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

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

From trained immunity in allergy to trained immunity‐based allergen vaccines.

Innate immune cells experience long lasting metabolic and epigenetic changes after an encounter with specific stimuli. This facilitates enhanced immune responses upon secondary exposition to both the same and unrelated pathogens, a process termed trained immunity. Trained immunity- based vaccines (TIbV) are vaccines able to induce innate immune memory, thus conferring heterologous protection against a broad range of pathogens. While trained immunity has been well documented in the con-text of infections and multiple immune- mediated diseases, the role of innate immune memory and its contribution to the initiation and maintenance of chronic allergic dis-eases remains poorly understood. Over the last years, different studies attempting to uncover the role of trained immunity in allergy have emerged. Exposition to en-vironmental factors impacting allergy development such as allergens or viruses in-duces the reprogramming of innate immune cells to acquire a more pro-inflammatory phenotype in the context of asthma or food allergy. Several studies have convincingly demonstrated that prevention of viral infections using TIbV contributes to reduce wheezing attacks in children, which represent a high- risk factor for asthma develop-ment later in life. Innate immune cells trained with specific stimuli might also acquire anti- inflammatory features and promote tolerance, which may have important impli-cations for chronic inflammatory diseases such as allergies. Recent findings showed that allergoid- mannan conjugates, which are next generation vaccines for allergen- specific immunotherapy (AIT), are able to reprogram monocytes into tolerogenic den-dritic cells by mechanisms depending on metabolic and epigenetic rewiring. A better understanding of the underlying mechanisms of trained immunity in allergy will pave the way for the design of novel trained immunity- based allergen vaccines as potential alternative strategies for the prevention and treatment of allergic diseases

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 prop-erties in different diseases. WIN55212-2, a non-selective synthetic cannabinoid, re-duces immediate anaphylactic reactions in a mouse model of peanut allergy, but its capacity to prevent peanut-allergic sensitization and the underlying mechanisms re-mains 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 epicuta-neously 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 quanti-fied. 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 sensi-tization and promotes tolerogenic responses, which might well pave the way for the development of novel prophylactic and therapeutic strategies for peanut allergy

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

The cannabinoid WIN55212-2 restores rhinovirus-induced epithelial barrier disruption

Carta al Editor. Received: 19 September 2020 | Revised: 20 November 2020 | Accepted: 5 December 2020Sección Deptal. de Química Orgánica (Óptica y Optometría)Fac. de Óptica y OptometríaTRUEMinisterio de Economía y Competitividad de España (MINECO)Swiss National Science FoundationChristine Kühne‐Center for Allergy Research and Education (CK‐CARE) (Suiza)Universidad Complutense de Madrid (España)inpres

Cannabinoides e inmunomodulación: nuevas estrategias terapéuticas dirigidas frente a células dendríticas para enfermedades inflamatorias

