Syk-Dependent Responses and Innate Cytosolic Defenses in Orientia tsutsugamushi-infected Macrophages

Abstract

Abstract Orientia tsutsugamushi is an obligately intracellular bacterium and the etiological agent of scrub typhus. Human studies and animal models of scrub typhus have shown robust type 1-skewed proinflammatory responses during severe infection. Macrophages play a critical role in initiating such responses, yet mechanisms of innate recognition for O. tsutsugamushi remain unclear. In this study, we investigated whether Syk-dependent C-type lectin receptors (CLRs) contribute to innate immune recognition and the generation of proinflammatory responses. To validate a role of CLRs in scrub typhus, we infected murine primary macrophages with O. tsutsugamushi in the presence of selective Syk inhibitors and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. We found that Mincle/Clec4a and Clec5a transcription was significantly abrogated upon Syk inhibition at 6 hours of infection. The effect of Syk inhibition on Mincle protein expression was validated via western blot. Syk-inhibited MF had diminished expression of type 1 cytokines and chemokines (Il12p40, Tnf, Il27p28, Cxcl1) during infection. Additionally, expression of innate immune cytosolic sensors (Mx1 and Oas 1–3) was highly induced in the brain of lethally infected mice. We established that Mx1 and Oas1 expression was reduced in Syk-inhibited MF, while Oas2, Oas3, and MerTK were not sensitive to Syk inhibition. This study reveals that Syk-dependent CLRs contribute to inflammatory responses to O. tsutsugamushi. It also provides the first evidence for Syk-dependent activation of intracellular defenses during infection, suggesting a role of pattern recognition receptor crosstalk in orchestrating macrophage-mediated responses to this poorly studied bacterium. Supported by the NIAID grants (R21 AI156536, R01 AI132674, and T32 AI007526). JF was the recipient of a NIAID Emerging and Tropical Infectious Diseases T32 fellowship.