Nickel allergy remains the most prevalent cause of allergic contact dermatitis worldwide, imposing a substantial socio-epidemiological and economic burden. Beyond its classical cutaneous presentation, systemic nickel allergy syndrome highlights the systemic dimension of Nickel hypersensitivity, wherein dietary nickel intake may provoke both gastrointestinal and cutaneous symptoms through mechanisms involving gut barrier impairment and mucosal immune priming. Recent evidence highlights the contribution of angiogenesis and lymph-angiogenesis to Nickel-induced allergic contact dermatitis, through crosstalk among keratinocytes, mast cells, endothelial cells, and pro-angiogenic mediators such as vascular endothelial growth factor. Against this background, we propose to revisit palmitoylethanolamide, an endogenous ALIAmide with well-documented anti-inflammatory, anti-angiogenic, and anti-allergic properties. Already studied in pain and inflammatory disorders and employed in veterinary dermatology, palmitoylethanolamide down-modulates mast cell degranulation, suppresses VEGF expression via PPAR-α/Akt/mTOR signaling, and enhances intestinal barrier integrity, acting as a promising "gatekeeper" molecule that reduces gut hyperpermeability characterizing systemic nickel allergy as well as other gut disorders with systemic consequences. This paper is presented as a viewpoint intended to highlight the untapped therapeutic potential of palmitoylethanolamide, suitable for both oral and topical administration, as a candidate to address the multifactorial pathophysiology of Nickel allergic contact dermatitis and systemic nickel allergy. Our purpose is not to provide definitive answers, but to stimulate scientific debate on its rational use within emerging gut-skin therapeutic strategies. We thus encourage future experimental and clinical studies to explore its potential integration within emerging gut-skin therapeutic paradigms.

Since its 1987 approval, rifaximin has emerged as a gut-targeted, minimally absorbed antibiotic achieving high intestinal levels. Rifaximin works by blocking bacterial RNA polymerase to stop bacterial growth, which helps treat both traveler’s diarrhea and irritable bowel syndrome with diarrhea. It effectively modulates gut microbiota and alleviates dysbiosis-related symptoms. Beyond antimicrobial activity, rifaximin modulates dysbiosis, strengthens the epithelial barrier, and exerts pregnane X receptor (PXR)-mediated anti-inflammatory and anti-angiogenic effects, with implications along the gut–liver and gut–brain axes. These pleiotropic properties support expanding uses in small intestinal bacterial overgrowth (SIBO), inflammatory bowel diseases (IBD), functional dyspepsia, bloating, and lactose intolerance. However, high cost and potential resistance with prolonged exposure highlight the need for antimicrobial stewardship. The aims of this review are to summarize licensed and emerging indications and delineate non-conventional mechanisms. Patient stratification and further controlled clinical studies are required to optimize rifaximin’s precision-medicine potential.

Colitis-associated colorectal cancer (CAC) arises from the interplay between chronic inflammation, epithelial dysregulation, angiogenesis, and microbiota alterations. Palmitoylethanolamide (PEA) is an endogenous lipid mediator with anti-inflammatory and anti-angiogenic properties, but despite its promise, insufficient bioavailability at the intestinal mucosa represents a major hurdle. We investigated whether local, sustained PEA delivery via an engineered Lactobacillus paracasei expressing N-acyl-phosphatidylethanolamine-specific phospholipase D (pNAPE-LP) could prevent CAC by targeting inflammatory, angiogenic, and microbial pathways. pNAPE-LP/palmitate markedly reduced mucosal damage and tumor burden compared with the AOM/DSS group, while naïve pLP and palmitate alone were ineffective. Treatment suppressed epithelial proliferation and angiogenesis, restored p53-wt expression, and significantly increased colonic PEA levels, confirming effective in situ biosynthesis. Mechanistically, pNAPE-LP inhibited activation of the Akt/mTOR/p70S6K pathway and reduced HIF-1α expression. Microbiota profiling revealed profound tumor-associated dysbiosis characterized by the collapse of dominant commensalsand expansion of pathobionts in the AOM/DSS group. pNAPE-LP induced a robust ecological remodeling, shifting beta-diversity toward healthy controls, restoring key genera associated with mucosal homeostasis, and suppressing taxa overrepresented in the tumor state. These effects were strictly dependent on PEA release following substrate administration, enabled by NAPE-PLD expression. Engineered pNAPE-LP acts as a living biotherapeutic that locally delivers PEA while simultaneously reprogramming inflammation, angiogenesis, oncogenic signaling, and microbiota composition. This multimodal mechanism effectively interrupts the pathogenic feedback loops driving CAC. Engineered PEA-producing probiotics represent a promising strategy for mucosa-targeted prevention of inflammation-associated colorectal cancer.

