Synthetic cathelicidin LL-37 reduces Mycobacterium avium subsp. paratuberculosis internalization and pro-inflammatory cytokines in macrophages
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-1β 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.
KeywordsMycobacterium avium subsp. paratuberculosis Macrophages Cathelicidin LL-37 IL-8
Immunofluorescence studies were conducted in the Live Cell Imaging Facility, Snyder Institute, University of Calgary.
KC acquired and analyzed all the data. PL and YT conducted the monocyte culture and qPCR data. RH conducted the macrophage imaging. RA created MAP A1-157 GFP, performed MAP culture and qPCR and live/dead assays. RA, JB, and HB provided critical scientific input. KC and EC conceived the experiment and wrote the manuscript.
This work was supported by the Margaret Gunn Endowment for Animal Research (UofC), NSERC Discovery (RGPAS-2017-507827) to EC and by the BEC.AR (Program for short term internship in biotechnology and agro-industry in BID country members sponsored by the Ministry of Education, Argentina) to KC.
Compliance with ethical statements
Conflict of interest
The authors declare that they have no conflicts of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Ahlstrom C, Barkema HW, Stevenson K, Zadoks RN, Biek R, Kao R, Trewby H, Haupstein D, Kelton DF, Fecteau G, Labrecque O, Keefe GP, McKenna SL, De Buck J (2015) Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level. BMC Genomics 16:161PubMedPubMedCentralGoogle Scholar
- Ahlstrom C, Barkema HW, Stevenson K, Zadoks RN, Biek R, Kao R, Trewby H, Haupstein D, Kelton DF, Fecteau G, Labrecque O, Keefe GP, McKenna SL, Tahlan K, De Buck J (2016b) Genome-wide diversity and phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian dairy cattle. PLoS One 11:e0149017PubMedPubMedCentralGoogle Scholar
- Bannantine JP, Stabel JR, Laws E, Cardieri MCD, Souza CD (2015) Mycobacterium avium subspecies paratuberculosis recombinant proteins modulate antimycobacterial functions of bovine macrophages. PLoS One 10:e0128966. https://doi.org/10.1371/journal.pone.0128966 CrossRefPubMedPubMedCentralGoogle Scholar
- Barkema HW, Orsel K, Nielsen SS, Koets AP, Rutten V, Bannantine JP, Keefe GP, Kelton DF, Wells SJ, Whittington RJ, Mackintosh CG, Manning EJ, Weber MF, Heuer C, Forde TL, Ritter C, Roche S, Corbett CS, Wolf R, Griebel PJ, Kastelic JP, De Buck J (2018) Knowledge gaps that hamper prevention and control of Mycobacterium avium subspecies paratuberculosis infection. Transbound Emerg Dis 65(Suppl 1):125–48PubMedGoogle Scholar
- Cobo ER, Kissoon-Singh V, Moreau F, Holani R, Chadee K (2017) MUC2 mucin and butyrate contribute to the synthesis of the antimicrobial peptide cathelicidin in response to Entamoeba histolytica- and dextran sodium sulfate-induced colitis. Infect Immun 85:e00905-16. https://doi.org/10.1128/IAI.00905-16
- Cooney MA, Steele JL, Steinberg H, Talaat AM (2014) A murine oral model for Mycobacterium avium subsp. paratuberculosis infection and immunomodulation with Lactobacillus casei ATCC 334. Front Cell Infect Microbiol 4:11.10.3389Google Scholar
- Donnellan S, Stone V, Johnston H, Giardiello M, Owen A, Rannard S, Aljayyoussi G, Swift B, Tran L, Watkins C, Stevenson K (2017) Intracellular delivery of nano-formulated antituberculosis drugs enhances bactericidal activity. J Inter Nanomed 2:146–156Google Scholar
- Hines ME, Stabel JR, Sweeney RW, Griffin F, Talaat AM, Bakker D, Benedictus G, Davis WC, de Lisle GW, Gardner IA, Juste RA, Kapur V, Koets A, McNair J, Pruitt G, Whitlock RH (2007) Experimental challenge models for Johne’s disease: a review and proposed international guidelines. Vet Microbiol 122:197–222PubMedGoogle Scholar
- Holani R, Marin M, Kastelic J, Cobo ER (2018) Host defense peptides as innate immunomodulators in the pathogenesis of colitis. In: Elsevier (ed) Antimicrobial peptides in gastrointestinal disease. Academic Press: Cambridge, MA, USA, pp 133–164Google Scholar
- Kuehnel MP, Goethe R, Habermann A, Mueller E, Rohde M, Griffiths G et al (2001) Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria. Cell Microbiol 3:551–566PubMedGoogle Scholar
- Lee SJ, Noh KT, Kang TH, Han HD, Shin SJ, Soh BY, Park JH, Shin YK, Kim HW, Yun CH, Park WS, Jung ID, Park YM (2014) The Mycobacterium avium subsp. paratuberculosis protein MAP1305 modulates dendritic cell-mediated T cell proliferation through Toll-like receptor-4. BMB Rep 47:115–120PubMedPubMedCentralGoogle Scholar
- Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, Ochoa MT, Schauber J, Wu K, Meinken C, Kamen DL, Wagner M, Bals R, Steinmeyer A, Zugel U, Gallo RL, Eisenberg D, Hewison M, Hollis BW, Adams JS, Bloom BR, Modlin RL (2006) Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 311:1770–1773PubMedGoogle Scholar
- Mookherjee N, Brown KL, Bowdish DM, Doria S, Falsafi R, Hokamp K, Roche FM, Mu R, Doho GH, Pistolic J, Powers JP, Bryan J, Brinkman FS, Hancock RE (2006) Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J Immunol 176:2455–2464PubMedGoogle Scholar
- Periasamy S, Tripathi BN, Singh N (2009) Mechanisms of Mycobacterium avium subsp. paratuberculosis induced apoptosis and necrosis in bovine macrophages. Vet Microbiol 165:392–401Google Scholar
- Rode AKO, Kongsbak M, Hansen MM, Lopez DV, Levring TB, Woetmann A, Odum N, Bonefeld CM, Geisler C (2017) Vitamin D counteracts Mycobacterium tuberculosis-induced cathelicidin downregulation in dendritic cells and allows Th1 differentiation and IFNgamma secretion. Front Immunol 8:656PubMedPubMedCentralGoogle Scholar
- Santos JC, Silva-Gomes S, Silva JP, Gama M, Rosa G, Gallo RL, Appelberg R (2014) Endogenous cathelicidin production limits inflammation and protective immunity to Mycobacterium avium in mice. Immun Inflammation Dis 2:1–12Google Scholar
- Young-Speirs M, Drouin D, Cavalcante PA, Barkema HW, Cobo ER (2018) Host defense cathelicidins in cattle: types, production, bioactive functions and potential therapeutic and diagnostic applications. Int J Antimicrob AgentsGoogle Scholar