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

Orientia tsutsugamushi is an obligately intracellular bacterium and an 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 (M phi) 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 the role of CLRs in scrub typhus, we infected murine bone marrow-derived M phi 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 h of infection. The effect of Syk inhibition on Mincle protein expression was validated via Western blot. Syk-inhibited M phi had diminished expression of type 1 cytokines/chemokines (Il12p40, Tnf, Il27p28, Cxcl1) during infection. Additionally, expression of innate immune cytosolic sensors (Mx1 and Oas1-3) was highly induced in the brain of lethally infected mice. We established that Mx1 and Oas1 expression was reduced in Syk-inhibited M phi, while Oas2, Oas3, and MerTK were not sensitive to Syk inhibition. This study reveals that Syk-dependent CLRs contribute to inflammatory responses against 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.