This chapter provides a detailed guide for isolating and dissecting enteric glial cells (EGCs) from human mucosal biopsies. EGCs play crucial roles in gut homeostasis and neuroimmune communication, making their isolation a valuable endeavor in neurogastroenterology research. The method outlined here aims to enable competent scientists, even those unfamiliar with the technique, to successfully isolate EGCs at their first attempt. It outlines a systematic approach for the collection, digestion, mechanical dissection, and purification of cells from human mucosal biopsies, primarily focused on gastrointestinal tissue.During the first step, tissue collection, the collaboration with clinical teams ensures timely acquisition of biopsies with minimal trauma. Biopsies are then promptly transferred to a cold solution to maintain viability during transport. The subsequent enzymatic digestion involves immersion of biopsies in a solution containing collagenase and Dispase II, optimizing conditions for maximal cell yield. Gentle agitation aids enzyme penetration without causing cellular stress. Mechanical dissection follows, where tissues are minced into uniform fragments using sterile instruments, maintaining sterile conditions to prevent contamination. Gentle trituration further disperses cells without introducing air bubbles. Finally, cell purification involves filtration to remove debris, followed by centrifugation to pellet cells. Careful resuspension in an appropriate medium ensures viability for downstream applications. Overall, this method offers a meticulous approach to isolate viable cells from human mucosal biopsies for further analysis.

Metabolic and mood disorders elicited by chronic exposure of high-fat diet (HFD) are often associated with intestinal dysbiosis and persistent low-grade inflammation in the small intestine. This leads to remodeling of the epithelial barrier with disruption of the neuroepithelial circuits that control energy homeostasis by the gut-brain axis. Therefore, therapies that restoreintestinal microbial niche and barrier function are promising candidates to counter peripheral metabolic challenges that affect behaviors controlled by the brain. The endogenous oleoylethanolamine (OEA) was found to shape the intestinal microbiota profile towards a "lean-like phenotype", ameliorating pathological profiles of metabolic diseases. Further, OEA displays beneficial effects in several cognitive paradigms and preserves the epithelial barrier integrity, acting as an intestinal "gate-keeper". Here, we developed an "intestinal OEA factory" for the in-situ and controlled release of OEA by using a probiotic-based delivery system. We engineered the Lactobacillus paracasei F19 (LP) to express the human N-acylphosphatidylethanolamine-preferring phospholipase D (NAPEpld) gene and to produce OEA in response to dietary ultra-low oleate supply. We treated 12-week HFD male mice with oleate-probiotic formulations and assessed their impact on metabolic and behavioral dysfunctions, and microbiota-gut-brain signaling after 8 weeks of treatment. NAPE-expressing LP (pNAPE-LP) led to significant reduced weight loss and improved metabolic dysfunction in HFD-treated mice. Further, a parallel improvement in depressive- and anxiety-like phenotypes was associated with the duodenal barrier function retrieval, the restoration of the Firmicutes/Bacteroidetes ratio, and an increase in beneficial bacteria, such as Lactobacillus, Prevotella, and Parabacteroides. The HFD-driven changes both in the enteric and central nervous system were prevented by pNAPE-LP/oleate treatment. Collectively, our data suggest that these effects were mediated by the oleate-dependent release of OEA by pNAPE-LP since no significant effects were observed in HFD mice treated with the native probiotic alone (pLP). This oleate-regulated delivery system of OEA is a safe and efficient probiotic-based strategy for the treatment of metabolic syndrome and related behavioral disorders.

