Mincle activation and type-1 proinflammatory response during scrub typhus: new insight into innate immune recognition of Orientia tsutsugamushi

Abstract O. tsutsugamushi is an obligate intracellular bacterium and etiologic agent of scrub typhus. The lungs harbor the greatest burden of infection, displaying overzealous, type 1-skewed proinflammatory responses. Here, we investigated whether the C-type lectin receptor (CLR) Mincle contributes to immune recognition and dysregulation. Following lethal infection in C57BL/6 mice, we analyzed pulmonary differential gene expression via the NanoString technology. Of 671 genes analyzed, we found 312 significantly induced genes (adj. p < 0.05) at the terminal phase of disease, among which Mincle (Clec4e) was the 4th greatest up-regulated gene (36 fold compared with mock). In addition, Mincle signaling partners (Fcgrs), Cxcr3, Ccr5, and their ligands, as well as Il27, were all highly up-regulated. To validate a role of Mincle in immune recognition, we exposed murine bone marrow-derived macrophages (MΦ) to live or heat-killed O. tsutsugamushi and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. While heat-killed bacteria transiently stimulated Mincle transcription that dissipated by 24 hpi, only live bacteria generated a sustained response; infection had limited impact on other tested CLRs. Mincle activation was validated by indirect immunofluorescence and western blot. Live, but not heat-killed, bacteria also generated sustained proinflammatory gene expression in MΦ (Cxcl9, Ccl2, Ccl5, Nos2, Il27). Our study provides the first evidence for selective activation of Mincle in sensing O. tsutsugamushi and a role of Mincle- and IL-27-related pathways in severe infection. Since there are no effective vaccines for scrub typhus, our study helps understand innate immune recognition of this poorly-studied bacterium.