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular, leída el 21/01/2021Dendritic cells (DCs) are professional antigen presenting cells that play a key role in the orchestration of immune responses by linking innate and adaptive immunity. The main function of DCs is to capture antigens in the periphery and transport them to the closer lymph nodes. There, they present the processed antigens to naïve T cells, inducing T cell activation and polarization into different phenotypes depending on the antigen, the environmental cues, costimulatory signals and cytokines. Changes in the energetic metabolism of DCs are essential for the regulation of their functional properties. At this regard, several studies have reported the involvement of autophagy, an essential catabolic process, in the control of DC functions. The endocannabinoid system (ECS) is a complex signalling network involved in many physiological processes such as neuronal development, brain plasticity, memory, cell survival, metabolism and immunity. The main components of the ECS include the endocannabinoid ligands, the cannabinoid receptors (CBRs) and the enzymes related to the ligand synthesis or degradation. Several studies showed that cannabinoid compounds display anti-inflammatory properties in different immune-mediated diseases. However, their potential capacity to regulate the orchestration of immune responses is not well understood, specifically in humans. Human DCs express all the components of the ECS. The ability of cannabinoids to immunomodulate the phenotype and function of DCs remain elusive. Considering all these aspects, the main objective of this Doctoral Thesis is to investigate the role and the underlying molecular mechanisms of synthetic cannabinoids in the control of human DC function and their potential implications in preclinical in vitro and in vivo models of immune-mediated diseases as potential novel strategies for the prevention and treatment of inflammatory disorders. In this work, we have developed and validated a novel HU210-Alexa488 fluorescence probe to visualize and quantify CB1-expressing cells in peripheral blood and tonsils by flow cytometry and confocal microscopy. We have also demonstrated for the first time that the synthetic cannabinoid WIN55212-2 promotes the generation of tolerogenic DCs with the capacity to generate functional FOXP3+ regulatory T (Treg) cells by mechanisms depending on CB1- and PPARα-mediated autophagy induction. We have validated the potential relevance of our findings in different preclinical human and mouse models. The synthetic cannabinoid WIN55212-2 exhibits in vivo protective and anti-inflammatory effects in LPS-induced sepsis model by mechanisms that rely on autophagy induction via activation of CB1 and PPARα. In a mouse model of peanut food allergy, the administration of WIN55212-2 during peanut challenge protects peanut-allergic mice from the immediate anaphylactic reaction. Moreover, WIN55212-2 administration promotes the generation of allergen-specific FOXP3+ regulatory T cells during late-phase responses. Finally, we have demonstrated that WIN55212-2 repairs rhinovirus-induced airway epithelial disruption by restoring the integrity of tight junction proteins without affecting epithelial cell-mediated innate immune response. In summary, in this Doctoral Thesis we uncover the previously unknown capacity of the synthetic cannabinoid WIN55212-2 to immunomodulate the phenotype and function of human DCs. We show for the first time the molecular mechanisms by which WIN55212-2 imprints tolerogenic features in human DCs, which include CB1- and PPARα-mediated autophagy induction and metabolic reprogramming. In addition, we have demonstrated the potential clinical applications of WIN55212-2 in different in vitro and in vivo preclinical human and mouse models such as sepsis, peanut allergy or rhinovirus-induced inflammation. The data presented in this Doctoral Thesis might well contribute to pave the way for the future development of novel cannabinoid-based strategies for the prevention and treatment of immune-mediated diseases.Las células dendríticas (DCs) son células presentadoras de antígeno profesionales que tienen un papel clave en la inducción de las respuestas inmunes conectando el sistema inmune innato con el adaptativo. La principal función de las DCs es reconocer antígenos en la periferia y transportarlos a los órganos linfoides más próximos. Allí, las DCs presentan los antígenos procesados a las células T vírgenes, induciendo su activación y diferenciación hacia distintos fenotipos dependiendo del antígeno, de las señales encontradas en el ambiente, de las señales co-estimuladoras y de las citoquinas. Cambios en el metabolismo energético de las DCs son esenciales en la regulación de su activación y función. Varios estudios han demostrado la implicación de la autofagia, un proceso catabólico esencial para las células, en el control de la función de las DCs. El sistema endocannabinoide (ECS) es una red compleja de señalización implicada en varios procesos fisiológicos como desarrollo neuronal, plasticidad cerebral, memoria, supervivencia celular, metabolismo e inmunidad. Los principales componentes del ECS son los ligandos endocannabinoides, los receptores de cannabinoides (CBRs) y las enzimas implicadas en la síntesis y degradación de los ligandos. Varios estudios han mostrado que los compuestos cannabinoides presentan propiedades anti-inflamatorias en diferentes enfermedades mediadas por el sistema inmune. Sin embargo, su capacidad para regular las respuestas inmunes no se conoce completamente, especialmente en humanos. Las DCs humanas expresan todos los componentes del ECS. La habilidad de los cannabinoides de inmunomodular el fenotipo y la función de las DCs se desconoce. Teniendo en cuenta estos aspectos, el objetivo de esta Tesis Doctoral es investigar el papel de los cannabinoides sintéticos en la regulación de la función de las DCs humanas, los mecanismos subyacentes y sus posibles implicaciones en modelos preclínicos in vitro e in vivo de enfermedades mediadas por el sistema inmune como posibles nuevas estrategias terapéuticas para la prevención y tratamiento de trastornos inflamatorios. En este trabajo hemos desarrollado y validado la sonda fluorescente HU210-Alexa488 para visualizar y cuantificar células que expresan CB1 en sangre periférica y amígdalas mediante citometría de flujo y microscopía confocal. Además, hemos demostrado por primera vez que el cannabinoide sintético WIN55212-2 induce DCs tolerogénicas con capacidad de generar células T reguladoras FOXP3+ funcionales mediante mecanismos dependientes de la inducción de autofagia vía CB1 y PPARα. La posible relevancia clínica de nuestros resultados fue estudiada en diferentes modelos preclinicos humanos y múridos tanto in vivo como in vitro. El cannabinoide sintético WIN55212-2 ejerce efectos protectores y anti-inflamatorios in vivo en un modelo de sepsis inducida por LPS mediante mecanismos que dependen de la inducción de autofagia vía activación de CB1 y PPARα. En un modelo de alergia a cacahuete, la administración de WIN55212-2 durante la provocación con cacahuete protege a los ratones alérgicos de la reacción anafiláctica inmediata. Además, la administración de WIN55212-2 induce la generación de células T reguladoras FOXP3+ específicas de alérgeno en la fase efectora tardía. Finalmente, WIN55212-2 repara la alteración del epitelio de las vías respiratorias inducida por rinovirus restaurando la integridad de las proteínas de las uniones estrechas sin afectar a la respuesta inmune innata inducida por las células epiteliales. En resumen, en esta Tesis Doctoral demostramos la capacidad del cannabinoide sintético WIN55212-2 de inmunomodular el fenotipo y la función de las DCs humanas. Hemos demostrado por primera vez los mecanismos moleculares mediante los cuales WIN55212-2 induce propiedades tolerogénicas en DCs humanas, que incluyen la inducción de autofagia mediante la activación de CB1 y PPARα y reprogramación metabólica. Además, hemos demostrado potenciales aplicaciones clínicas de WIN55212-2 en diferentes modelos preclínicos humanos y de ratón como sepsis, alergia al cacahuete o inflamación inducida por rinovirus. Los resultados presentados en esta Tesis Doctoral podrían sentar las bases que contribuyan en el desarrollo de futuras estrategias basadas en cannabinoides para la prevención y el tratamiento de enfermedades mediadas por el sistema inmune.Fac. de Ciencias QuímicasTRUEunpu

