Cannabinoid WIN55,212-2 reprograms monocytes and macrophages to inhibit LPS-induced inflammation.

Introduction: Chronic or uncontrolled activation of myeloid cells including monocytes, macrophages and dendritic cells (DCs) is a hallmark of immune-mediated inflammatory disorders. There is an urgent need for the development of novel drugs with the capacity to impair innate immune cell overactivation under inflammatory conditions. Compelling evidence pointed out cannabinoids as potential therapeutic tools with anti-inflammatory and immunomodulatory capacity. WIN55,212-2, a non-selective synthetic cannabinoid agonist, displays protective effects in several inflammatory conditions by mechanisms partially depending on the generation of tolerogenic DCs able to induce functional regulatory T cells (Tregs). However, its immunomodulatory capacity on other myeloid cells such as monocytes and macrophages remains incompletely understood. Methods: Human monocyte-derived DCs (hmoDCs) were differentiated in the absence (conventional hmoDCs) or presence of WIN55,212-2 (WIN-hmoDCs). Cells were stimulated with LPS, cocultured with naive T lymphocytes and their cytokine production and ability to induce T cell responses were analysed by ELISA or flow cytometry. To evaluate the effect of WIN55,212-2 in macrophage polarization, human and murine macrophages were activated with LPS or LPS/IFNγ, in the presence or absence of the cannabinoid. Cytokine, costimulatory molecules and inflammasome markers were assayed. Metabolic and chromatin immunoprecipitation assays were also performed. Finally, the protective capacity of WIN55,212-2 was studied in vivo in BALB/c mice after intraperitoneal injection with LPS. Results: We show for the first time that the differentiation of hmoDCs in the presence of WIN55,212-2 generates tolerogenic WIN-hmoDCs that are less responsive to LPS stimulation and able to prime Tregs. WIN55,212-2 also impairs the pro-inflammatory polarization of human macrophages by inhibiting cytokine production, inflammasome activation and rescuing macrophages from pyroptotic cell death. Mechanistically, WIN55,212-2 induced a metabolic and epigenetic shift in macrophages by decreasing LPS-induced mTORC1 signaling, commitment to glycolysis and active histone marks in pro-inflammatory cytokine promoters. We confirmed these data in ex vivo LPS-stimulated peritoneal macrophages (PMΦs), which were also supported by the in vivo anti-inflammatory capacity of WIN55,212-2 in a LPS-induced sepsis mouse model. Conclusion: Overall, we shed light into the molecular mechanisms by which cannabinoids exert anti-inflammatory properties in myeloid cells, which might well contribute to the future rational design of novel therapeutic strategies for inflammatory disorders.

Cannabinoid WIN55212-2 impairs peanut-allergic sensitization and promotes the generation of allergen-specific regulatory T cells.

BACKGROUND: Cannabinoids are lipid-derived mediators with anti-inflammatory properties in different diseases. WIN55212-2, a non-selective synthetic cannabinoid, reduces immediate anaphylactic reactions in a mouse model of peanut allergy, but its capacity to prevent peanut-allergic sensitization and the underlying mechanisms remains largely unknown. OBJECTIVE: To investigate the capacity of WIN55212-2 to immunomodulate peanut-stimulated human dendritic cells (DCs) and peanut-allergic sensitization in mice. METHODS: Surface markers and cytokines were quantified by flow cytometry, ELISA and qPCR in human monocyte-derived DCs (hmoDCs) and T-cell cocultures after stimulation with peanut alone or in the presence of WIN55212-2. Mice were epicutaneously sensitized with peanut alone or peanut/WIN55212-2. After peanut challenge, drop in body temperature, haematocrit, clinical symptoms, peanut-specific antibodies in serum and FOXP3+ regulatory (Treg) cells in spleen and lymph nodes were quantified. Splenocytes were stimulated in vitro with peanut to analyse allergen-specific T-cell responses. RESULTS: WIN55212-2 reduced peanut-induced hmoDC activation and promoted the generation of CD4+ CD127- CD25+ FOXP3+ Treg cells, while reducing the induction of IL-5-producing T cells. In vivo, WIN55212-2 impaired the peanut-induced migration of DCs to lymph nodes and their maturation. WIN55212-2 significantly reduced the induction of peanut-specific IgE and IgG1 antibodies in serum during epicutaneous peanut sensitization, reduced the clinical symptoms score upon peanut challenge and promoted the generation of allergen-specific FOXP3+ Treg cells. CONCLUSIONS: The synthetic cannabinoid WIN55212-2 interferes with peanut sensitization and promotes tolerogenic responses, which might well pave the way for the development of novel prophylactic and therapeutic strategies for peanut allergy.

