Evaluation of the innate immune response of caprine neutrophils against Mycobacterium avium subspecies paratuberculosis in vitro

Abstract

Neutrophils constitute an essential component of the innate immune response, readily killing most bacteria through phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs) among other mechanisms. These cells play an unclear role in mycobacterial infections such as Mycobacterium avium subspecies paratuberculosis (Map), the etiological agent of paratuberculosis, and its response is particularly understudied in ruminants. Herein, a wide set of techniques were adapted, or newly developed, to study the in vitro response of caprine neutrophils after Map infection. Immunofluorescence was used to demonstrate, simultaneously, chemotaxis, phagocytosis, degranulation, and NETs. The quantification of neutrophil phagocytic activity against Map at a 1:10 multiplicity of infection (MOI), through flow cytometry, showed values that varied from 4.54 to 5.63% of phagocyting neutrophils. By immunofluorescence, a 73.3 ± 14.5% of the fields showed NETs, and the mean release of DNA, attributable to NETosis, calculated through a fluorometric method, was 16.2 ± 3.5%. In addition, the RNA expression of TGF-β, TNF and IL-1β cytokines, measured through reverse transcription qPCR, was significantly higher in the two latter. Overall, neutrophil response was proportional to the number of bacteria. This work confirms that the simultaneous study of several neutrophil mechanisms, and the combination of different methodologies, are essential to reach a comprehensive understanding of neutrophil response against pathogens, demonstrates that, in vitro, caprine neutrophils display a strong innate response against Map, using their entire repertoire of effector functions, and sets the basis for further in vitro and in vivo studies on the role of neutrophils in paratuberculosis.

Cathepsin S Is More Abundant in Serum of Mycobacterium avium subsp. paratuberculosis-Infected Dairy Cows

“…Furthermore, human CTSC is essential for optimal NET formation [64]. In a strong innate response to MAP, caprine neutrophils use all their effector functions-phagocytosis, chemotaxis, degranulation and NET release [65]. Oral vaccination against MAP in a rabbit infection model stimulated the phagocytosis rate of neutrophils and additionally increased NET release against MAP and non-related pathogens [66].…”

Non-specific effects of inactivated Mycobacterium bovis oral and parenteral treatment in a rabbit scabies model

“…It also warns on the timing of the optimal response regarding experimental designs because it seems possible that the desirable long-term effects cannot be seen with shorter experiment follow-ups. Additionally, it seems that the oral route might be a good delivery way [ 40 , 41 ], thus supporting the beneficial effects of use of immune enhancers in the diet [ 1 ]. This is also consistent with a mechanism developed and operating in natural conditions by chance when the right conditions concur.…”

No Evidence of Neutrophil Response Modulation in Goats after Immunization against Paratuberculosis with a Heat-Inactivated Vaccine

“…A new effector function, the formation of neutrophil extracellular traps (NETs), was first described in 2004 [ 19 ], and a regulatory role of neutrophils (the expression and production of both pro- and anti-inflammatory cytokines during the early stages of infection) is being revealed [ 20 , 21 , 22 ]. In this regard, the advancements in cell function assays have made it possible to demonstrate that neutrophils employ their “classic” effector functions against Map and, above all, that they efficiently kill this bacterium [ 16 , 18 , 23 ]. NETosis against Map has recently been described in vitro in several ruminant species: cattle [ 16 ], sheep [ 24 ], and goats [ 23 ].…”

“…In this regard, the advancements in cell function assays have made it possible to demonstrate that neutrophils employ their “classic” effector functions against Map and, above all, that they efficiently kill this bacterium [ 16 , 18 , 23 ]. NETosis against Map has recently been described in vitro in several ruminant species: cattle [ 16 ], sheep [ 24 ], and goats [ 23 ]. In vivo induction of NETs by M. tuberculosis ( Mtb ) has already been proven in a guinea pig model [ 25 ], and even though its presence in PTB infection in vivo is still unknown, an extracellular Map DNase (MAP3916c) which destroys NETs in vitro has been shown to be necessary for Map virulence, suggesting that this mechanism occurs in vivo as well [ 26 ].…”

“…In vivo induction of NETs by M. tuberculosis ( Mtb ) has already been proven in a guinea pig model [ 25 ], and even though its presence in PTB infection in vivo is still unknown, an extracellular Map DNase (MAP3916c) which destroys NETs in vitro has been shown to be necessary for Map virulence, suggesting that this mechanism occurs in vivo as well [ 26 ]. With regard to the modulating role of neutrophils, an increase in proinflammatory cytokine expression (IL-1β and TNF) [ 23 ] and production (IL-1β and IL8) [ 16 ] by caprine and bovine neutrophils, respectively, has been observed after Map exposure ex vivo.…”