A Palmitoylethanolamide Producing Lactobacillus paracasei Improves Clostridium difficile Toxin A-Induced Colitis

Abstract

Genetically engineered probiotics, able to in situ deliver therapeutically active compounds while restoring gut eubiosis, could represent an attractive therapeutic alternative in Clostridium difficile infection (CDI). Palmitoylethanolamide is an endogenous lipid able to exert immunomodulatory activities and restore epithelial barrier integrity in human models of colitis, by binding the peroxisome proliferator–activated receptor-α (PPARα). The aim of this study was to explore the efficacy of a newly designed PEA-producing probiotic (pNAPE-LP) in a mice model of C. difficile toxin A (TcdA)-induced colitis. The human N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), a key enzyme involved in the synthesis of PEA, was cloned and expressed in a Lactobacillus paracasei that was intragastrically administered to mice 7 days prior the induction of the colitis. Bacteria carrying the empty vector served as negative controls (pLP).In the presence of palmitate, pNAPE-LP was able to significantly increase PEA production by 27,900%, in a time- and concentration-dependent fashion. Mice treated with pNAPE-LP showed a significant improvement of colitis in terms of histological damage score, macrophage count, and myeloperoxidase levels (−53, −82, and −70.4%, respectively). This was paralleled by a significant decrease both in the expression of toll-like receptor-4 (−71%), phospho-p38 mitogen-activated protein kinase (−72%), hypoxia-inducible factor-1-alpha (−53%), p50 (−74%), and p65 (−60%) and in the plasmatic levels of interleukin-6 (−86%), nitric oxide (−59%), and vascular endothelial growth factor (−71%). Finally, tight junction protein expression was significantly improved by pNAPE-LP treatment as witnessed by the rescue of zonula occludens-1 (+304%), Ras homolog family member A-GTP (+649%), and occludin expression (+160%). These protective effects were mediated by the specific release of PEA by the engineered probiotic as they were abolished in PPARα knockout mice and in wild-type mice treated with pLP. Herein, we demonstrated that pNAPE-LP has therapeutic potential in CDI by inhibiting colonic inflammation and restoring tight junction protein expression in mice, paving the way to next generation probiotics as a promising strategy in CDI prevention.

N-Palmitoyl-D-Glucosamine Inhibits TLR-4/NLRP3 and Improves DNBS-Induced Colon Inflammation through a PPAR-α-Dependent Mechanism

“…Oral administration of micronized PGA improved the DAI score and resulted in macroscopic amelioration of intestinal inflammation, as shown by the increased expression of tight junction proteins (ZO-1 and occludin) in the colonic mucosa and lower histological damage score. Similar to the homologous ALIAmide PEA, which was successful in several colitis models treatment in mice and humans [ 21 , 22 , 23 , 24 , 25 ], PGA revealed an anti-inflammatory effect through a PPAR-α-dependent mechanism since in the presence of PPAR-α antagonist MK886, PGA-mediated effects were abolished. We demonstrated that PGA caused a significant decrease of pro-inflammatory mediators, such as iNOS and NLRP3 protein expression in colonic tissues as well as PGE 2 and IL-1β cytokines release in mice plasma.…”

Role of the Endocannabinoid System in the Regulation of Intestinal Homeostasis

“…They demonstrated that this approach was effective in a mouse model of Clostridium difficile colitis where colonic damage, inflammatory mediator release, and tight junction protein expression were all improved. 121 It was also similarly effective in DSS colitis. 121 The effects of the probiotic bacterium were abolished in PPARa knockout mice, suggesting they were mediated by PEA.…”

“…121 It was also similarly effective in DSS colitis. 121 The effects of the probiotic bacterium were abolished in PPARa knockout mice, suggesting they were mediated by PEA. However, because NAPE-PLD can synthesize a variety of NAEs and these were not assessed, it remains to be determined whether the effects observed are solely mediated by PEA.…”

Emerging mechanisms by which endocannabinoids and their derivatives modulate bacterial populations within the gut microbiome

“…In addition to their effects on central host metabolism, certain NAE species such as PEA also exhibit anti-inflammatory properties in experimental colitis models through incompletely defined mechanisms [ 90 – 96 ]. As a strategy to augment local levels of PEA, a second group genetically engineered the probiotic Lactobacillus paracasei substrain Paracasei F19 to synthesize and secrete PEA when the strain was supplied exogenous palmitic acid [ 97 ]. Using a chemically induced model of colitis, the authors demonstrated that co-administration of the PEA-producing L. paracasei with palmitate significantly increased intestinal concentrations of PEA and resulted in attenuated colitis development.…”