Polarized lung inflammation and Tie2/angiopoietin-mediated endothelial dysfunction during severe Orientia tsutsugamushi infection

Abstract

Orientia tsutsugamushi infection can cause acute lung injury and high mortality in humans; however, the underlying mechanisms are unclear. Here, we tested a hypothesis that dysregulated pulmonary inflammation and Tie2-mediated endothelial malfunction contribute to lung damage. Using a murine model of lethal O. tsutsugamushi infection, we demonstrated pathological characteristics of vascular activation and tissue damage: 1) a significant increase of ICAM-1 and angiopoietin-2 (Ang2) proteins in inflamed tissues and lung-derived endothelial cells (EC), 2) a progressive loss of endothelial quiescent and junction proteins (Ang1, VE-cadherin/CD144, occuludin), and 3) a profound impairment of Tie2 receptor at the transcriptional and functional levels. In vitro infection of primary human EC cultures and serum Ang2 proteins in scrub typhus patients support our animal studies, implying endothelial dysfunction in severe scrub typhus. Flow cytometric analyses of lung-recovered cells further revealed that pulmonary macrophages (MΦ) were polarized toward an M1-like phenotype (CD80+CD64+CD11b+Ly6G-) during the onset of disease and prior to host death, which correlated with the significant loss of CD31+CD45- ECs and M2-like (CD206+CD64+CD11b+Ly6G-) cells. In vitro studies indicated extensive bacterial replication in M2-type, but not M1-type, MΦs, implying the protective and pathogenic roles of M1-skewed responses. This is the first detailed investigation of lung cellular immune responses during acute O. tsutsugamushi infection. It uncovers specific biomarkers for vascular dysfunction and M1-skewed inflammatory responses, highlighting future therapeutic research for the control of this neglected tropical disease.

Fig 1. Endothelial cell (EC) activation and vascular damage in the lungs of O. tsutsugamushi-infected mice.

Female C57BL/6J mice were inoculated with 1.325 x 10⁶ of O. tsutsugamushi Karp strain (4–5 mice/group) or PBS (3–4 mice/group). At days 2, 6, or 9 post-infection, equivalent lung portions were collected for analyses. (A) Frozen lung sections were co-stained for Orientia bacteria (red), ICAM-1 (green), and DAPI (blue, top row, scale bar = 50 μm) with close-up views of the boxed areas in the bottom row (bar = 20 μm). (B) Lung sections were co-stained for VE-cadherin (adherens junctions, red), FITC-labeled I-B₄ lectins (green), and DAPI (blue, top row, bars = 50 μm). Close-up views of the boxed areas located the bottom row (bar = 20 μm). (C) Quantification of fluorescent ICAM-1 and VE-Cadherin staining (four images per time point). (D) Lung-derived cells were analyzed via flow cytometry for the percentage of ICAM-1⁺ cells among gated CD31⁺CD45^- ECs (4–5 mice/group in infected groups; 3 mice/group in PBS groups). *, p < 0.05; **, p < 0.01; ***, p <0.001; and ****, p < 0001 compared to PBS controls. Graphs are shown as mean +/- SEM. Flow cytometric and qRT-PCR data were analyzed by using one-way ANOVA with Tukey’s Post Hoc. At least 4 independent mouse infection experiments and 2 independent in vitro experiments were performed with similar trends; shown are representative data.

Fig 2. Elevated Ang2 expression and decreased Ang1 expression during O. tsutsugamushi infection.

Mice were infected as in Fig 1. (A) Frozen lung sections were co-stained for Ang1 (a marker for endothelial quiescence, green), Ang2 (an endothelial stress marker, red), and DAPI (blue) showing images at a low magnification (top row, scale bar = 50 μm) and close-up views of the boxed areas (bottom row, bar = 20 μm). (B) Quantification of fluorescent Ang1 and Ang2 staining (four images per time point). (C) Human serum Ang2 proteins in the control subjects (CNT) or scrub typhus patients (8/group) with different anti-Orientia IFA antibody titers were measured by ELISA. Shown are data from two independent experiments. *, p < 0.05; **, p < 0.01 and ***; p< 0.001; ****; p < 0001 compared to the controls. Graphs are shown as mean +/- SEM. Serum ELISA and qRT-PCR groups were analyzed via one-way ANOVA with Tukey’s Post Hoc. At least 3 independent mouse infection experiments were performed with similar trends; shown are representative data.

Fig 3. Reduced Tie2 expression and activation in the lungs of O. tsutsugamushi infected mice.

(A) Frozen lung tissue sections were stained for the Tie2 receptor (red) and DAPI (blue, bar = 50 μm). (B) Lung tissue homogenates (40 μg/lane) were measured by Western blots for the levels of phospho-Tie2 (pTie2) and total Tie2 proteins and compared with the GAPDH controls. (C) TIE2 mRNA levels in mouse lungs were measured via qRT-PCR; data are presented as relative mRNA values normalized to β-actin. **, p < 0.01 compared to the controls. Graph shown as mean +/- SEM. Serum ELISA and qRT-PCR groups were analyzed via one-way ANOVA with Tukey’s Post Hoc.

Fig 4. Polarized MΦ activation in infected mouse lungs.

Mice were infected with O. tsutsugamushi (4–5 mice/group) or PBS (3–4 mice/group) for lung tissues collection at indicated days of infection, as in Fig 1. (A) Flow cytometric analyses of lung-derived cells, gated on CD11b⁺Ly6G^- MΦs and MΦ subsets, are shown for the D9 samples. The percentages and total numbers of (B) MΦs (CD64⁺CD11b⁺Ly6G^-), (C) M1-type MΦs (CD80⁺CD206^- CD64⁺CD11b⁺Ly6G^-), as well as (D) M2-type MΦs (CD206⁺CD80^- CD64⁺CD11b⁺Ly6G^-) are shown, respectively. *, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p 0.0001 compared to the PBS controls.

Fig 5. Transcription of M1 and M2 associated genes in the lung of infected mice.

Lung tissues were measured for the expression of M1-related genes (A) and M2-related genes (B), respectively. Data are presented as relative to β-actin values. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to the PBS controls. Graphs are shown as mean +/- SEM. One-way ANOVA with Tukey’s Post Hoc was used for statistical analysis. Two independent mouse infection experiments were performed with similar trends; shown are representative data.

Fig 6. Enhanced bacterial growth in M2-polarized MΦs.

Bone marrow-derived MΦs were generated from C57BL/6J mice, polarized into M1 or M2 types by pre-treatment of cells with LPS (100 ng/ml) or rIL-4 (10 ng/ml), or given media only for non-polarized M0 MΦs. MΦs were then infected with bacteria (5 MOI). (A) Bacterial loads at 3, 48, and 72 hpi (n = 5) were determined by qPCR. Data are presented as the Orientia 47-kDa gene copy per pg of DNA. (B) Cells were co-stained for Orientia (green), IBA-1 (a MΦ marker, red), and DAPI (blue) at 72 hpi. *, p < 0.05; **, p < 0.01; ****; p < 0001. Data for qPCR was analyzed using two-way ANOVA with Tukey multiple comparison test.