Orientia tsutsugamushi selectively stimulates the C-type lectin receptor Mincle and type 1-skewed proinflammatory immune responses

Orientia tsutsugamushi is an obligately intracellular bacterium and the etiological agent of scrub typhus. The lung is a major target organ of infection, displaying type 1-skewed proinflammatory responses. Lung injury and acute respiratory distress syndrome are common complications of severe scrub typhus; yet, their underlying mechanisms remain unclear. In this study, we investigated whether the C-type lectin receptor (CLR) Mincle contributes to immune recognition and dysregulation. Following lethal infection in mice, we performed pulmonary differential expression analysis with NanoString. Of 671 genes examined, we found 312 significantly expressed genes at the terminal phase of disease. Mincle (Clec4e) was among the top 5 greatest up-regulated genes, accompanied with its signaling partners, type 1-skewing chemokines (Cxcr3, Ccr5, and their ligands), as well as Il27. To validate the role of Mincle in scrub typhus, we exposed murine bone marrow-derived macrophages (MΦ) to live or inactivated O. tsutsugamushi and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. We found that while heat-killed bacteria stimulated transitory Mincle expression, live bacteria generated a robust response in MΦ, which was validated by indirect immunofluorescence and western blot. Notably, infection had limited impact on other tested CLRs or TLRs. Sustained proinflammatory gene expression in MΦ (Cxcl9, Ccl2, Ccl5, Nos2, Il27) was induced by live, but not inactivated, bacteria; infected Mincle-/- MΦ significantly reduced proinflammatory responses compared with WT cells. Together, this study provides the first evidence for a selective expression of Mincle in sensing O. tsutsugamushi and suggests a potential role of Mincle- and IL-27-related pathways in host responses to severe infection. Additionally, it provides novel insight into innate immune recognition of this poorly studied bacterium.
Author summary Scrub typhus is a life-threatening disease caused by the bacterium Orientia tsutsugamushi. Severe disease often involves the lung, where diffuse alveolar damage can lead to significant morbidity and mortality. It is thought that dysregulated immune responses contribute to the development of severe disease; however, it remains unknown as to how the immune system senses and responds to O. tsutsugamushi. In this study, we used a lethal mouse model of scrub typhus, along with cultured macrophages and neutrophils, to examine immune sensors and inflammatory responses which may be involved during infection. We found that O. tsutsugamushi selectively stimulated the C-type lectin receptor Mincle in the lungs of infected mice, which was accompanied by increased markers of proinflammatory and type 1-responses. We also found that macrophages treated with live or inactivated bacteria increased Mincle expression alongside type 1 and M1 proinflammatory markers, some of which were markedly decreased in Mincle-/- cells. Activation of Mincle and its associated inflammatory profile by both live and inactivated O. tsutsugamushi may generate an overzealous immune response that contributes to acute tissue damage. This study provides novel insight into immune recognition of O. tsutsugamushi and a new framework for evaluating immune dysregulation in scrub typhus.