, impaired phagocytic functions and failed to transfer efficient protection against a secondary infection into monocytopenic CCR2-deficient mice. In conclusion, our work unveiled key dysfunctions of intravascular inflammatory Mo during the recovery phase of sepsis and provided new insights to improve patient protection against secondary infections.

ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.

CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious drug target for the treatment of inflammation. A number of nonpeptidic, competitive CCR2 antagonists have been developed, but none has yet been approved for clinical use. Our aim was to identify a short peptide that showed allosteric antagonism against human and mouse CCR2. On the basis of sequence analysis and 3-dimensional modeling, we identified an original 7-d-amino acid peptidic CCR2 inhibitor that we have called extracellular loop 1 inverso (ECL1i), d(LGTFLKC). In vitro, ECL1i selectively and potently inhibits CC chemokine ligand type 2 (CCL2)-triggered chemotaxis (IC50, 2 µM) but no other conventional CCL2-associated events. We used the classic competitive CCR2 antagonist, BMS22 {2-[(isopropylaminocarbonyl)amino]-N-[2-[[cis-2-[[4-(methylthio)benzoyl]amino]cyclohexyl]amino]-2-oxoethyl]-5-(trifluoromethyl)benzamide}, as positive control and inhibited CCL2-dependent chemotaxis with an IC50 of 18 nM. As negative control, we used a peptide with the same composition as ECL1i, but in a different sequence, d(FKLTLCG). In vivo, ECL1i (4 mg/kg) interfered with CCR2-positive cell recruitment and attenuated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This study establishes ECL1i as the first allosteric inhibitor of CCR2 with functional selectivity. ECL1i is a promising new agent in therapeutic development, and it may, by its selective effect, increase our understanding of CCR2 signaling pathways and functions.-Auvynet, C., Baudesson de Chanville, C., Hermand, P., Dorgham, K., Piesse, C., Pouchy, C., Carlier, L., Poupel, L., Barthélémy, S., Felouzis, V., Lacombe, C., Sagan, S., Salomon, B., Deterre, P., Sennlaub, F., Combadière, C. ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.

Respective pharmacological features of neuropathic-like pain evoked by intrathecal BDNF versus sciatic nerve ligation in rats.

Numerous reported data support the idea that Brain Derived Neurotrophic Factor (BDNF) is critically involved in both depression and comorbid pain. The possible direct effect of BDNF on pain mechanisms was assessed here and compared with behavioral/neurobiological features of neuropathic pain caused by chronic constriction injury to the sciatic nerve (CCI-SN). Sprague-Dawley male rats were either injected intrathecally with BDNF (3.0 ng i.t.) or subjected to unilateral CCI-SN. Their respective responses to anti-hyperalgesic drugs were assessed using the Randall-Selitto test and both immunohistochemical and RT-qPCR approaches were used to investigate molecular/cellular mechanisms underlying hyperalgesia in both models. Long lasting hyperalgesia and allodynia were induced by i.t. BDNF in intact healthy rats like those found after CCI-SN. Acute treatment with the BDNF-TrkB receptor antagonist cyclotraxin B completely prevented i.t. BDNF-induced hyperalgesia and partially reversed this symptom in both BDNF-pretreated and CCI-SN lesioned rats. Acute administration of the anticonvulsant pregabalin, the NMDA receptor antagonist ketamine, the opioid analgesics morphine and tapentadol or the antidepressant agomelatine also transiently reversed hyperalgesia in both i.t. BDNF injected- and CCI-SN lesioned-rats. Marked induction of microglia activation markers (OX42, Iba1, P-p38), proinflammatory cytokine IL-6, NMDA receptor subunit NR2B and BDNF was found in spinal cord and/or dorsal root ganglia of CCI-SN rats. A long lasting spinal BDNF overexpression was also observed in BDNF i.t. rats, indicating an autocrine self-induction, with downstream long lasting TrkB-mediated neuropathic-like pain. Accordingly, TrkB blockade appeared as a relevant approach to alleviate not only i.t. BDNF- but also nerve lesion-evoked neuropathic pain.

