Altered Toll-Like Receptor 9 Signaling in Mycobacterium avium subsp. paratuberculosis-Infected Bovine Monocytes Reveals Potential Therapeutic Targets

Abstract

Mycobacterium aviumsubsp.paratuberculosisis the causative agent of Johne's disease in cattle. The complex, multifaceted interaction ofM. aviumsubsp.paratuberculosiswith its host includes dampening the ability of infected cells to respond to stimuli that promoteM. aviumsubsp.paratuberculosisclearance. By disrupting host defenses,M. aviumsubsp.paratuberculosiscreates an intracellular environment that favors the establishment and maintenance of infection. Toll-like receptors (TLRs) are important sensors that initiate innate immune responses to microbial challenge and are also immunotherapeutic targets. For example, TLR9 contributes to host defense againstM. aviumsubsp.paratuberculosis, and its agonists (CpG oligodeoxynucleotides [ODNs]) are under investigation for treatment of Johne's disease and other infections. Here we demonstrate thatM. aviumsubsp.paratuberculosisinfection changes the responsiveness of bovine monocytes to TLR9 stimulation.M. aviumsubsp.paratuberculosisinhibits classical TLR9-mediated responses despite a 10-fold increase in TLR9 expression and maintained uptake of CpG ODNs. Other TLR9-mediated responses, such as oxidative burst, which occur through noncanonical signaling, remain functional. Kinome analysis verifies that classic TLR9 signaling is blocked byM. aviumsubsp.paratuberculosisinfection and that signaling instead proceeds through a Pyk2-mediated mechanism. Pyk2-mediated signaling does not hinder infection, as CpG ODNs fail to promoteM. aviumsubsp.paratuberculosisclearance. Indeed, Pyk2 signaling appears to be an important aspect ofM. aviumsubsp.paratuberculosisinfection, as Pyk2 inhibitors significantly reduce the number of intracellularM. aviumsubsp.paratuberculosisbacteria. The actions ofM. aviumsubsp.paratuberculosison TLR9 signaling may represent a strategy to generate a host environment which is better suited for infection, revealing potential new targets for therapeutic intervention.