C-Type Lectin Receptor Mediated Modulation of T2 Immune Responses to Allergens

Purpose of Review: Allergic diseases represent a major health problem of increasing prevalence worldwide. In allergy, dendritic cells (DCs) contribute to both the pathophysiology and the induction of healthy immune responses to the allergens. Different studies have reported that some common allergens contain glycans in their structure. C-type lectin receptors (CLRs) expressed by DCs recognize carbohydrate structures and are crucial in allergen uptake, presentation, and polarization of T cell responses. This review summarizes the recent literature regarding the role of CLRs in the regulation of type 2 immune responses to allergens. Recent Findings: In this review, we highlight the capacity of CLRs to recognize carbohydrates in common allergens triggering different signaling pathways involved in the polarization of CD4+ T cells towards specific Th2 responses. Under certain conditions, specific CLRs could also promote tolerogenic responses to allergens, which might well be exploited to develop novel therapeutic approaches of allergen-specific immunotherapy (AIT), the single treatment with potential disease-modifying capacity for allergic disease. At this regard, polymerized allergens conjugated to non-oxidized mannan (allergoid-mannan conjugated) are next-generation vaccines targeting DCs via CLRs that promote regulatory T cells, thus favoring allergen tolerance both in preclinical models and clinical trials. Summary: A better understanding of the role of CLRs in the development of allergy and in the induction of allergen tolerance might well pave the way for the design of novel strategies for allergic diseases.MICINNDepto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu

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

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.MINECODepto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu

Candida albicans V132 induces trained immunity and enhances the responses triggered by the polybacterial vaccine MV140 for genitourinary tract infections

Introduction: Recurrent urinary tract infections (RUTIs) and recurrent vulvovaginal candidiasis (RVVCs) represent major healthcare problems all over the world. Antibiotics and antifungals are widely used for such infectious diseases, which is linked with microbial resistances and microbiota deleterious effects. The development of novel approaches for genitourinary tract infections (GUTIs) such as trained immunity-based vaccines (TIbV) is therefore highly required. MV140 is a sublingual whole-cell heat-inactivated polybacterial preparation with demonstrated clinical efficacy for RUTIs. The sublingual heat-inactivated Candida albicans vaccine V132 has been developed for RVVCs. We previously showed that the combination of MV140 and V132 promotes potent Th1/Th17 and regulatory T-cell responses against antigens contained in the formulation and unrelated antigens. The specific contribution of each preparation to such effects and the underlying molecular mechanisms remain incompletely understood. Methods: PBMC and monocytes were isolated from healthy donors and in vitro stimulated with V132, MV140 or MV140/V132. After 6 days of resting, cells were reestimulated with LPS and MV140. Analysis of cytokine production by ELISA, Seahorse assays for functional metabolic experiments and chromatin immunoprecipitation assays were performed. BALB/c mice were intraperitoneally and sublingually immunized with V132. Results: We uncover that V132 induces trained immunity in human PBMCs and purified monocytes, significantly increasing the responses triggered by purified monocytes, significantly increasing the responses triggered by subsequent stimulation with MV140. Mechanistically, V132 drives metabolic rewiring towards increased glycolysis and oxidative phosphorylation and induces epigenetic reprogramming that enhances the transcription of the pro-inflammatory genes IL6 and TNFA. Splenocytes and peritoneal cells from V132-immunize mice show increased responses upon in vitro stimulation with MV140. Remarkably, splenocytes from sublingually V132-immunized and MV140 in vivo treatment mice show stronger Th17 responses than mice exposed to excipients upon in vitro stimulation with MV140. Conclusion: Overall, we provide novel mechanistic insights into how V132- induced trained immunity enhances both innate and adaptive immune responses triggered by MV140, which might open the door for new interventions for GUTIs with important clinical implications.MINECOThe Netherlands Organization for Scientific ResearchNetherlands Organization for Scientific ResearchERC Advanced GrantDepto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu