MHC Class II Antigen Presentation by Lymphatic Endothelial Cells in Tumors Promotes Intratumoral Regulatory T cell-Suppressive Functions.

Several solid malignancies trigger lymphangiogenesis, facilitating metastasis. Tumor-associated lymphatic vessels significantly contribute to the generation of an immunosuppressive tumor microenvironment (TME). Here, we have investigated the ability of tumoral lymphatic endothelial cells (LEC) to function as MHC class II-restricted antigen-presenting cells in the regulation of antitumor immunity. Using murine models of lymphangiogenic tumors engrafted under the skin, we have shown that tumoral LECs upregulate MHC class II and the MHC class II antigen-processing machinery, and that they promote regulatory T-cell (Treg) expansion ex vivo. In mice with LEC-restricted lack of MHC class II expression, tumor growth was severely impaired, whereas tumor-infiltrating effector T cells were increased. Reduction of tumor growth and reinvigoration of tumor-specific T-cell responses both resulted from alterations of the tumor-infiltrating Treg transcriptome and phenotype. Treg-suppressive functions were profoundly altered in tumors lacking MHC class II in LECs. No difference in effector T-cell responses or Treg phenotype and functions was observed in tumor-draining lymph nodes, indicating that MHC class II-restricted antigen presentation by LECs was required locally in the TME to confer potent suppressive functions to Tregs. Altogether, our study suggests that MHC class II-restricted antigen-presenting tumoral LECs function as a local brake, dampening T cell-mediated antitumor immunity and promoting intratumoral Treg-suppressive functions.

Effects of Beta-Blockers on Melanoma Microenvironment and Disease Survival in Human.

Background: The regulation of melanoma by noradrenergic signaling has gain attention since pre-clinical and clinical studies suggested a benefit of using beta-blockers to control disease progression. We need to confirm that human melanoma recapitulates the mechanisms described from pre-clinical models. Methods: The sources and targets of norepinephrine in the microenvironment of 20 human melanoma samples was investigated using immunostaining. The effect of an exposure to beta-blockers on immune cell type distribution and expression of immune response markers was assessed with immunostaining on 212 human primary melanoma. A statistical analysis explored the effect of an exposure to beta-blockers on progression free survival, melanoma related survival, and overall survival on the 286 eligible patients. Results: Tumor cells and macrophages may be a source of norepinephrine in melanoma microenvironment. Tumors from patients exposed to wide spectrum beta-blockers recapitulate the increased infiltration of T-lymphocytes and the increased production of granzyme B observed in pre-clinical models. An exposure to beta-blockers is associated with a better outcome in our cohort of melanoma patients. Conclusion: This study shows the association between an exposure to wide spectrum beta-blockers and markers of an effective anti-tumor immune response as well as the protective effect of beta-blockers in human melanoma patients.

The Janus Kinase inhibitor tofacitinib impacts human dendritic cell differentiation and favours M1 macrophage development.

Several cytokines signalling via Janus Kinase (JAK) proteins have been implicated in the pathogenesis of immune-mediated inflammatory diseases, including psoriasis and rheumatoid arthritis (RA). Tofacitinib, a small JAK inhibitor, is approved for the treatment of RA and has demonstrated good efficacy in psoriasis phase III clinical trials. In this work, we analysed the in vitro effects of tofacitinib on the functions of human dendritic cells (DCs) and macrophages. When assessing the effects of tofacitinib on monocyte-derived DCs, we observed reduced differentiation of monocytes into immature DCs, as evidenced by a decreased transcription of CD209 and CD80. Phenotype assessment in the presence of tofacitinib suggested a switch towards a M1-like macrophage phenotype, as evidenced by the expression of M1 markers such as iNOS, as well as cytokines typically expressed by M1 cells, including IL-12 and IL-23. Of note, Arginase1 and CD200R, typically expressed by M2 cells, were absent on tofacitinib-treated DCs. Furthermore, tofacitinib affected the response of differentiated DCs to maturation stimuli such as LPS and IFNγ, resulting in a partial up-regulation of IL-23 and down-regulation of IL-12, as assessed by qPCR. When investigating macrophage development, we found that tofacitinib inhibited the ability of monocytes to differentiate and polarize into regulatory M2 macrophages, while rather enhancing the ability to develop into inflammatory M1-like macrophages, as evidenced by decreased expression of the M2 marker CD200R and enhanced production of IL-12 and IL-23. In conclusion, tofacitinib impacts the differentiation of human DCs and macrophages, it particularly favours generation of M1-like pro-inflammatory macrophages.

