Bovine Neutrophils Release Extracellular Traps and Cooperate With Macrophages in Mycobacterium avium subsp. paratuberculosis clearance In 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. Neutrophils released extracellular traps against mycobacteria when cultured alone and in the presence of macrophages without direct cell contact, but resulted inhibited in direct contact. 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.

Oral vaccination stimulates neutrophil functionality and exerts protection in a Mycobacterium avium subsp. paratuberculosis infection model

“…PMNs from healthy animals are also capable of releasing ETs against pathogens such as S. aureus 35 , E. coli 36 , and Map 34 or Mbv 34 , so we assessed if ex vivo NET formation was affected by vaccination and challenge against mycobacterial and non-mycobacterial microorganisms and NETosis levels were represented on a heat-map (Fig. 3a ).…”

“…However, their role in defense against Map has only been suggested by recent transcriptomic studies describing the impairment of PMN recruitment and activation during PTB 31 – 33 . PMNs from healthy cattle have shown to exert extracellular trap (ET) formation and killing effects against Map in vitro 34 . The idea that proper PMN activation or training by PTB vaccines could eventually prevent or mitigate the effects of Map infection is a hypothesis that should be tested.…”

The Bovine Tuberculoid Granuloma

“…Neutrophils are part of the innate immune response and are regarded as the first line of defense to invading pathogens. Neutrophils are short-lived, terminally differentiated, effector cells with three main antimicrobial functions: phagocytosis, degranulation and release of material in the form of neutrophil extracellular traps (NETs) [74,75] Upon arrival to the site of infection, neutrophils may phagocytose invading pathogens, including mycobacteria through Fc and complement receptors. Complex signaling pathways promoted by the phagocytic process, lead to the fusion of protease containing granules with the phagosome, triggering the oxidative burst [76].…”

Antimicrobial Activity of Neutrophils Against Mycobacteria

“…The major antimicrobial strategy employed by neutrophils involves the ingestion of pathogens into phagosomes, followed by the degranulation of antimicrobial peptides and proteins and the production of toxic reactive oxygen species inside the phagosome ( 8 , 114 ). Neutrophils are known to phagocytose both pathogenic and non-pathogenic mycobacteria ( 7 , 30 , 36 , 112 , 115 – 121 ), with ingestion of mycobacteria increasing in the presence of serum ( 112 , 115 – 117 ). The mechanism of phagocytosis differs depending on whether neutrophils bind opsonized or non-opsonized mycobacteria.…”

“…Several mycobacteria, including M. tuberculosis , M. bovis BCG, M. abscessus, M. avium subsp. paratuberculosis and M. leprae , have been shown to induce NETs in vitro ( 121 , 129 , 146 , 148 , 176 , 178 – 181 ). How the interaction of mycobacteria with neutrophils leads to the formation of NETs appears to differ depending on the bacterial species.…”