The Gene Signature of Activated M-CSF-Primed Human Monocyte-Derived Macrophages Is IL-10-Dependent.

During inflammatory responses, monocytes are recruited into inflamed tissues, where they become monocyte-derived macrophages and acquire pro-inflammatory and tissue-damaging effects in response to the surrounding environment. In fact, monocyte-derived macrophage subsets are major pathogenic cells in inflammatory pathologies. Strikingly, the transcriptome of pathogenic monocyte-derived macrophage subsets resembles the gene profile of macrophage colony-stimulating factor (M-CSF)-primed monocyte-derived human macrophages (M-MØ). As M-MØ display a characteristic cytokine profile after activation (IL10high TNFlow IL23low IL6low), we sought to determine the transcriptional signature of M-MØ upon exposure to pathogenic stimuli. Activation of M-MØ led to the acquisition of a distinctive transcriptional profile characterized by the induction of a group of genes (Gene set 1) highly expressed by pathogenic monocyte-derived macrophages in COVID-19 and whose presence in tumor-associated macrophages (TAM) correlates with the expression of macrophage-specific markers (CD163, SPI1) and IL10. Indeed, Gene set 1 expression was primarily dependent on ERK/p38 and STAT3 activation, and transcriptional analysis and neutralization experiments revealed that IL-10 is not only required for the expression of a subset of genes within Gene set 1 but also significantly contributes to the idiosyncratic gene signature of activated M-MØ. Our results indicate that activation of M-CSF-dependent monocyte-derived macrophages induces a distinctive gene expression profile, which is partially dependent on IL-10, and identifies a gene set potentially helpful for macrophage-centered therapeutic strategies.

Cannabinoids induce functional Tregs by promoting tolerogenic DCs via autophagy and metabolic reprograming.

The generation of functional regulatory T cells (Tregs) is essential to keep tissue homeostasis and restore healthy immune responses in many biological and inflammatory contexts. Cannabinoids have been pointed out as potential therapeutic tools for several diseases. Dendritic cells (DCs) express the endocannabinoid system, including the cannabinoid receptors CB1 and CB2. However, how cannabinoids might regulate functional properties of DCs is not completely understood. We uncover that the triggering of cannabinoid receptors promote human tolerogenic DCs that are able to prime functional FOXP3+ Tregs in the context of different inflammatory diseases. Mechanistically, cannabinoids imprint tolerogenicity in human DCs by inhibiting NF-κB, MAPK and mTOR signalling pathways while inducing AMPK and functional autophagy flux via CB1- and PPARα-mediated activation, which drives metabolic rewiring towards increased mitochondrial activity and oxidative phosphorylation. Cannabinoids exhibit in vivo protective and anti-inflammatory effects in LPS-induced sepsis and also promote the generation of FOXP3+ Tregs. In addition, immediate anaphylactic reactions are decreased in peanut allergic mice and the generation of allergen-specific FOXP3+ Tregs are promoted, demonstrating that these immunomodulatory effects take place in both type 1- and type 2-mediated inflammatory diseases. Our findings might open new avenues for novel cannabinoid-based interventions in different inflammatory and immune-mediated diseases.

Allergoid-mannan conjugates reprogram monocytes into tolerogenic dendritic cells via epigenetic and metabolic rewiring.