Drug-induced expression of the RNA-binding protein HuR attenuates the adaptive response to BRAF inhibition in melanoma.

Strategies that aim to limit the adaptive response to pathway inhibition in BRAF-mutated melanoma face the inherent limit of signaling redundancy and multiplicity of possible bypass mechanisms. Drug-induced expression of selected RNA-binding proteins, like the ubiquitously expressed HuR, has the potential to differentially stabilize the expression of many genes involved in the compensatory mechanisms of adaptive response. Here, we detect in BRAF-mutated melanoma cell lines having a higher propensity for adaptive response and in non-responding melanoma tumors, a larger proportion of HuRLow cells in the expression distribution of HuR. Using knockdown experiments, we demonstrate, through expression profiling and phenotypic assays, that increasing the proportion of HuRLow cells favors the adaptive response to BRAF inhibition, provided that the HuRLow state stays reversible. The MAPK dependency of melanoma cells appears to be diminished as the proportion of HuRLow cells increases. In single-cell assays, we demonstrate that the HuRLow cells display plasticity in their growth expression profile. Importantly, the adaptive over-proliferating cells emerge in the subpopulation containing the HuRLow cells. Therapeutic concentrations of lithium salts, although they moderately increase the global expression of HuR, are sufficient to suppress the HuRLow cells, induce an overall less resistant expression profile and attenuate in a HuR-dependent manner the adaptive response of melanoma cells in ex vivo assays. The therapeutic effectiveness of this approach is also demonstrated in vivo in mice xenografts. This study has immediate clinical relevance for melanoma therapy and opens a new avenue of strategies to prevent the adaptive response to targeted cancer therapy.

IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation.

IL-17E (IL-25) is a member of the IL-17 cytokine family involved in the promotion of type 2 immune responses. Recently, IL-17E has been reported to be up-regulated in distinct skin inflammatory diseases such as psoriasis and atopic and contact dermatitis. We assessed the role played by IL-17E in skin inflammation. Here, we show that IL-17E induces skin inflammation in vivo, characterized by the expression of innate immune response genes and the recruitment of innate immune cells, particularly neutrophils. Genetic deletion or IL-17E neutralization ameliorated skin inflammation induced by imiquimod application or tape stripping, with reductions in neutrophil and macrophage infiltration as assessed by t-distributed stochastic neighbor embedding-guided multiparameter flow cytometry analysis, in mice. In humans, IL-17E promotes the recruitment of neutrophils via activation of macrophages in a p38-dependent mechanism. In addition, IL-17E is up-regulated in neutrophil-rich inflammatory skin diseases, such as pyoderma gangrenosum and acute generalized exanthematous pustulosis. Our data show a role for IL-17E in skin inflammation that is unrelated to the development of type 2 immune reactions. We propose that IL-17E is an important common denominator of chronic skin inflammation, promoting innate immune cell recruitment and activation.

[Repurposing of betablockers in dermatology]. / Bêtabloquants : un nouveau traitement dermatologique ?

Betablockers are used for decades in the treatment of cardiovascular diseases. Recently, the repurposing of the wide spectrum betablocker propranolol revolutionized the infantile hemangioma management. Besides, betablockers have been shown to significantly improve the prognosis of melanoma patients. Blocking the adrenergic receptors blocks the stress-induced tumorigenic pathways. Propranolol inhibits melanoma by downregulating the tumor angiogenesis but also tumor cell proliferation, invasiveness and local immune suppression. These antitumor effects have been observed in other cancers. The repurposing of propranolol has already changed the practice of pediatricians and dermatologists and may soon help oncologists treat various cancers.

Les bêtabloquants sont utilisés depuis des décennies pour les maladies cardiovasculaires. Le propranolol, bêtabloquant à large spectre, a récemment révolutionné la prise en charge de l'hémangiome infantile. Par ailleurs, ces molécules semblent améliorer le pronostic des patients atteints de mélanome. Le blocage des récepteurs adrénergiques inhibe les différentes voies protumorales induites par le stress. Les bêtabloquants freinent le développement du mélanome en inhibant la néoangiogenèse et la prolifération des cellules tumorales, l'invasion et l'immunosuppression locale. Ces effets antitumoraux sont aussi observés dans d'autres cancers. La réorientation des bêtabloquants a déjà changé la pratique quotidienne des pédiatres et dermatologues et pourrait bientôt concerner les oncologues.

