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Paired-end RNA Sequencing on human dermal microvascular endothelial cells (HMEC-1) infected or not with Borrelia burgdorferi (MOI= 200 bacteria/cell) examined at different times post-infection (namely 4, 24 or 48 h post-infection)

ABSTRACT: Purpose: When we infect human dermal microvascular endothelial cells (HMEC-1) infected or not with Borrelia burgdorferi (MOI= 200 bacteria/cell) and examine the host cell kinematics and dynamics over the course of 3 days post-infection we observe a change in those at early times post-infection, but at late times (> 20 h post-infection) cells from infected or not wells behave similarly. To get a sense of the biochemical signals that might be driving the changes in host cell mechanics, we performed RNA-Sequencing and gene expression profiling for uninfected HMEC-1 cells or cells infected for 4, 24 or 48 h with Borrelia burgdorferi at an MOI of 200 bacteria/cell. Methods and Results: By analyzing the differentially expressed genes between all conditions we find a significant number of up- or down-regulated genes infected and uninfected cells just at 4 h post-infection. Through pathway enrichemnet analysis we find that compared to cells not exposed to infection infected cells showed significant upregulation to genes related IL-17 and NF-kB and TNF related signaling pathways. Interestingly these changes are not present at 24 or 48 h post-infection where cells from infected wells are more similar gene expression-wise to uninfected cells. Conclusions: The changes in kinematics and dynamics of HMEC-1 cells exposed to Borrelia burgdorferi (MOI= 200 bacteria/cell) at early times post-infection are associated with changes in innate immune signaing of the host cells. At later times post-infection were these changes in gene expression are not present anymore, host cell kinematics and dynamics are identical between infected and uninfected cells. Thus it is possible that innate immune singaling might be underlying the changes that host cells undergo in their motion and traction forces at early infection.

ORGANISM(S): Homo sapiens

PROVIDER: GSE174545 | GEO | 2021/05/18

REPOSITORIES: GEO

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Project description:The overall goal of these experiments was to determine how human cells respond to ontegrin binding by Borrelia burgdorferi. Lyme disease is the most common vector-borne infection in the Northern hemisphere, yet little is understood about the pathogenic mechanisms of the causative agent, Borrelia burgdorferi. Cells in culture were infected with the bacteria that do and do not express the beta3 integrin ligand P66, which are otherwise isogenic. As additional controls, uninfected cells were also included in the analyses. The cell line used for primary data analyses provided here was Ea.hy926, a macrovascular line (Edgell, C. J.,et al. 1990. Endothelium specific Weibel-Palade bodies in a continuous human cell line, EA.hy926. In vitro Cell. & Dev. Biol. 26:1167-1172, and Edgell, C. J., et al. 1983. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc. Natl. Acad. Sci. 80:3734-3737). In addition, the human microvascular endothelial cell line HMEC (Ades et al, 1992. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol. 99:683-690) was used. Epithelial cells HEK-293 (which does not express b3 integrins) and HEK-293 transfected to express integrin ?v?3 were also analyzed. Infection times were 1 hour or 3 hours for cells lifted with EDTA and washed prior to bacterial infection, or 3, 6, and 24 hours for samples in which the bacteria were added to adherent cells. RNA was harvested and reverse transcribed. Labeled cDNAs were used to probe 2 color arrays. In each of at least three biological replicate experiments, for each time point, the following comparisons were made. First, cells infected with B. burgdorferi with a marker inserted adjacent to the p66 gene (p66+) were compared to uninfected cells. Second, cells infected with the p66 gene interrupted by the marker (Coburn, J. and Cugini, C. (2003) Targeted mutation of P66 of Borrelia burgdorferi disrupts attachment to integrin ?v?3. Proc. Natl. Acad. Sci. 100:7301-7306) the mutant-infected cells were compared to the uninfected cells. In some experiments, cells infected with p66+ were compared to cells infected with the p66- mutant. Arrays used were HEEBO arrays purchased from Microarrays Inc. (Nashville, TN). Ea.hy926 or HMEC endothelial cell lines, or HEK-293 epithelial cells, or transfected HEK-293 derivative expressing integrin avb3

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Paired-end RNA Sequencing on human dermal microvascular endothelial cells (HMEC-1) infected or not with Borrelia burgdorferi (MOI= 200 bacteria/cell) examined at different times post-infection (namely 4, 24 or 48 h post-infection)

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