Metabolic syndrome (MetS) represents a growing clinical challenge worldwide, significantly increasing cardiovascular disease and type 2 diabetes risk, and all-cause mortality. Defined by a constellation of risk factors, including central obesity, dyslipidemia, hypertension, and insulin resistance, MetS is frequently associated with hepatic involvement such as metabolic dysfunction-associated fatty liver disease (MAFLD) and non-alcoholic steatohepatitis (NASH). A growing body of evidence also highlights intestinal barrier dysfunction called "leaky-gut syndrome" and gut microbiota dysbiosis as key contributors to the chronic low-grade inflammation that underlies MetS. Current therapies, including statins, GLP-1 receptor agonists, SGLT2 inhibitors, and antihypertensives, target individual components of the syndrome but fail to address its multifactorial pathophysiology. Additionally, these therapies often demonstrate tolerability issues, limited long-term adherence and compliance, and potential side effects particularly in hepatic and metabolic contexts. ALIAmides such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are emerging as promising adjunctive agents for MetS. By activating PPARs, modulating immune responses, and restoring gut barrier integrity, these compounds provide a multi-targeted strategy that addresses the complex pathophysiology of MetS. This review summarizes current evidence on ALIAmides, highlighting their potential to complement standard care and improve long-term metabolic outcomes.

Background Achalasia is characterized by symptoms of esophageal obstruction, preventing food consumption. However, weight loss is observed only in a subset of patients, and data from literature is conflicting. Aims Our study aimed at evaluating predictors of weight loss in achalasia patients and at verifying the impact of treatment on nutritional status. Methods 123 achalasia patients, eligible for laparoscopic Heller myotomy, were studied. Demographic, clinical and nutritional data (calorie intake and macronutrient composition) were recorded at baseline and one-year post-treatment. Significant weight loss/gain was considered for variation of 10 % of body weight at baseline and post-treatment, respectively. Results 57.7 % of patients reported weight loss at presentation. These subjects had shorter disease duration, worse symptoms, lower BMI and consumed fewer calories than patients without weight loss. Post-treatment, we observed a considerable improvement in Eckardt score and BMI values. Almost 50 % of the population reported significant weight gain, particularly in individuals with weight loss at baseline. Caloric intake also rose significantly, positively affecting BMI categories. Conclusion We showed that achalasia-induced weight loss is associated with symptoms’ severity and disease duration. Conversely, over 50 % of treated patients were in the overweight/obese category, highlighting the need for individualized nutritional interventions in achalasia patients.

Chronic intestinal inflammation and neo-angiogenesis are interconnected in colorectal carcinoma (CRC) pathogenesis. Molecules reducing inflammation and angiogenesis hold promise for CRC prevention and treatment. N-Palmitoyl-d-glucosamine (PGA), a natural glycolipid analog with anti-inflammatory properties, has shown efficacy against acute colitis. Micronized PGA (mPGA) formulations exhibit superior anti-inflammatory activity. This study investigates the in vivo anti-angiogenic and protective effects of mPGA in a mouse model of colitis-associated CRC induced by azoxymethane/dextran sodium sulfate (AOM/DSS). CRC was induced in C57BL/6J mice using intraperitoneal azoxymethane followed by three cycles of 2.5% dextran sodium sulfate (DSS) in drinking water. Mice were treated with mPGA (30-150 mg/kg) with or without the PPARα inhibitor MK886 (10 mg/kg). At Day 70 post-azoxymethane injection, mice underwent anesthetized endoscopic colon evaluation. Post-mortem analysis of tumorigenesis and angiogenesis was performed using histological, immunohistochemical, and immunoblotting techniques. mPGA improved disease progression and survival rates in a dose- and PPARα-dependent manner in AOM/DSS-exposed mice. It reduced polyp formation, decreased pro-angiogenic CD31, pro-proliferative Ki67, and pro-inflammatory TLR4 expression levels, and inhibited VEGF and MMP-9 secretion by disrupting the pAkt/mTOR/HIF1α pathway. mPGA increased colon PEA levels, restoring anti-tumoral PPARα and wtp53 protein expression. Given its lack of toxicity, mPGA shows potential as a nutritional intervention to counteract inflammation-related angiogenesis in CRC.

