<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>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), 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

Activation of inflammasome by rickettsiae in THP-1 derived macrophages and caspase-1-dependent secretion of IL-1β.

<p>Human THP-1 cells were differentiated to macrophages using PMA, and infected with <i>R</i>. <i>australis</i> at an MOI of 5. A, Infection with <i>R</i>. <i>australis</i> induced a significant increase in IL-1β production compared to uninfected cells at 24 h p.i.. B, Inhibition of caspase-1 significantly reduced the secretion levels of IL-1β by <i>R</i>. <i>autralis</i>-infected THP-1 derived macrophages. The fold change in IL-1β by treated cells vs. untreated controls was calculated and compared. Data represent two independent experiments with consistent results. Each experiment included at least 4 replicates. *, <i>p</i><0.05.</p

<i>R</i>. <i>australis</i> activated inflammasome in BMMs.

<p>A, WT BMMs were isolated, cultivated and infected with <i>R</i>. <i>australis</i> at an MOI of 10. At 24 h p.i., culture supernatant and cell lysates were collected. Cell lysates were processed for detection of activation of caspase-1. B, BMMs of B6N and caspase-1/11-double knockout mice were isolated and infected with <i>R</i>. <i>australis</i> as described above. The secretion levels of IL-1β and IL-18 were determined by ELISA. *, <i>p</i><0.05.</p

Kinetics and dose-dependent mechanisms of secretion of IL-1β and IL-18 by <i>Rickettsia</i>-infected BMMs.

<p>WT BMMs were isolated, cultivated, and infected with <i>R</i>. <i>australis</i> at MOI of 2 or 6. Cell culture supernatants were harvested at 4 hour intervals over 24 h p.i.. Secretion of IL-1β (A) and IL-18 (B) was determined by ELISA. Data represent two independent experiments with consistent results. Each experiment included at least 4 replicates. *, <i>p</i><0.05; ns, not significantly different.</p

NLRP3 was involved in recognition of rickettsiae by inflammasome at the early stage of infection in BMMs.

<p>WT and NLRP3^-/- BMMs were isolated, cultivated, and infected with <i>R</i>. <i>australis</i> at MOIs of 6 and 2. The transcriptional levels of NLRP3 in WT BMMs at different time intervals of infection (at MOI of 6) were determined by RT-PCR as described in Materials and Methods (A). At 12 h (B) p.i., the secretion levels of IL-1β by WT and NLRP3^-/- BMMs were determined by ELISA. The cleavage of caspase-1 was determined by detection of the active unit p10 in the processed supernatant at 12 h p.i. (C). Data represent mean ± SD for at least 3 replicates each group. *, <i>p</i><0.05 for a significant difference between WT and NLRP3^-/- mice; ns, not significantly different.</p