Study of the implication of the Beta-3 adrenergic receptor in macrophages in inflammatory context

Introduction : Le cancer colorectal (CCR) est responsable de 500 000 morts par an dans le monde et représente la 2ème cause de mortalité par cancer dans les pays industrialisés. En dépit des progrès réalisés, il existe un réel besoin de développer de nouvelles thérapies pour améliorer la survie des patients. Un des principaux facteurs favorisant la survenue et la progression du CCR est le stress se traduisant notamment par la sécrétion de catécholamines activant les récepteurs β-adrénergiques (β1-, β2- et β3-AR) au niveau de la tumeur. Diverses études et observations ont démontré que l’activation des β-AR pouvait favoriser la prolifération tumorale de manière directe (via différents mécanismes comme la prolifération cellulaire) ou indirecte (via une action sur la composante immunitaire). Parmi les cellules immunitaires présentes au sein de la tumeur, les macrophages tumoraux (TAM) peuvent représenter jusqu’à 50% du volume tumoral. Ces derniers y sont retrouvés sous leurs différents phénotypes (M1-like antitumoral et M2-like pro-tumoral). Divers travaux ont fait état de la présence des β-AR à la surface des macrophages où leur effet semble être en faveur d’une polarisation vers un phénotype pro-tumoral. En outre, parmi les 3 sous-types de récepteurs, de nombreux arguments soulignent une implication majoritaire du β3-AR dans ces effets par rapport aux β1- et β2-AR, tandis que seule une surexpression du β3-AR a été observée dans les biopsies de tumeurs du côlon. Objectifs/Méthodes : Nous nous sommes donc attachés à mettre au point une stratégie méthodologique pour l’étude du β3-AR dans le macrophage en condition d’inflammation tissulaire présentant une importante composante macrophagique. Nous avons ensuite étudié les effets du β3-AR sur la prolifération de lignées de cancer colorectal puis sur la polarisation de macrophages humains ainsi que de TAM. Enfin, après avoir étudié la signalisation de ce récepteur chez les macrophages M1 et M2, nous avons observé les effets d’une inhibition pharmacologique du β3-AR sur la polarisation de TAM et sur la progression de tumeurs murines et humaines in vivo. Résultats : Nous avons confirmé que le β3-AR est présent et fonctionnel à la surface des macrophages où son activation résulte en un puissant effet antioxydant et anti-inflammatoire via l’inhibition de NOX2 et l’induction de l’expression de la catalase. Ces effets semblent passer par une signalisation Gs/PKA/Src/Erk1/2 induisant l’activation de PPARγ. Dans nos travaux, nous avons aussi pu voir que le β3-AR n’a pas d’effet prolifératif sur des lignées humaines de CCR. Nous avons également démontré que le β3-AR favorise la polarisation des macrophages vers un phénotype M2 et diminue la polarisation de ces derniers vers un phénotype M1. L’étude des signalisations de ce récepteur chez ces deux phénotypes a indiqué que les voies Gs/PKA/Src/ERK1/2 (M1) et Src/PI3K/ERK1/2 (M2) étaient impliquées. Enfin, l’inhibition du β3-AR a prévenu la progression de tumeurs murines (CT-26) et humaines (SW480) in vivo, via un effet anti-M2-like et pro-M1-like sur les TAM. En conclusion, ces résultats suggèrent que l’inhibition du β3-AR, à travers ses effets sur la polarisation des macrophages, pourrait être une stratégie prometteuse afin d’améliorer la prise en charge des patients souffrant de CCR.Background: Colorectal cancer (CRC) is responsible for 500.000 deaths per year worldwide and represents the 2nd cause of death by cancer in industrialized countries. Despite the progress made, there is a real need for new therapies to increase patients’ survival. Stress is one of the main factors, which contributes to the occurrence and the progression of CRC, by secreting catecholamines that activate β-adrenergic receptors (β1-, β2- and β3-AR) within the tumor. Several studies and observations have showed that the activation of β-ARs could directly increase tumor proliferation (via mechanisms such as cell proliferation), or indirectly (via an action on immune cells). Among immune cells within the tumor, tumor-associated macrophages (TAMs) represent up to 50% of the tumor mass where they exhibit their different phenotypes (M1-like anti-tumor and M2-like pro-tumor). Several studies report the presence of β-ARs in macrophages where they seem to favour a pro-tumor polarization. Furthermore, among the three subtypes of β-ARs, most of the studies seem to describe a major implication of the β3-AR compared to β1- and β2-AR. Moreover, only the β3-AR was found to be overexpressed in CRC biopsies. Objectives/Methods: We thus aimed to develop a model to study the β3-AR in macrophages within inflammatory macrophage-dependent conditions. Then, we studied the effects of the β3-AR on colorectal cancer cells’ proliferation and human macrophages and TAMs polarization. Finally, after the study of the signaling pathways of this receptor within M1 and M2 macrophages, we assessed the effects of a pharmacological inhibition of the β3-AR on TAM polarization and tumor progression. Results: We confirmed that the β3-AR is expressed and functional in human macrophages where its activation leads to potent antioxidant and anti-inflammatory effects through NOX2 inhibition and catalase expression. These effects appear to be subsequent to a Gs/PKA/Src/Erk1/2 signaling leading to the activation of PPARγ. In this work, we also saw that the β3-AR does not produce any effect on human CRC cell lines’ proliferation. We also showed that the β3-AR increases macrophage polarization towards the M2 phenotype while it decreases the M1 polarization. The study of β3-AR signaling in M1 and M2 macrophages exhibited Gs/PKA/Src/ERK1/2 and Src/PI3K/ERK1/2 pathways respectively. Finally, a pharmacological inhibition of the β3-AR prevented murine (CT-26) and human (SW480) tumors progression in vivo, through anti-M2-like and pro-M1-like effects on TAM polarization. In conclusion, these results suggest that the inhibition of the β3-AR, through its effects on macrophages polarization, could represent a promising strategy in order to improve CRC patient care