Background SARS-CoV-2 belongs to the coronaviridae family and infects human cells by directly interacting with the angiotensin-converting enzyme-2 (ACE-2) through the viral Spike Protein (SP). While vaccines are crucial, much attention has been directed towards managing the symptoms of acute respiratory distress syndrome. Our present study highlights the potential in counteracting lung inflammation triggered by SARS-CoV-2 SP of the intranasal administration of the engineered probiotic Lactobacillus paracasei F19 expressing the enzyme NAPE-PLD (pNAPE-LP) able to in situ release palmitoylethanolamide (PEA) under a super-low boost of palmitate. Methods C57BL/6J mice undergo prophylactic treatment with intranasal pNAPE-LP/palmitate for 7 days before a 7 days challenge with intranasal SARS-CoV-2 SP. Then the capability of pNAPE-LP of colonizing the lungs and actively release PEA in situ have been determined by immunofluorescence, western blot and HPLC-MS. Moreover, the innate immune system downregulation and the histological damage rescue exerted by pNAPE-LP have been tested by immunofluorescence, hematoxylin and eosin staining, western blot analysis and ELISA test for the release of the pro-inflammatory mediators. Results pNAPE-LP effectively colonizes mice lungs and releases the anti-inflammatory compound PEA. Moreover, pNAPE-LP exhibits a protective effect on alveolar morphology, innate immune cells infiltration and in the reduction of neutrophil count, effectively reducing lung injury induced by SARS-CoV-2 SP. This is achieved by mitigating TLR4-mediated NLRP3 activation and the downstream pro-inflammatory products such as ILs, TNFα, C-reactive protein and the myeloperoxidase activity. Interestingly we observed a global reduction ACE2 expression in the lungs. Conclusion pNAPE-LP actively protect from severe inflammatory-related symptoms in SP-challenged mice. Also, it can downregulate the expression of ACE-2 receptors at the lung site potentially preventing the spreading of the infection.

While current anti-Spike protein (SP) vaccines have been pivotal in managing the pandemic, their limitations in delivery, storage, and the inability to provide mucosal immunization (preventing infections) highlight the ongoing necessity for research and innovation. To tackle these constraints, our research group developed a bacterial-based vaccine using a non-pathogenic E. coli Nissle 1917 (EcN) strain genetically modified to express the SARS-CoV-2 spike protein on its surface (EcN-pAIDA1-SP). We intranasally delivered the EcN-pAIDA1-SP in two doses and checked specific IgG/IgA production as well as the key immune mediators involved in the process. Moreover, following the initial and booster vaccine doses, we exposed both immunized and non-immunized mice to intranasal delivery of SARS-CoV-2 SP to assess the effectiveness of EcN-pAIDA1-SP in protecting lung tissue from the inflammation damage. We observed detectable levels of anti-SARS-CoV-2 spike IgG in serum samples and IgA in bronchoalveolar lavage fluid two weeks after the initial treatment, with peak concentrations in the respective samples on the 35th day. Moreover, immunoglobulins displayed a progressively enhanced avidity index, suggesting a selective binding to the spike protein. Finally, the pre-immunized group displayed a decrease in proinflammatory markers (TLR4, NLRP3, ILs) following SP challenge, compared to the non-immunized groups, along with better preservation of tissue morphology. Our probiotic-based technology provides an effective immunobiotic tool to protect individuals against disease and control infection spread.