BACKGROUND: Allergoid-mannan conjugates are novel vaccines for allergen-specific immunotherapy being currently assayed in phase 2 clinical trials. Allergoid-mannan conjugates target dendritic cells (DCs) and generate functional forkhead box P3 (FOXP3)-positive Treg cells, but their capacity to reprogram monocyte differentiation remains unknown. OBJECTIVE: We studied whether allergoid-mannan conjugates could reprogram monocyte differentiation into tolerogenic DCs and the underlying molecular mechanisms. METHODS: Monocytes from nonatopic and allergic subjects were differentiated into DCs under conventional protocols in the absence or presence of allergoid-mannan conjugates. ELISA, real-time quantitative PCR, coculture, flow cytometry, and suppression assay were performed. Metabolic and epigenetic techniques were also used. RESULTS: Monocyte differentiation from nonatopic and allergic subjects into DCs in the presence of allergoid-mannan conjugates yields stable tolerogenic DCs. Lipopolysaccharide-stimulated mannan-tolDCs show a significantly lower cytokine production, lower TNF-α/IL-10 ratio, and higher expression of the tolerogenic molecules PDL1, IDO, SOCS1, SOCS3, and IL10; and they induce higher numbers of functional FOXP3+ Treg cells than conventional DC counterparts. Mannan-tolDCs shift glucose metabolism from Warburg effect and lactate production to mitochondrial oxidative phosphorylation. They also display epigenetic reprogramming involving specific histone marks within tolerogenic loci and lower expression levels of histone deacetylase genes. Mannan-tolDCs significantly increase the expression of the anti-inflammatory miRNA-146a/b and decrease proinflammatory miRNA-155. CONCLUSIONS: Allergoid-mannan conjugates reprogram monocyte differentiation into stable tolerogenic DCs via epigenetic and metabolic reprogramming. Our findings shed light on the novel mechanisms by which allergoid-mannan conjugates might contribute to allergen tolerance induction during allergen-specific immunotherapy.

The Role of Cannabinoids in Allergic Diseases: Collegium Internationale Allergologicum (CIA) Update 2020.

The human endocannabinoid system (ECS) is a complex signalling network involved in many key physiological processes. The ECS includes the cannabinoid receptors, the endocannabinoid ligands, and the enzymes related to their synthesis and degradation. Other cannabinoids encompass the phytocannabinoids from Cannabis sativaL.(marijuana) and the synthetic cannabinoids. Alterations in the ECS are associated with different diseases, including inflammatory and immune-mediated disorders such as allergy. Allergy is a global health problem of increasing prevalence with high socio-economic impact. Different studies have convincingly demonstrated that cannabinoids play a role in allergy, but their actual contribution is still controversial. It has been shown that cannabinoids exert anti-inflammatory properties in the airways and the skin of allergic patients. Other studies reported that cannabinoids might exacerbate asthma and atopic dermatitis mainly depending on CB2-mediated signalling pathways. A better understanding of the molecular mechanisms involved in the mode of action of specific cannabinoids and cannabinoid receptors on relevant immune cells under different biological contexts might well contribute to the design of novel strategies for the prevention and treatment of allergic diseases. Future research in this promising emerging field in the context of allergy is warranted for the upcoming years.

Alum impairs tolerogenic properties induced by allergoid-mannan conjugates inhibiting mTOR and metabolic reprogramming in human DCs.

BACKGROUND: Polymerized allergoids conjugated to mannan (PM) are suitable vaccines for allergen-specific immunotherapy (AIT). Alum remains the most widely used adjuvant in AIT, but its way of action is not completely elucidated. The better understanding of the mechanisms underlying alum adjuvanticity could help to improve AIT vaccine formulations. OBJECTIVE: We sought to investigate the potential influence of alum in the tolerogenic properties imprinted by PM at the molecular level. METHODS: Flow cytometry, ELISAs, cocultures, intracellular staining and suppression assays were performed to assess alum and PM effects in human dendritic cells (DCs). BALB/c mice were immunized with PM alone or adsorbed to alum. Allergen-specific antibodies, splenocyte cytokine production and splenic forkhead box P3 (FOXP3)+ regulatory T (Treg) cells were quantified. Metabolic and immune pathways were also studied in human DCs. RESULTS: Alum decreases PD-L1 expression and IL-10 production induced by PM in human DCs and increases pro-inflammatory cytokine production. Alum impairs PM-induced functional FOXP3+ Treg cells and promotes Th1/Th2/Th17 responses. Subcutaneous immunization of mice with PM plus alum inhibits in vivo induction of Treg cells promoted by PM without altering the capacity to induce functional allergen-specific blocking antibodies. Alum inhibits mTOR activation and alters metabolic reprogramming by shifting glycolytic pathways and inhibiting reactive oxygen species (ROS) production in PM-activated DCs, impairing their capacity to generate functional Treg cells. CONCLUSION: We uncover novel mechanisms by which alum impairs the tolerogenic properties induced by PM, which might well contribute to improve the formulation of novel vaccines for AIT.