Immunomodulatory Role of Cathelicidin at Colonic Epithelium

Cathelicidin, a small, cationic and amphiphilic host defense peptide, is expressed by hematopoietic and non-hematopoietic cells. Initially considered an antimicrobial, cathelicidin are more than simply natural antibiotics. Due to their ability to interact with multiple receptors, cathelicidin can modulate immune responses by a variety of host cells. However, their immunomodulatory effects vary based on their micro-environment, including tissue type (hematopoietic vs. non-hematopoietic), availability of microbes or associated factor(s) and effective concentration of cathelicidin. Since most studies are performed in leukocytes, ability of cathelicidin to regulate immune responses by intestinal epithelium remains largely unknown. The objective was to elucidate immunomodulatory potential of cathelicidin in colonic epithelium, under homeostasis as well as during bacterial colitis. In the first study, we determined how cathelicidin synergized with lipopolysaccharide or Salmonella enterica Typhimurium to enhance production of neutrophil chemokine CXCL8 (human) or murine CXCL1 (functional homologue of CXCL8). Further, we identified a two-signal mechanism triggered by the complex of LPS-LL37, in which nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling was involved in CXCL8 mRNA synthesis, whereas p38 mitogen-activated protein kinase (p38MAPK) was involved in CXCL8 mRNA stabilization. In a Citrobacter rodentium model of colitis, we corroborated increased synthesis of CXCL1 chemokine, effective neutrophil recruitment and reduced bacterial burden in Camp+/+ versus Camp-/- (cathelicidin null) mice. In the second study, under physiological conditions, cathelicidin induced synthesis of Toll-like receptor (TLR) negative regulator, Toll-interacting protein (Tollip). This induced Tollip production inhibited TLR-mediated apoptosis in colonic epithelium, both in vitro by cytokines tumour necrosis factor- α and interferon- γ as well as in vivo in a C. rodentium model of colitis. In conclusion, cathelicidin regulated colonic innate immunity via reduction of unwarranted inflammation by inhibiting TLR responses through Tollip, while promoting a neutrophil response to infection

Cathelicidin modulates synthesis of Toll-like Receptors (TLRs) 4 and 9 in colonic epithelium

Cathelicidin are innate antimicrobial peptides with broad immunomodulatory functions; however, their role in regulating intestinal defenses is not well characterized. This study aimed to investigate the role of cathelicidin modulating expression of Toll-like receptors (TLRs) 4 and 9 in colonic epithelium in response to bacterial patterns. We demonstrated herein that intestinal epithelial cells, when primed by bacterial lipopolysaccharide (LPS), responded to cathelicidin by increased transcription and protein synthesis of TLR4. This cathelicidin-induced response required the interaction of LPS-TLR4 and activation of MAPK signalling pathways. However, cathelicidin blocked TLR9 responses induced by TLR9 ligand CpG oligodeoxynucleotide (CpG ODN) in these colonic epithelial cells. Modulations of TLRs triggered by cathelicidin in intestinal epithelium occurred mainly in the apical compartment of intestinal cells. Activation of TLR4 by ligands in combination with cathelicidin promoted CXCL8 chemokine secretion and epithelial antimicrobial defenses against Escherichia coli. We concluded that cathelicidin selectively modulated synthesis of TLR4 and 9 in intestinal epithelium, but only when cells were exposed to virulence factors, mostly from apical surfaces. Enhanced TLR4 expression promoted by cathelicidin in intestinal epithelium may be crucial for controlling enteric infectious diseases.Fil: Marin, Maia Solange. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Holani, Ravi. University of Calgary; CanadáFil: Shah, Chaitanya B.. University of Calgary; CanadáFil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Cobo, Eduardo Ruben. University of Calgary; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Human cathelicidin improves colonic epithelial defenses against Salmonella typhimurium by modulating bacterial invasion, TLR4 and pro-inflammatory cytokines

The intestinal mucosa contributes to frontline gut defenses by forming a barrier (physical and biochemical) and preventing the entry of pathogenic microbes. One innate role of the human colonic epithelium is to secrete cathelicidin, a peptide with broad antimicrobial and immunomodulatory functions. In this study, the effect of cathelicidin in the maintenance of epithelial integrity, Toll-like receptor recognition, bacterial invasion and initiation of inflammatory response against Salmonella typhimurium is investigated in cultured human colonic epithelium. We found exogenous human cathelicidin restores the epithelial integrity in S. typhimurium-infected colonic epithelial (T84) cells by mostly post-translational effects associated with reorganization of zonula occludens (ZO)-1 tight junction proteins. Endogenous cathelicidin prevents S. typhimurium internalization as shown in colonic epithelial cells genetically deficient in the only human cathelicidin, LL-37 (shLL-37). Moreover, supplementation of shLL-37 cells with synthetic LL-37 reduces the grade of S. typhimurium internalization in a dose-dependent manner. Mechanistically, shLL-37 cells have lower gene expression of TLR4 and pro-inflammatory cytokine IL-1β in response to S. typhimurium. Thus, human cathelicidin aids in the early colonic epithelial defenses against enteric S. typhimurium by preventing bacterial invasion and maintaining epithelial barrier integrity, likely to occur due to the production of sensing TLR4 and pro-inflammatory cytokines.Fil: Marin, Maia Solange. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; ArgentinaFil: Holani, Ravi. University of Calgary; CanadáFil: Blyth, Graham A. D.. University of Calgary; CanadáFil: Drouin, Dominique. University of Calgary; CanadáFil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; ArgentinaFil: Cobo, Eduardo R.. University of Calgary; Canad

Sequence edition of single domains modulates the final immune and antimicrobial potential of a new generation of multidomain recombinant proteins

Combining several innate immune peptides into a single recombinant antimicrobial and immunomodulatory polypeptide has been recently demonstrated. However, the versatility of the multidomain design, the role that each domain plays and how the sequence edition of the different domains affects their final protein activity is unknown. Parental multidomain antimicrobial and immunomodulatory protein JAMF1 and several protein variants (JAMF1.2, JAMF2 and AM2) have been designed and recombinantly produced to explore how the tuning of domain sequences affects their immunomodulatory potential in epithelial cells and their antimicrobial capacity against Gram-positive and Gram-negative bacteria. The replacement of the sequence of defensin HD5 and phospholipase sPLA2 by shorter active fragments of both peptides improves the final immunomodulatory (IL-8 secretion) and antimicrobial function of the multidomain protein against antimicrobial-resistant Klebsiella pneumoniae and Enterococcus spp. Further, the presence of Jun and Fos leucine zippers in multidomain proteins is crucial in preventing toxic effects on producer cells. The generation of antimicrobial proteins based on multidomain polypeptides allows specific immunomodulatory and antimicrobial functions, which can be easily edited by modifying of each domain sequence.info:eu-repo/semantics/publishedVersio