Spinal cord transection-induced allodynia in rats--behavioral, physiopathological and pharmacological characterization.

In humans, spinal cord lesions induce not only major motor and neurovegetative deficits but also severe neuropathic pain which is mostly resistant to classical analgesics. Better treatments can be expected from precise characterization of underlying physiopathological mechanisms. This led us to thoroughly investigate (i) mechanical and thermal sensory alterations, (ii) responses to acute treatments with drugs having patent or potential anti-allodynic properties and (iii) the spinal/ganglion expression of transcripts encoding markers of neuronal injury, microglia and astrocyte activation in rats that underwent complete spinal cord transection (SCT). SCT was performed at thoracic T8-T9 level under deep isoflurane anaesthesia, and SCT rats were examined for up to two months post surgery. SCT induced a marked hyper-reflexia at hindpaws and strong mechanical and cold allodynia in a limited (6 cm2) cutaneous territory just rostral to the lesion site. At this level, pressure threshold value to trigger nocifensive reactions to locally applied von Frey filaments was 100-fold lower in SCT- versus sham-operated rats. A marked up-regulation of mRNAs encoding ATF3 (neuronal injury) and glial activation markers (OX-42, GFAP, P2×4, P2×7, TLR4) was observed in spinal cord and/or dorsal root ganglia at T6-T11 levels from day 2 up to day 60 post surgery. Transcripts encoding the proinflammatory cytokines IL-1ß, IL-6 and TNF-α were also markedly but differentially up-regulated at T6-T11 levels in SCT rats. Acute treatment with ketamine (50 mg/kg i.p.), morphine (3-10 mg/kg s.c.) and tapentadol (10-20 mg/kg i.p.) significantly increased pressure threshold to trigger nocifensive reaction in the von Frey filaments test, whereas amitriptyline, pregabalin, gabapentin and clonazepam were ineffective. Because all SCT rats developed long lasting, reproducible and stable allodynia, which could be alleviated by drugs effective in humans, thoracic cord transection might be a reliable model for testing innovative therapies aimed at reducing spinal cord lesion-induced central neuropathic pain.

Differential involvement of the endocannabinoid system in short- and long-term expression of incentive learning supported by nicotine in rats.

RATIONALE: We previously reported that the CB1 cannabinoid receptor antagonist, rimonabant, impaired the acquisition and the short-term (24 h), but not long-term (3 weeks), expression of conditioned place preference (CPP) induced by nicotine in rats. OBJECTIVE: To assess the time interval of efficacy of a single pretest injection of rimonabant to abolish nicotine-CPP, and the effects of chronic CB1 receptor blockade on long-term expression of nicotine-CPP. MATERIALS AND METHODS: Wistar rats were conditioned to nicotine (0.06 mg/kg, subcutaneous) using an unbiased one-compartment procedure. Two test sessions were conducted 24 h (without injection) and 1, 2, or 3 weeks later. Rimonabant (3 mg/kg, intraperitoneal) or vehicle was administered daily between the two test sessions. In addition, the CB1-stimulated [(35)S]GTP-gamma-S binding was assessed in rats from the 3-week experiment. RESULTS: The capacity of a single injection of rimonabant (3 mg/kg, 30 min pretest) to block the expression of nicotine-CPP disappeared within 1 week after conditioning. Daily administrations of rimonabant for 6, 13, or 20 days post-acquisition did not impair nicotine-CPP but allowed an additional pretest injection of rimonabant to retain its capacity to abolish long-term expression of nicotine-CPP. The CB1 receptor-mediated G-protein signaling was not altered in various brain areas of rats given rimonabant for 3 weeks. CONCLUSIONS: The endocannabinoid system is essential to the expression of nicotine-CPP during less than 1 week after conditioning but not later. However, endocannabinoid-dependent mechanisms are critically involved in the development of the neuroadaptive changes responsible for the shift from CB1-dependent to CB1-independent expression of nicotine incentive learning.