As of October 2022, the COVID-19 pandemic continues to pose a major public health conundrum, with increased rates of symptomatic infections in vaccinated individuals. An ideal vaccine candidate for the prevention of outbreaks should be rapidly scalable, easy to administer, and able to elicit a potent mucosal immunity. Towards this aim, we proposed an engineered Escherichia coli (E. coli) Nissle 1917 (EcN) strain with SARS-CoV-2 spike protein (SP)-coding plasmid, which was able to expose SP on its cellular surface by a hybridization with the adhesin involved in diffuse adherence 1 (AIDA1). In this study, we presented the effectiveness of a 16-week intragastrically administered, engineered EcN in producing specific systemic and mucosal immunoglobulins against SARS-CoV-2 SP in mice. We observed a time-dependent increase in anti-SARS-CoV-2 SP IgG antibodies in the sera at week 4, with a titre that more than doubled by week 12 and a stable circulating titre by week 16 (+309% and +325% vs. control; both p < 0.001). A parallel rise in mucosal IgA antibody titre in stools, measured via intestinal and bronchoalveolar lavage fluids of the treated mice, reached a plateau by week 12 and until the end of the immunization protocol (+300, +47, and +150%, at week 16; all p < 0.001 vs. controls). If confirmed in animal models of infection, our data indicated that the engineered EcN may be a potential candidate as an oral vaccine against COVID-19. It is safe, inexpensive, and, most importantly, able to stimulate the production of both systemic and mucosal anti-SARS-CoV-2 spike-protein antibodies.

Enteric glia are a unique population of peripheral neuroglia associated with the enteric nervous system (ENS) throughout the digestive tract. The emerging data from the latest glial biology studies unveiled enteric glia as a heterogenic population with plastic and adaptative abilities that display phenotypic and functional changes upon distinct extrinsic cues. This aspect is essential in the dynamic signaling that enteric glia engage with neurons and other neighboring cells within the intestinal wall, such as epithelial, endocrine, and immune cells to maintain local homeostasis. Likewise, enteric glia sense signals from luminal microbes, although the extent of this active communication is still unclear. In this minireview, we discuss the recent findings that support glia-microbes crosstalk in the intestine in health and disease, pointing out the critical aspects that require further investigation.

Developing drugs for brain infection by Naegleria fowleri is an unmet medical need. We used a combination of cheminformatics, target-, and phenotypic-based drug discovery methods to identify inhibitors that target an essential N. fowleri enzyme, sterol 14-demethylase (NfCYP51). A total of 124 compounds preselected in silico were tested against N. fowleri. Nine primary hits with EC50 ≤ 10 μM were phenotypically identified. Cocrystallization with NfCYP51 focused attention on one primary hit, miconazole-like compound 2a. The S-enantiomer of 2a produced a 1.74 Å cocrystal structure. A set of analogues was then synthesized and evaluated to confirm the superiority of the S-configuration over the R-configuration and the advantage of an ether linkage over an ester linkage. The two compounds, S-8b and S-9b, had an improved EC50 and KD compared to 2a. Importantly, both were readily taken up into the brain. The brain-to-plasma distribution coefficient of S-9b was 1.02 ± 0.12, suggesting further evaluation as a lead for primary amoebic meningoencephalitis.

Improving clinical outcomes and delaying disease recrudescence in Ulcerative Colitis (UC) patients is crucial for clinicians. In addition to traditional and new pharmacological therapies that utilize biological drugs, the development of medical devices that can ameliorate UC and facilitate the remission phase should not be overlooked. Drug-based therapy requires time to be personalized and to evaluate the benefit/risk ratio. However, the increasing number of diagnosed UC cases worldwide necessitates the exploration of new strategies to enhance clinical outcomes. By incorporating medical devices alongside pharmacological treatments, clinicians can provide additional support to UC patients, potentially improving their condition and slowing down the recurrence of symptoms. Chemically identified as an azelaic acid derivative and palmitoylethanolamide (PEA) analog, adelmidrol is a potent anti-inflammatory and antioxidant compound. In this study, we aimed to evaluate the effect of an intrarectal administration of 2% adelmidrol (Ade) and 0.1% hyaluronic acid (HA) gel formulation in both the acute and resolution phase of a mouse model of colitis induced via DNBS enema. We also investigated its activity in cultured human colon biopsies isolated from UC patients in the remission phase at follow-up when exposed in vitro to a cytomix challenge. Simultaneously, with its capacity to effectively alleviate chronic painful inflammatory cystitis when administered intravesically to urological patients such as Vessilen, the intrarectal administration of Ade/HA gel has shown remarkable potential in improving the course of colitis. This treatment approach has demonstrated a reduction in the histological damage score and an increase in the expression of ZO-1 and occludin tight junctions in both in vivo studies and human specimens. By acting independently on endogenous PEA levels and without any noticeable systemic absorption, the effectiveness of Ade/HA gel is reliant on a local antioxidant mechanism that functions as a “barrier effect” in the inflamed gut. Building on the findings of this preliminary study, we are confident that the Ade/HA gel medical device holds promise as a valuable adjunct in supporting traditional anti-UC therapies.

