RESUMO
Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic diarrheic intestinal infections in domestic and wild ruminants (paratuberculosis or Johne's disease) for which there is no effective treatment. Critical in the pathogenesis of MAP infection is the invasion and survival into macrophages, immune cells with ability to carry on phagocytosis of microbes. In a search for effective therapeutics, our objective was to determine whether human cathelicidin LL-37, a small peptide secreted by leuckocytes and epithelial cells, enhances the macrophage ability to clear MAP infection. In murine (J774A.1) macrophages, MAP was quickly internalized, as determined by confocal microscopy using green fluorescence protein expressing MAPs. Macrophages infected with MAP had increased transcriptional gene expression of pro-inflammatory TNF-α, IFN-γ, and IL-1ß cytokines and the leukocyte chemoattractant IL-8. Pretreatment of macrophages with synthetic LL-37 reduced MAP load and diminished the transcriptional expression of TNF-α and IFN-γ whereas increased IL-8. Synthetic LL-37 also reduced the gene expression of Toll-like receptor (TLR)-2, key for mycobacterial invasion into macrophages. We concluded that cathelicidin LL-37 enhances MAP clearance into macrophages and suppressed production of tissue-damaging inflammatory cytokines. This cathelicidin peptide could represent a foundational molecule to develop therapeutics for controlling MAP infection.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Citocinas/metabolismo , Macrófagos/microbiologia , Mycobacterium avium subsp. paratuberculosis/efeitos dos fármacos , Paratuberculose/microbiologia , Animais , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Bovinos , Linhagem Celular , Citocinas/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/tratamento farmacológico , Paratuberculose/imunologia , CatelicidinasRESUMO
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-1ß 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.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Colo/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Colo/imunologia , Colo/microbiologia , Células HT29 , Humanos , Interleucina-1beta/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Receptor 4 Toll-Like/imunologia , Proteína da Zônula de Oclusão-1/metabolismo , CatelicidinasRESUMO
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.