The Role of Regulatory T Cells in Allergic Diseases: Collegium Internationale Allergologicum (CIA) Update 2024.

BACKGROUND: Allergy represents a major health problem of increasing prevalence worldwide with a high socioeconomic impact. Our knowledge on the molecular mechanisms underlying allergic diseases and their treatments has significantly improved over the last years. The generation of allergen-specific regulatory T cells (Tregs) is crucial in the induction of healthy immune responses to allergens, preventing the development and worsening of allergic diseases. SUMMARY: In the last decades, intensive research has focused on the study of the molecular mechanisms involved in Treg development and Treg-mediated suppression. These mechanisms are essential for the induction of sustained tolerance by allergen-specific immunotherapy (AIT) after treatment discontinuation. Compelling experimental evidence demonstrated altered suppressive capacity of Tregs in patients suffering from allergic rhinitis, allergic asthma, food allergy, or atopic dermatitis, as well as the restoration of their numbers and functionality after successful AIT. KEY MESSAGE: The better understanding of the molecular mechanisms involved in Treg generation during allergen tolerance induction might well contribute to the development of novel strategies for the prevention and treatment of allergic diseases.

A tumor-associated heparan sulfate-related glycosaminoglycan promotes the generation of functional regulatory T cells.

Functional Tregs play a key role in tumor development and progression, representing a major barrier to anticancer immunity. The mechanisms by which Tregs are generated in cancer and the influence of the tumor microenvironment on these processes remain incompletely understood. Herein, by using NMR, chemoenzymatic structural assays and a plethora of in vitro and in vivo functional analyses, we demonstrate that the tumoral carbohydrate A10 (Ca10), a cell-surface carbohydrate derived from Ehrlich's tumor (ET) cells, is a heparan sulfate-related proteoglycan that enhances glycolysis and promotes the development of tolerogenic features in human DCs. Ca10-stimulated human DCs generate highly suppressive Tregs by mechanisms partially dependent on metabolic reprogramming, PD-L1, IL-10, and IDO. Ca10 also reprograms the differentiation of human monocytes into DCs with tolerogenic features. In solid ET-bearing mice, we found positive correlations between Ca10 serum levels, tumor size and splenic Treg numbers. Administration of isolated Ca10 also increases the proportion of splenic Tregs in tumor-free mice. Remarkably, we provide evidence supporting the presence of a circulating human Ca10 counterpart (Ca10H) and show, for the first time, that serum levels of Ca10H are increased in patients suffering from different cancer types compared to healthy individuals. Of note, these levels are higher in prostate cancer patients with bone metastases than in prostate cancer patients without metastases. Collectively, we reveal novel molecular mechanisms by which heparan sulfate-related structures associated with tumor cells promote the generation of functional Tregs in cancer. The discovery of this novel structural-functional relationship may open new avenues of research with important clinical implications in cancer treatment.

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.

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. 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.

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 context of infections and multiple immune-mediated diseases, the role of innate immune memory and its contribution to the initiation and maintenance of chronic allergic diseases remains poorly understood. Over the last years, different studies attempting to uncover the role of trained immunity in allergy have emerged. Exposition to environmental factors impacting allergy development such as allergens or viruses induces 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 development later in life. Innate immune cells trained with specific stimuli might also acquire anti-inflammatory features and promote tolerance, which may have important implications 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 dendritic 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.

Mechanisms in AIT: Insights 2021.

BACKGROUND: Allergen-specific immunotherapy (AIT) is currently the only treatment with potential long-term disease-modifying effects for patients suffering from allergic diseases such as allergic rhinitis, allergic asthma, venom allergy, or IgE-mediated food allergy. A better understanding of the molecular mechanisms underlying immune responses during successful AIT is of utmost importance and it may help to develop more effective and safer treatments. MATERIALS AND METHODS: PubMed literature review was performed using keywords such as allergen-specific immunotherapy; regulatory T cells; regulatory B cells; regulatory innate lymphoid cells; and allergen-specific antibody from years 2018 to 2021. RESULTS: The proposed mechanism of long-term tolerance induction in AIT, even upon treatment discontinuation, involves basophils, mast cells, innate lymphoid cells, dendritic cells, allergen-specific regulatory T and B cells, downregulation of effector type 2 responses, decrease in the production of IgE and increase in production of allergen-specific blocking antibodies, such as IgG2 and IgG4. CONCLUSION: We summarize the most recent advances related to mechanisms involved in the restoration of healthy immune responses to allergens during AIT. Our knowledge in this regard has significantly improved over the last years, which might well contribute to design novel and improved therapeutic approaches.

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 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.

Effects of non-steroidal anti-inflammatory drugs and other eicosanoid pathway modifiers on antiviral and allergic responses: EAACI task force on eicosanoids consensus report in times of COVID-19.

Non-steroidal anti-inflammatory drugs (NSAIDs) and other eicosanoid pathway modifiers are among the most ubiquitously used medications in the general population. Their broad anti-inflammatory, antipyretic, and analgesic effects are applied against symptoms of respiratory infections, including SARS-CoV-2, as well as in other acute and chronic inflammatory diseases that often coexist with allergy and asthma. However, the current pandemic of COVID-19 also revealed the gaps in our understanding of their mechanism of action, selectivity, and interactions not only during viral infections and inflammation, but also in asthma exacerbations, uncontrolled allergic inflammation, and NSAIDs-exacerbated respiratory disease (NERD). In this context, the consensus report summarizes currently available knowledge, novel discoveries, and controversies regarding the use of NSAIDs in COVID-19, and the role of NSAIDs in asthma and viral asthma exacerbations. We also describe here novel mechanisms of action of leukotriene receptor antagonists (LTRAs), outline how to predict responses to LTRA therapy and discuss a potential role of LTRA therapy in COVID-19 treatment. Moreover, we discuss interactions of novel T2 biologicals and other eicosanoid pathway modifiers on the horizon, such as prostaglandin D2 antagonists and cannabinoids, with eicosanoid pathways, in context of viral infections and exacerbations of asthma and allergic diseases. Finally, we identify and summarize the major knowledge gaps and unmet needs in current eicosanoid research.