Beta3 adrenergic receptor stimulation in human macrophages inhibits NADPHoxidase activity and induces catalase expression via PPARγ activation

IF 4.521International audienceThe beta3 adrenergic receptor (β3-AR) stimulation plays a protective role against preterm labor by blocking myometrial contraction, cytokine production, remodeling and apoptosis. We previously demonstrated that macrophage-induced ROS production in the myometrium was a key element leading to the induction of all these labor-associated features. We thus aimed to investigate if the β3-AR could be expressed in human macrophages and could trigger its protective role in the myometrium by directly inhibiting ROS production. Using lipopolysaccharide (LPS)-stimulated myometrial samples and cell co-culture experiments, we demonstrated that β3-AR stimulation inhibits the activation of the NADPH oxidase, leading to the subsequent inhibition of ROS production by macrophages. This antioxidant effect was associated with a potent anti-inflammatory response in macrophages. Furthermore, we observed that β3-AR leads to the expression of catalase not only in macrophages but also in myometrial cells, thereby preventing the transactivation of myometrial cells by hydrogen peroxide. Pharmacological experiments allowed us to demonstrate that these effects were driven by an Erk1/2-mediated activation of the antioxidant transcription factor PPARγ. These results suggest that β3-AR protective effects in the myometrium could be due to its dual antioxidant properties. Further, the effects observed in a macrophage could highlight new applications in chronic inflammatory diseases

Lipoproteins LDL versus HDL as nanocarriers to target either cancer cells or macrophages

International audienceIn this work, we have explored natural unmodified low- and high-density lipoproteins (LDL and HDL) as selective delivery vectors in colorectal cancer therapy. We show in vitro in cultured cells and in vivo (NanoSPECT/CT) in the CT-26 mice colorectal cancer model that LDLs are mainly taken up by cancer cells, while HDLs are preferentially taken up by macrophages. We loaded LDLs with cisplatin and HDLs with the heat shock protein-70 inhibitor AC1LINNC, turning them into a pair of “Trojan horses” delivering drugs selectively to their target cells as demonstrated in vitro in human colorectal cancer cells and macrophages, and in vivo. Coupling of the drugs to lipoproteins and stability was assessed by mass and raman spectrometry analysis. Cisplatin vectorized in LDLs led to better tumor growth suppression with strongly reduced adverse effects such as a renal or liver toxicity. AC1LINNC vectorized into HDLs induced a strong oxidative burst in macrophages and innate anti-cancer immune response. Cumulative anti-tumor effect was observed for both drug-loaded lipoproteins. Altogether, our data show that lipoproteins from patient’s blood can be used as natural nanocarriers allowing cell specific targeting, paving the way toward more efficient, safer and personalized use of chemo-and immunotherapeutic drugs in cance

Selecting the first chemical molecule inhibitor of HSP110 for colorectal cancer therapy.