Adelmidrol is a promising palmitoylethanolamide (PEA) analog which displayed up-and-coming anti-inflammatory properties in several inflammatory conditions. Recent studies demonstrated that Adelmidrol is an in vitro enhancer of PEA endogenous production, through the so called "entourage" effect. The present study investigated the ability of Adelmidrol (1 and 10 mg/Kg per os) to increase the endogenous level of PEA in the duodenum and colon of mice after 21-day oral administration in the presence and absence of PPAR-γ inhibitor (1 mg/kg). The level of PEA was analyzed by HPLC-MS. The expression of PEA-related enzymatic machinery was evaluated by western blot and RT-PCR analysis. Our findings demonstrated that Adelmidrol significantly increased PEA levels in the duodenum and colon in a dose/time-dependent manner. We also revealed that Adelmidrol up regulated the enzymatic machinery responsible for PEA metabolism and catabolism. Interestingly, the use of the selective irreversible PPAR-γ antagonist did not affect either PEA intestinal levels or expression/transcription of PEA metabolic enzymes following Adelmidrol administration. The "entourage effect" with Adelmidrol as an enhancer of PEA was thus PPAR-γ-independent. The findings suggest that Adelmidrol can maximize a PEA therapeutic-based approach in several intestinal morbidities.

Crohn's disease (CD) is a chronic inflammatory gastrointestinal disorder requiring lifelong medications. The currently approved drugs for CD are associated with relevant side effects and several studies suggest an increased use of nutraceuticals among CD patients, seeking for what is perceived as a more "natural" approach in controlling this highly morbid condition. Nutraceuticals are foods or foods' components with beneficial health properties that could aid in CD treatment for their anti-inflammatory, analgesic and immunoregulatory activities that come along with safety, high tolerability, easy availability and affordability. Depending on their biological effect, nutraceuticals' support could be employed in different subsets of CD patients, both those with active disease, as adjunctive immunomodulatory therapies, and/or in quiescent disease to provide symptomatic relief in patients with residual functional symptoms. Despite the increasing interest of the general public, both limited research and lack of education from healthcare professionals regarding their real clinical effectiveness account for the increasing number of patients turning to unconventional sources. Professionals should recognize their widespread use and the evidence base for or against their efficacy to properly counsel IBD patients. Overall, nutraceuticals appear to be safe complements to conventional therapies; nonetheless, little quality evidence supports a positive impact on underlying inflammatory activity.

Engineered probiotics represent a cutting-edge therapy in intestinal inflammatory disease (IBD). Genetically modified bacteria have provided a new strategy to release therapeutically operative molecules in the intestine and have grown into promising new therapies for IBD. Current IBD treatments, such as corticosteroids and immunosuppressants, are associated with relevant side effects and a significant proportion of patients are dependent on these therapies, thus exposing them to the risk of relevant long-term side effects. Discovering new and effective therapeutic strategies is a worldwide goal in this research field and engineered probiotics could potentially provide a viable solution. This review aims at describing the proceeding of bacterial engineering and how genetically modified probiotics may represent a promising new biotechnological approach in IBD treatment.