IL4-induced gene 1 promotes tumor growth by shaping the immune microenvironment in melanoma.

Amino acid catabolizing enzymes emerged as a crucial mechanism used by tumors to dampen immune responses. The L-phenylalanine oxidase IL-4 induced gene 1 (IL4I1) is expressed by tumor-associated myeloid cells of most solid tumors, including melanoma. We previously provided the only evidence that IL4I1 accelerates tumor growth by limiting the CD8+ T cell mediated immune response, in a mouse model of melanoma cell transplantation. Here, we explored the role of IL4I1 in Ret mice, a spontaneous model of melanoma. We found that IL4I1 was expressed by CD11b+ myeloid cells and that its activity correlated with disease aggressiveness. IL4I1 did not enhance tumor cell proliferation or angiogenesis, but orchestrated the remodeling of the immune compartment within the primary tumor. Indeed, the inactivation of IL4I1 limited the recruitment of polymorphonuclear myeloid-derived suppressor cells and enhanced the infiltration by Th1 and cytotoxic T cells, thus delaying tumor development and metastatic dissemination. Accordingly, human primary melanomas that were poorly infiltrated by IL4I1+ cells exhibited a higher density of CD8+ T cells. Collectively, our findings strengthen the rationale for therapeutic targeting of IL4I1 as one of the key immune regulators.

Propranolol induces a favourable shift of anti-tumor immunity in a murine spontaneous model of melanoma.

In a previous study on a xenograft model of melanoma, we showed that the beta-adrenergic receptor antagonist propranolol inhibits melanoma development by modulating angiogenesis, proliferation and cell survival. Stress hormones can influence tumor development in different ways and norepinephrine was shown to downregulate antitumor immune responses by favoring the accumulation of immunosuppressive cells, impairing the function of lymphocytes. We assessed the effect of propranolol on antitumor immune response in the MT/Ret mouse model of melanoma. Propranolol treatment delayed primary tumor growth and metastases development in MT/Ret mice. Consistent with our previous observations in human melanoma xenografts, propranolol induces a decrease in cell proliferation and vessel density in the primary tumors and in metastases. In this immunocompetent model, propranolol significantly reduced the infiltration of myeloid cells, particularly neutrophils, in the primary tumor. Inversely, cytotoxic tumor infiltrating lymphocytes were more frequent in the tumor stroma of treated mice. In a consistent manner, we observed the same shift in the proportions of infiltrating leukocytes in the metastases of treated mice. Our results suggest that propranolol, by decreasing the infiltration of immunosuppressive myeloid cells in the tumor microenvironment, restores a better control of the tumor by cytotoxic cells.

Inhibition of human melanoma growth by a non-cardioselective ß-blocker.

Recently, retrospective studies provided conflicting results on the benefit of ß-adrenoceptor-blockers (ß-blockers) on melanoma progression. Most of these studies did not define the ß-blocker used, making it difficult to understand the source of discrepancies between results. Therefore, we investigated the effect of non-cardioselective and cardioselective ß-blockers on melanoma progression at the cellular, molecular, and tumor levels. Here we show that the non-cardioselective ß-blocker propranolol hydrochloride (propranolol) inhibits proliferation and induces apoptosis in primary cell cultures derived from a primary and a metastasis of human melanoma and in melanoma cell lines. In contrast, the cardioselective ß-blocker metoprolol tartrate hardly affects melanoma cell survival or proliferation. We further highlight that a daily treatment with propranolol slows down tumor development in immunodeficient mice transplanted with human melanoma cells. RNA microarrays, quantitative PCR, and histochemistry analyses showed that propranolol regulates the expression of different genes involved in tumor angiogenesis, cell death, or proliferation. Thus, our results suggest that non-cardioselective ß-blockers affect melanoma progression, and bring first clues about the pathways involved in this antitumor effect.

Distribution and identity of neurons expressing the oxytocin receptor in the mouse spinal cord.

Oxytocin can influence various spinal functions. However, little is known about the spinal neuronal networks responsible for oxytocin effects. The aim of this study was to localize and characterize spinal neurons expressing oxytocin receptors. We used an oxytocin receptor-reporter mouse in which the fluorescent protein Venus is expressed under the control of the oxytocin receptor gene promoter. At all segmental levels, Venus-expressing neurons were most numerous in the substantia gelatinosa, mingled with protein kinase Cγ interneurons in the innermost layer of the inner lamina II, which, in contrast to the outer two thirds of this layer, does not receive nociceptive input. Venus-expressing neurons were also observed in the intermediolateral and sacral parasympathetic nuclei, where they represented about 5% of presumed preganglionic neurons identified by choline acetyltransferase immunoreactivity. Finally, Venus immunoreactivity was detected in lumbar and sacral dorsal gray commissures as well as in isolated neurons scattered in different regions of the dorsal horn. Altogether, our results establish the location of neurons putatively involved in oxytocin modulation of spinal functions, in particular of sexual functioning and nociception.

