Evaluation of the innate immune response of caprine neutrophils against Mycobacterium avium susbspecies 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 mecha‑ nisms. 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 neu‑ trophils after Map infection. Immunofuorescence was used to demonstrate, simultaneously, chemotaxis, phagocy‑ tosis, degranulation, and NETs. The quantifcation of neutrophil phagocytic activity against Map at a 1:10 multiplicity of infection (MOI), through fow cytometry, showed values that varied from 4.54 to 5.63% of phagocyting neutrophils. By immunofuorescence, a 73.3±14.5% of the felds showed NETs, and the mean release of DNA, attributable to NETo‑ sis, calculated through a fuorometric 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 signifcantly higher in the two latter. Overall, neutrophil response was proportional to the number of bacteria. This work confrms that the simultaneous study of several neutrophil mechanisms, and the combination of diferent 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 efector functions, and sets the basis for further in vitro and in vivo studies on the role of neutrophils in paratuberculosis.