Similar to canine inflammatory enteropathy, inflammatory bowel disease (IBD) is a chronic idiopathic condition characterized by remission periods and recurrent flares in which diarrhea, visceral pain, rectal bleeding/bloody stools, and weight loss are the main clinical symptoms. Intestinal barrier function alterations often persist in the remission phase of the disease without ongoing inflammatory processes. However, current therapies include mainly anti-inflammatory compounds that fail to promote functional symptoms-free disease remission, urging new drug discoveries to handle patients during this step of the disease. ALIAmides (ALIA, autacoid local injury antagonism) are bioactive fatty acid amides that recently gained attention because of their involvement in the control of inflammatory response, prompting the use of these molecules as plausible therapeutic strategies in the treatment of several chronic inflammatory conditions. N-palmitoyl-D-glucosamine (PGA), an under-researched ALIAmide, resulted in being safe and effective in preclinical models of inflammation and pain, suggesting its potential engagement in the treatment of IBD. In our study, we demonstrated that micronized PGA significantly and dose-dependently reduces colitis severity, improves intestinal mucosa integrity by increasing the tight junction proteins expression, and downregulates the TLR-4/NLRP3/iNOS pathway via PPAR-α receptors signaling in DNBS-treated mice. The possibility of clinically exploiting micronized PGA as support for the treatment and prevention of inflammation-related changes in IBD patients would represent an innovative, effective, and safe strategy.

Despite its possible therapeutic potential against COVID-19, the exact mechanism(s) by which palmitoylethanolamide (PEA) exerts its beneficial activity is still unclear. PEA has demonstrated analgesic, anti-allergic, and anti-inflammatory activities. Most of the anti-inflammatory properties of PEA arise from its ability to antagonize nuclear factor-κB (NF-κB) signalling pathway via the selective activation of the PPARα receptors. Acting at this site, PEA can downstream several genes involved in the inflammatory response, including cytokines (TNF-α, Il-1β) and other signal mediators, such as inducible nitric oxide synthase (iNOS) and COX2. To shed light on this, we tested the anti-inflammatory and immunomodulatory activity of ultramicronized(um)-PEA, both alone and in the presence of specific peroxisome proliferator-activated receptor alpha (PPAR-α) antagonist MK886, in primary cultures of murine alveolar macrophages exposed to SARS-CoV-2 spike glycoprotein (SP). SP challenge caused a significant concentration-dependent increase in proinflammatory markers (TLR4, p-p38 MAPK, NF-κB) paralleled to a marked upregulation of inflammasome-dependent inflammatory pathways (NLRP3, Caspase-1) with IL-6, IL-1β, TNF-α over-release, compared to vehicle group. We also observed a significant concentration-dependent increase in angiotensin-converting enzyme-2 (ACE-2) following SP challenge. um-PEA concentration-dependently reduced all the analyzed proinflammatory markers fostering a parallel downregulation of ACE-2. Our data show for the first time that um-PEA, via PPAR-α, markedly inhibits the SP induced NLRP3 signalling pathway outlining a novel mechanism of action of this lipid against COVID-19.

Given the abundancy of angiotensin converting enzyme 2 (ACE‐2) receptors density, beyond the lung, the intestine is considered as an alternative site of infection and replication for severe acute respiratory syndrome by coronavirus type 2 (SARS‐CoV‐2). Cannabidiol (CBD) has recently been proposed in the management of coronavirus disease 2019 (COVID‐19) respiratory symptoms because of its anti‐inflammatory and immunomodulatory activity exerted in the lung. In this study, we demonstrated the in vitro PPAR‐γ‐dependent efficacy of CBD (10−9‐10−7 M) in preventing epithelial damage and hyperinflammatory response triggered by SARS‐CoV‐2 spike protein (SP) in a Caco‐2 cells. Immunoblot analysis revealed that CBD was able to reduce all the analyzed proinflammatory markers triggered by SP incubation, such as tool‐like receptor 4 (TLR‐4), ACE‐2, family members of Ras homologues A‐GTPase (RhoA‐GTPase), inflammasome complex (NLRP3), and Caspase‐1. CBD caused a parallel inhibition of interleukin 1 beta (IL‐1β), IL‐6, tumor necrosis factor alpha (TNF‐α), and IL‐18 by enzyme‐linked immunosorbent assay (ELISA) assay. By immunofluorescence analysis, we observed increased expression of tight‐junction proteins and restoration of transepithelial electrical resistance (TEER) following CBD treatment, as well as the rescue of fluorescein isothiocyanate (FITC)–dextran permeability induced by SP. Our data indicate, in conclusion, that CBD is a powerful inhibitor of SP protein enterotoxicity in vitro.