Bovine neutrophils release extracellular traps and cooperate with macrophages inMycobacterium aviumsubsp.paratuberculosisclearancein vitro

Abstract

Mycobacterium avium subsp. paratuberculosis (Map) is the underlying pathogen causing bovine paratuberculosis (PTB), an enteric granulomatous disease that mainly affects ruminants and for which an effective treatment is needed. Macrophages are the primary target cells for Map, which survives and replicates intracellularly by inhibiting phagosome maturation. Neutrophils are present at disease sites during the early stages of the infection, but seem to be absent in the late stage, in contrast to healthy tissue. Although neutrophil activity has been reported to be impaired following Map infection, their role in PTB pathogenesis has not been fully defined. Neutrophils are capable of releasing extracellular traps consisting of extruded DNA and proteins that immobilize and kill microorganisms, but this mechanism has not been evaluated against Map. Our main objective was to study the interaction of neutrophils with macrophages during an in vitro mycobacterial infection. For this purpose, neutrophils and macrophages from the same animal were cultured alone or together in the presence of Map or Mycobacterium bovis Bacillus-Calmette-Guérin (BCG). Extracellular trap release, mycobacteria killing as well as IL-1β and IL-8 release were assessed. Extracellular trap formation was highest in neutrophils against Map in the presence of macrophages, but without direct cell contact, indicating a paracrine activation. Macrophages were extremely efficient at killing BCG, but ineffective at killing Map. In contrast, neutrophils showed similar killing rates for both mycobacteria. Co-cultures infected with Map showed the expected killing effect of combining both cell types, whereas co-cultures infected with BCG showed a potentiated killing effect beyond the expected one, indicating a potential synergistic cooperation. In both cases, IL-1β and IL-8 levels were lower in co-cultures, suggestive of a reduced inflammatory reaction. These data indicate that cooperation of both cell types can be beneficial in terms of decreasing the inflammatory reaction while the effective elimination of Map can be compromised. These results suggest that neutrophils are effective at Map killing and can exert protective mechanisms against Map that seem to fail during PTB disease after the arrival of macrophages at the infection site.

Effect of Paratuberculosis Vaccination on the in Vitro Release of Neutrophil Extracellular Traps in a Sheep Model

“…The release of NETs is characterized by the immobilization and elimination of pathogens, although their presence could act as a double-edged weapon related to severe collateral tissue damage [30,43,44]. Previous studies have demonstrated the generation of NETs in response to SA [33,45,46], EC [31,47] and Map [35]. In this study, infection of ovine neutrophils with SA resulted in the highest percentage of extracellular DNA.…”

“…Besides, the action of NETs during paratuberculosis pathogenesis has not been fully elucidated. Bovine neutrophils have been demonstrated to release NETs in response to in vitro Map infection [35]. Here, although in a lesser extent than SA and EC, Map infection also promoted the release of NETs in ovine neutrophils.…”

“…Furthermore, it has been reported that NETs are unable to kill M. tuberculosis, but they trap the bacteria preventing their dissemination and stimulating the formation of granulomas [32]. Thus, it could be inferred that role of NETs during paratuberculosis infection might be related with the immobilization and, in the short term, the elimination of Map enhancing the susceptibility of trapped bacteria to other antimicrobial mechanisms, since it has been observed that NETs release is reduced in the presence of monocytederived macrophages through the clearance of their components by phagocytosis [35].…”

“…In addition, neutrophil extracellular traps (NETs), based on the extracellular release of chromatin bers [30], have demonstrated to trap and eliminate a wide range of extracellular pathogens (i.e. Staphylococcus aureus and Escherichia coli), but also intracellular pathogens such as Toxoplasma gondii, Mycobacterium tuberculosis and Map, preventing their systemic spread [31][32][33][34][35].…”

Mycobacterial infection-induced miR-206 inhibits protective neutrophil recruitment via the CXCL12/CXCR4 signalling axis

“…We chose to assess the neutrophil responses as alterations in Cxcr4/Cxcl12 signalling would likely impact the mobility and responsiveness of neutrophils to pathogens. Recent studies on the role of the neutrophils in early infection suggest a host-protective response against mycobacteria [40,59], and we hypothesised that these cells may be responsible for the reduced disease burden in miR-206 knockdown embryos. It is, however, likely that the alteration of targets Elmo1 and Timp3 may be contributing to this effect and that role of these genes in mycobacterial infection must be further examined, especially in regard to their involvement in the miR-206/Cxcr4/Cxcl12 signalling axis.…”