Distinct roles of Hoxa2 and Krox20 in the development of rhythmic neural networks controlling inspiratory depth, respiratory frequency, and jaw opening.

BACKGROUND: Little is known about the involvement of molecular determinants of segmental patterning of rhombomeres (r) in the development of rhythmic neural networks in the mouse hindbrain. Here, we compare the phenotypes of mice carrying targeted inactivations of Hoxa2, the only Hox gene expressed up to r2, and of Krox20, expressed in r3 and r5. We investigated the impact of such mutations on the neural circuits controlling jaw opening and breathing in newborn mice, compatible with Hoxa2-dependent trigeminal defects and direct regulation of Hoxa2 by Krox20 in r3. RESULTS: We found that Hoxa2 mutants displayed an impaired oro-buccal reflex, similarly to Krox20 mutants. In contrast, while Krox20 is required for the development of the rhythm-promoting parafacial respiratory group (pFRG) modulating respiratory frequency, Hoxa2 inactivation did not affect neonatal breathing frequency. Instead, we found that Hoxa2-/- but not Krox20-/- mutation leads to the elimination of a transient control of the inspiratory amplitude normally occurring during the first hours following birth. Tracing of r2-specific progenies of Hoxa2 expressing cells indicated that the control of inspiratory activity resides in rostral pontine areas and required an intact r2-derived territory. CONCLUSION: Thus, inspiratory shaping and respiratory frequency are under the control of distinct Hox-dependent segmental cues in the mammalian brain. Moreover, these data point to the importance of rhombomere-specific genetic control in the development of modular neural networks in the mammalian hindbrain.

Exposure to retinoic acid at the onset of hindbrain segmentation induces episodic breathing in mice.

Hyperpnoeic episodic breathing (HEB), a cyclic waxing and waning of breathing, has been widely reported in pre-term neonates, patients with Joubert syndrome and adults (Cheyne-Stokes respiration) with congestive heart failure and brainstem infarction. We now provide a developmental mouse model of neonatal HEB. We used retinoic acid (RA) (0.5-10 mg/kg of maternal weight) to alter embryonic development of the respiratory neuronal network at the onset of hindbrain segmentation (7.5 days post-coitum). HEB was observed in vivo after RA treatment during post-natal days 1-7 but not in control animals. HEB persisted after reduction of the chemoafferent input by hypocapnic hyperoxia (100% O(2)). A large increase and decrease of the rhythm resembling an HEB episode was induced in vitro by stimulating the parafacial respiratory oscillator in treated but not in control neonates. Post-natal localization of the superior cerebellar peduncle and adjacent dorsal tegmentum was found to be abnormal in the pons of RA-treated juvenile mice. Thus, early developmental specifications in the rostral hindbrain are required for the development of neurones that stabilize the function of the respiratory rhythm generator, thereby preventing HEB during post-natal maturation.

Developmental gene control of brainstem function: views from the embryo.

The respiratory rhythm is generated within the hindbrain reticular formation, rostrally in the vicinity of the facial nucleus and caudally within the vagal/glossopharyngeal domain. This is probably one of the best models to understand how genes have been selected and conserved to control adaptive behaviour in vertebrates. The para-facial region is well understood with respect to the transcription factors that underlie antero-posterior specification of neural progenitors in the embryo. Hox paralogs and Hox-regulating genes kreisler and Krox-20 govern transient formation of developmental compartments, the rhombomeres, in which rhythmic neuronal networks develop. Hox are master genes selecting and coordinating the developmental fate of reticular and motor neurons thereby specifying patterns of motor activities operating throughout life. Neuronal function and development are also tightly linked in the vagal/glossopharyngeal domain. At this level, bdnf acts as a neurotrophin of peripheral chemoafferent neural populations and as a neuromodulator of the central rhythmogenic respiratory circuits. A general view is now emerging on the role of developmental transcription and trophic factors allowing the coordinated integration of different neuronal types to produce, and eventually refine, respiratory rhythmic pattern in a use-dependent manner.