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.

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.

Current perspective on eicosanoids in asthma and allergic diseases: EAACI Task Force consensus report, part I.

Eicosanoids are biologically active lipid mediators, comprising prostaglandins, leukotrienes, thromboxanes, and lipoxins, involved in several pathophysiological processes relevant to asthma, allergies, and allied diseases. Prostaglandins and leukotrienes are the most studied eicosanoids and established inducers of airway pathophysiology including bronchoconstriction and airway inflammation. Drugs inhibiting the synthesis of lipid mediators or their effects, such as leukotriene synthesis inhibitors, leukotriene receptors antagonists, and more recently prostaglandin D2 receptor antagonists, have been shown to modulate features of asthma and allergic diseases. This review, produced by an European Academy of Allergy and Clinical Immunology (EAACI) task force, highlights our current understanding of eicosanoid biology and its role in mediating human pathology, with a focus on new findings relevant for clinical practice, development of novel therapeutics, and future research opportunities.

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.

Ascaris lumbricoides Cystatin Prevents Development of Allergic Airway Inflammation in a Mouse Model.

Severe helminth infections are negatively associated to allergic diseases like asthma; therefore, the immunomodulatory properties of parasite-derived components have been analyzed, raising the possibility of their use as anti-inflammatory molecules. We evaluated the immunomodulatory properties of Ascaris lumbricoides recombinant cysteine protease inhibitor (rAl-CPI) in a mouse model of allergic airway inflammation induced by the house dust mite (HDM) Blomia tropicalis and its effects on human monocyte-derived dendritic cells (HmoDCs). The B. tropicalis sensitized/challenged mice developed extensive cellular airway inflammatory response, which was significantly reduced upon treatment with rAl-CPI prior to B. tropicalis sensitization, affecting particularly the perivascular/peribronchial infiltrate cells, eosinophils/neutrophils, and goblet cells. A significant decrease of Th2 cytokines, total, and specific IgE antibodies was observed in rAl-CPI treated mice. The antibody response was biased to IgG, mainly IgG2a. Administration of rAl-CPI-alone and rAl-CPI before mite sensitization were associated with a significant increase of regulatory T cells (Tregs) in spleen and elevated IL-10 levels in BAL and splenocytes culture supernatants, which was partially affected by anti-IL10 receptor use. In vitro, rAl-CPI showed a modulatory effect on HmoDCs, lowering the expression of HLA-DR, CD83, and CD86, while inducing IL-10 and IL-6 production. This suggests an inhibition of HmoDC maturation and a possible link with the inhibition of the allergic response observed in the murine model.

Development of a Fluorescent Bodipy Probe for Visualization of the Serotonin 5-HT1A Receptor in Native Cells of the Immune System.

Serotonin (5-HT) modulates key aspects of the immune system. However, its precise function and the receptors involved in the observed effects have remained elusive. Among the different serotonin receptors, 5-HT1A plays an important role in the immune system given its presence in cells involved in both the innate and adaptive immune responses, but its actual levels of expression under different conditions have not been comprehensively studied due to the lack of suitable tools. To further clarify the role of 5-HT1A receptor in the immune system, we have developed a fluorescent small molecule probe that enables the direct study of the receptor levels in native cells. This probe allows direct profiling of the receptor expression in immune cells using flow cytometry. Our results show that important subsets of immune cells including human monocytes and dendritic cells express functional 5-HT1A and that its activation is associated with anti-inflammatory signaling. Furthermore, application of the probe to the experimental autoimmune encephalomyelitis model of multiple sclerosis demonstrates its potential to detect the specific overexpression of the 5-HT1A receptor in CD4+ T cells. Accordingly, the probe reported herein represents a useful tool whose use can be extended to study the levels of 5-HT1A receptor in ex vivo samples of different immune system conditions.

A Fluorescent Probe to Unravel Functional Features of Cannabinoid Receptor CB1 in Human Blood and Tonsil Immune System Cells.

The human endogenous cannabinoid system (ECS) regulates key physiological processes and alterations in its signaling pathways, and endocannabinoid levels are associated with diseases such as neurological and neuropsychiatric conditions, cancer, pain and inflammation, obesity, and metabolic and different immune related disorders. Immune system cells express the G-protein coupled cannabinoid receptor 1 (CB1), but its functional role has not been fully understood, likely due to the lack of appropriate tools. The availability of novel tools to investigate the role of CB1 in immune regulation might contribute to identify CB1 as a potential novel therapeutic target or biomarker for many diseases. Herein, we report the development and validation of the first fluorescent small molecule probe to directly visualize and quantify CB1 in blood and tonsil immune cells by flow cytometry and confocal microscopy. We coupled the cannabinoid agonist HU210 to the fluorescent tag Alexa Fluor 488, generating a fluorescent probe with high affinity for CB1 and selectivity over CB2. We validate HU210-Alexa488 for the rapid, simultaneous, and reproducible identification of CB1 in human monocytes, T cells, and B cells by multiplexed flow cytometry. This probe is also suitable for the direct visualization of CB1 in tonsil tissues, allowing the in vivo identification of tonsil CB1-expressing T and B cells. This study provides the first fluorescent chemical tool to investigate CB1 expression and function in human blood and tonsil immune cells, which might well pave the way to unravel essential features of CB1 in different immune and ECS-related diseases.