<i>R</i>. <i>massiliae</i> and <i>R</i>. <i>conorii</i> (ISF) infection resulted in plaques in Vero cell monolayers and replicated efficiently in the human dermal microvascular cell line, HMEC-1 cells.

<p>(A) <i>R</i>. <i>massiliae</i> and <i>R</i>. <i>conorii</i> (ISF) were grown in Vero cells at 34°C for 5 and 7 days to quantify the number of plaques present as revealed by crystal violet staining (B). <i>R</i>. <i>massiliae</i> and <i>R</i>. <i>conorii</i> (ISF) were grown in HMEC-1 cells for times indicated. Data are representative of three independent experiments (A and B).</p

<i>R</i>. <i>conorii</i> (ISF) induced significant secretion of IL-8 and IL-6, while <i>R</i>. <i>massiliae</i> induced significant production of MCP-1 in infected HMEC-1 cells.

<p>The production levels of MCP-1 (A), IL-8 (B) and IL-6 (C) in the supernatant of <i>Rickettsia</i>-infected HMEC-1 cells was assessed by ELISA. Bar graphs indicate the average and standard error of three independent experiments. White bars represent uninfected cells, grey bars represent <i>R</i>. <i>massiliae</i>-infected cells, and <i>R</i>. <i>conorii</i> (ISF)—infected cells are represented by black bars. *, <i>p</i> < 0.05, ** n.s. = non-statistically significant.</p

<i>R</i>. <i>massiliae</i> induced upregulation of MCP-1 mRNA levels and <i>R</i>. <i>conorii</i> (ISF) induced IL-8 mRNA levels in HMEC-1 cells.

<p>Real-time quantitative PCR analysis of the expression of MCP-1 mRNA (A) and IL-8mRNA (B) in HMEC-1 cells infected at an MOI of 5. Bar graphs indicate the average and standard error of three independent experiments. White bars represent uninfected cells, grey bars represent <i>R</i>. <i>massiliae-</i> infected cells, and <i>R</i>. <i>conorii</i> (ISF)-infected cells are represented by black bars. *, <i>p</i> <0.05.</p

<i>R</i>. <i>conorii</i> (ISF), but not <i>R</i>. <i>massiliae</i>, increased endothelial cell monolayer permeability.

<p>(A) Representative ECIS graph demonstrating the loss of electrical resistance across an HMEC-1 monolayer in real-time. (B) Average resistance of endothelial cell monolayers after infection with rickettsiae for designated time points. Bar graphs (B) indicate the average and standard error of three independent experiments. White bars represent uninfected cells, grey bars represent <i>R</i>. <i>massiliae</i>-infected cells, and <i>R</i>. <i>conorii</i> (ISF)—infected cells are represented by black bars. *, <i>p</i> < 0.05.</p

<i>R</i>. <i>conorii</i> (ISF), but not <i>R</i>. <i>massiliae</i>, induced cell death at 72 HPI in HMEC-1 cells: (A) Cells were infected at an MOI of 5 and stained with Live/Dead fixable dye for 30 minutes before fixation in paraformaldehyde.

<p>For flow cytometry experiments, ⩾10,000 cells were analyzed. (B) Annexin V staining of HMEC-1 cells following 72 hours of infection with <i>R</i>. <i>massiliae</i> or <i>R</i>. <i>conorii</i> (ISF). Cell counts were normalized to mode. (C) Lactate dehydrogenase (LDH) activity assay of monolayer supernatants demonstrating significantly increased levels of endothelial cell cytotoxicity after infection with <i>R</i>. <i>conorii</i> (ISF) for 72 hours. Bar graphs (A and C) indicate the average and standard error of three independent experiments. White bars represent uninfected cells, grey bars represent <i>R</i>. <i>massiliae</i>-infected cells, <i>R</i>. <i>conorii</i> (ISF)-infected cells are represented by black bars, and cells treated with staurosporine are represented by the checkered bar. *, <i>p</i> < 0.05.</p

Endothelial cell death induced by <i>R</i>. <i>conorii</i> (ISF) is partially dependent on caspase-1.

<p>Cells were treated with caspase-1 and caspase-4 inhibitors and then infected with <i>R</i>. <i>conorii</i> (ISF) for 72 hours. Fresh medium and caspase inhibitors were added daily, and the removed supernatant was used immediately for the LDH activity assay. The bar graph indicates the average and standard error of three independent experiments. *, <i>p</i> < 0.05, inh = inhibitor, Rc = <i>R</i>. <i>conorii</i> (ISF), casp = caspase.</p