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

Giuseppe Esposito,Chiara Corpetti,Marcella Pesce,Alessandro Del Re,Jie Lu,Giovanni Sarnelli,Giuseppe Annunziata,Luisa Seguella,Walter Sanseverino,Martina Vincenzi,Roberta Lattanzi

doi:10.3389/fphar.2021.639728

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.

Highlights

Clostridium (Clostridioides) difficile infection (CDI) represents the leading cause of nosocomial diarrhea in North America and Europe and has been labeled as an urgent public health threat by the US Center for Disease Control and Prevention (CDC) (Lessa et al, 2015)
Palmitoylethanolamide (PEA) is an endogenous, on demandreleased N-acylethanolamine belonging to the family of bioactive autacoid local injury antagonist amides (ALIAmides), a group of lipid molecules involved in the regulation of several physiological processes, ranging from analgesia, neuroprotection, and inflammation (Petrosino and Di Marzo, 2017)
By selectively binding the peroxisome proliferator–activated receptor-α (PPARα) (Sarnelli et al, 2016b), PEA exerts a wide range of anti-inflammatory effects, downregulating inducible nitric oxide synthase, cycloxigenase-2 (COX-2), tumor necrosis factor-α (TNF-α) expression, and the NF-κB signaling pathway, with downstream regulation of pro-inflammatory cytokines and immune cell infiltration in inflamed tissues

Summary

Introduction

Clostridium (Clostridioides) difficile infection (CDI) represents the leading cause of nosocomial diarrhea in North America and Europe and has been labeled as an urgent public health threat by the US Center for Disease Control and Prevention (CDC) (Lessa et al, 2015). RhoA-GTPase proteins are physiologically involved in actin cytoskeleton and tight junctions’ assembly (Terry et al, 2010), resulting in the disruption of the epithelial barrier, and a profound inflammatory response with release of pro-inflammatory cytokines and extensive neutrophil infiltration, through activation of the nuclear factor-kappa B (NF-κB) signaling pathway (Kim et al, 2006). By selectively binding the peroxisome proliferator–activated receptor-α (PPARα) (Sarnelli et al, 2016b), PEA exerts a wide range of anti-inflammatory effects, downregulating inducible nitric oxide synthase (iNOS), cycloxigenase-2 (COX-2), tumor necrosis factor-α (TNF-α) expression, and the NF-κB signaling pathway, with downstream regulation of pro-inflammatory cytokines and immune cell infiltration in inflamed tissues. To date its potential in models of Clostridium difficile–induced colitis remains to be established (Esposito et al, 2014; Pesce et al, 2018)

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