Pro-survival stress-inducible chaperone HSP110 is the only HSP for which a mutation has been found in a cancer. Multicenter clinical studies demonstrated a direct association between HSP110 inactivating mutation presence and excellent prognosis in colorectal cancer patients. Here, we have combined crystallographic studies on human HSP110 and in silico modeling to identify HSP110 inhibitors that could be used in colorectal cancer therapy. Two molecules (foldamers 33 and 52), binding to the same cleft of HSP110 nucleotide-binding domain, were selected from a chemical library (by co-immunoprecipitation, AlphaScreening, Interference-Biolayer, Duo-link). These molecules block HSP110 chaperone anti-aggregation activity and HSP110 association to its client protein STAT3, thereby inhibiting STAT3 phosphorylation and colorectal cancer cell growth. These effects were strongly decreased in HSP110 knockdown cells. Foldamer's 33 ability to inhibit tumor growth was confirmed in two colorectal cancer animal models. Although tumor cell death (apoptosis) was noted after treatment of the animals with foldamer 33, no apparent toxicity was observed, notably in epithelial cells from intestinal crypts. Taken together, we identified the first HSP110 inhibitor, a possible drug-candidate for colorectal cancer patients whose unfavorable outcome is associated to HSP110

Lactobacillus stress protein GroEL prevents colonic inflammation.

BACKGROUND: We previously showed that supernatants of Lactobacillus biofilms induced an anti-inflammatory response by affecting the secretion of macrophage-derived cytokines, which was abrogated upon immunodepletion of the stress protein GroEL. METHODS: We purified GroEL from L. reuteri and analysed its anti-inflammatory properties in vitro in human macrophages isolated from buffy coats, ex vivo in explants from human biopsies and in vivo in a mouse model of DSS induced intestinal inflammation. As a control, we used GroEL purified (LPS-free) from E. coli. RESULTS: We found that L. reuteri GroEL (but not E. coli GroEL) inhibited pro-inflammatory M1-like macrophages markers, and favored M2-like markers. Consequently, L. reuteri GroEL inhibited pro-inflammatory cytokines (TNFα, IL-1β, IFNγ) while favouring an anti-inflammatory secretome. In colon tissues from human biopsies, L. reuteri GroEL was also able to decrease markers of inflammation and apoptosis (caspase 3) induced by LPS. In mice, we found that rectal administration of L. reuteri GroEL (but not E. coli GroEL) inhibited all signs of haemorrhagic colitis induced by DSS including intestinal mucosa degradation, rectal bleeding and weight loss. It also decreased intestinal production of inflammatory cytokines (such as IFNγ) while increasing anti-inflammatory IL-10 and IL-13. These effects were suppressed when animals were immunodepleted in macrophages. From a mechanistic point of view, the effect of L. reuteri GroEL seemed to involve TLR4, since it was lost in TRL4(-/-) mice, and the activation of a non-canonical TLR4 pathway. CONCLUSIONS: L. reuteri GroEL, by affecting macrophage inflammatory features, deserves to be explored as an alternative to probiotics

HSP110 translocates to the nucleus upon genotoxic chemotherapy and promotes DNA repair in colorectal cancer cells

International audienceA multicenter clinical study demonstrated the presence of a loss-of-function HSP110 mutation in about 15% of colorectal cancers, which resulted from an alternative splicing and was produced at the detriment of wild-type HSP110. Patients expressing low levels of wild-type HSP110 had excellent outcomes (i.e. response to an oxaliplatin-based chemotherapy). Here, we show in vitro, in vivo, and in patients' biopsies that HSP110 co-localizes with DNA damage (γ-H2AX). In colorectal cancer cells, HSP110 translocates into the nucleus upon treatment with genotoxic chemotherapy such as oxaliplatin. Furthermore, we show that HSP110 interacts with the Ku70/Ku80 heterodimer, an essential element of the non-homologous end joining (NHEJ) repair machinery. We also demonstrate by evaluating the resolved 53BP1 foci that depletion in HSP110 impairs repair steps of the NHEJ pathway, which is associated with an increase in DNA double-strand breaks and in the cells' sensitivity to oxaliplatin. HSP110-depleted cells sensitization to oxaliplatin-induced DNA damage is abolished upon re-expression of HSP110. Confirming a role for HSP110 in DNA non-homologous repair, SCR7 and NU7026, two inhibitors of the NHEJ pathway, circumvents HSP110-induced resistance to chemotherapy. In conclusion, HSP110 through its interaction with the Ku70/80 heterodimer may participate in DNA repair, thereby inducing a protection against genotoxic therapy