The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

Abstract

Background: Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like receptors (TLRs) TLR2 and TLR4 and have been shown to generate Neutrophil extracellular traps (NETs) upon exposure to Eimeria bovis. The present work investigated the role of TLR2 and TLR4 in the recognition and uptake of E. bovis sporozoites, IL-8 production and neutrophil extracellular trap (NET) formation. Methods: TLR expression was performed by flow cytometric analysis on PMN exposed to live carboxyfluorescein succinimidyl ester (CFSE)-stained sporozoites. Supernatants of PMN exposed to different E. bovis sporozoite preparations and antigens in the absence or presence of TLR antibodies were assessed for IL-8 secretion. Cells were exposed to sporozoite preparations and assessed for the activation of transcription factor NF-κB using a luciferase reporter assay. Immunofluorescence analysis was done to investigate TLR2 and TLR4 surface expression and NET formation on bovine PMN exposed to vital sporozoites. Results: we observed significantly increased TLR2 and TLR4 expression with a mean increase in expression that was greater for TLR2 than TLR4. This upregulation neither inhibited nor promoted sporozoite phagocytosis by bovine PMN. Live sporozoites together with anti-TLR2 mAb resulted in a significant enhancement of IL-8 production. NF-κB activation was more strongly induced in TLR2-HEK cells than in TLR4/MD2-HEK cells exposed to heat-killed sporozoites and antigens. Immunofluorescence analysis showed TLR-positive signals on the surface of PMN and concomitant NET formation. Conclusions: This is the first report on E. bovis-induced concomitant TLR2 and TLR4 expression during bovine PMN-derived NETosis.

Receptor-Mediated NETosis on Neutrophils

“…Additionally, in the natural immune confrontation between fungus and host, the receptors TLR2 and TLR4 are essential for ROS-dependent NETosis induction by Fonsecaea pedrosoi ( F. pedrosoi ) ( 29 ). Regarding parasites, Eimeria bovis ( E. bovis ) can increase TLR2 and TLR4 expression on the PMN and induce TLR2/4-dependent NF-κB activation, resulting in NETosis ( 30 ). Regarding bacteria, Streptococcus suis serotype 2 (SS2) can be recognized by TLR2 and/or TLR4, initiating NETosis in a NOX - dependent manner.…”

Significance of Mast Cell Formed Extracellular Traps in Microbial Defense

“…It is now clear that neutrophils recognize several pathogens using TLR-2 and produce NETs in turn; since MCs express TLR-2, the receptor is likely involved in production of MCETs, but it has not been specifically investigated. [140,141] including MCETs is likely to be a late response but effective one against the presence of intruding microorganisms, however, the release of DNA into extracellular space may orchestrate the immune responses such as the production of anti-citrullinated protein antibodies in seropositive rheumatoid arthritis. Not surprisingly, since many AMPs attached to DNA web should be normally restricted in cytoplasmic granules, their release may have harmful effects such as degrading ECM or activating tissue-destructive mechanisms [93,94].…”

A Review of the Neutrophil Extracellular Traps (NETs) from Cow, Sheep and Goat Models

“…Neutrophils release ETs as a defense strategy against pathogens [ 24 , 27 , 28 , 31 , 32 , 65 , 67 , 68 , 70 , 113 , 114 , 115 ]. These parasites include Eimeria bovis [ 31 , 32 , 67 ], Neospora caninum [ 59 ], trypomastigotes [ 116 ] and Haemonchus contortus [ 64 ]. Pathogens can be entrapped and killed within NET-like structures in a ROS-dependent or independent manner [ 32 , 59 , 63 ].…”

“…Porphyromonas gingivalis [55] V. cholera [45] Aeromonas hydrophila [56] E. arloingi sporozoites, B. besnoiti, C. parvum, Spermatozoa, H. contortus, N. caninum, D (-) lactic acid, M. bovis, E. ninakohlyakimovae, T. gondii, S. uberis [24,[31][32][33][34][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72] Formation of NETs contributes to the pathogenesis of inflammatory and autoimmune diseases in man. Pathologies associated with NET formation have been reported in systemic lupus erythematosus, rheumatoid arthritis [73], vasculitis [74,75], diabetes [76,77], atherosclerosis and cancer [78].…”

“…Neutrophils release ETs as a defense strategy against pathogens [24,27,28,31,32,65,67,68,70,[113][114][115]. These parasites include Eimeria bovis [31,32,67], Neospora caninum [59], trypomastigotes [116] and Haemonchus contortus [64].…”