Borrelia burgdorferi modulates the physical forces and immunity signaling in endothelial cells

Abstract

Borrelia burgdorferi (Bb), a vector-borne bacterial pathogen and the causative agent of Lyme disease, can spread to distant tissues in the human host by traveling in and through monolayers of endothelial cells (ECs) lining the vasculature. To examine whether Bb alters the physical forces of ECs to promote its dissemination, we exposed ECs to Bb and observed a sharp and transient increase in EC traction and intercellular forces, followed by a prolonged decrease in EC motility and physical forces. All variables returned to baseline at 24 h after exposure. RNA sequencing analysis revealed an upregulation of innate immune signaling pathways during early but not late Bb exposure. Exposure of ECs to heat-inactivated Bb recapitulated only the early weakening of EC mechanotransduction. The differential responses to live versus heat-inactivated Bb indicate a tight interplay between innate immune signaling and physical forces in host ECs and suggest their active modulation by Bb.

Keywords: Biophysics; Cell biology; Immunology; Microbiology; Transcriptomics.

Graphical abstract

Figure 1

ECs internalize Bb in a dose-dependent manner (A) Darkfield (left) and fluorescence image (right) of Bb constitutively expressing GFP (Bb-GFP) grown to a concentration of 5 × 10⁷ bacteria/mL in BSK-H media. (B and C) Barplots of percentage of ECs infected with Bb versus MOI (mean ± SD) (B) and corresponding histograms of the logarithm of Bb-GFP fluorescence intensity per cell for ECs infected with different MOIs of Bb (N = 3 replicate wells shown in different colors) (C). The histogram of control unexposed cells is shown in red. Based on the autofluorescence of the control group, a gate is defined showing what is considered non-exposed (left, GFP-) and exposed (right, GFP+). (D) Images of fixed samples of ECs exposed to Bb-GFP at a MOI = 11 at 4 hpe. Inside/outside staining was used to confirm internal localization of Bb. Left to right: brightfield image superimposed to maximum intensity projection of EC nuclei (blue), Bb-GFP fluorescence, antibody fluorescence of non-internalized adhering bacteria and overlay of the last two channels. White circle indicates an internalized spirochete. (E–G) Barplots of all Bb-GFP spirochetes per host cell nucleus (E), inside Bb-GFP spirochetes per host cell nucleus (F), and invasion efficiency (inside/outside Bb) (mean ± SD, WRST: ∗∗p<0.01, ns: non signficant) at different time points after exposure. N = 20 fields of view were segmented and analyzed. See also Figure S1 and Video S1.

Figure 2

ECs slow down during the early stages of Bb-exposure but their motility is recovered at later time points (A) Representative time-lapse epifluorescence microscopy images of ECs in monolayer during exposure to Bb at a MOI =200. Columns show: phase contrast image; Hoechst-stained EC nuclei; Bb-GFP fluorescence; cellular displacements. Rows show different time points after exposure. (B) Plot of mean host cell speed versus time (h) relative to the time point when host cells were exposed to Bb (mean ± SD, N = 3 recordings). Magenta dashed line corresponds to the time immediately after addition of Bb. (C) Boxplots of mean EC speed for ECs before Bb-exposure (tracked for 4 h) and after exposure to Bb at MOI = 200 up to 4 hpe. Different colors correspond to different recordings and circles depict mean spread in the whole field of view (mean ± SD, WRST: ∗p<0.05). See also Figure S2 and Video S2.

Figure 3

ECs weaken their traction stresses during the early but not late stages of Bb exposure (A) Representative phase contrast image (first column), Bb-GFP fluorescence (second column) and cellular traction stress map (third column, Pa) for ECs in monolayer at different time points (rows) after exposure to Bb-GFP (MOI =200). TFM was performed for ECs residing on 3 kPa ECM. The fourth and fifth columns show the corresponding phase contrast image and cellular traction stress map for cells not exposed to Bb. (B) Normalized strain energy (mechanical work) imparted by ECs during a TFM recording (mean ± SEM, N = 3 independent experiments). Strain energy has been normalized with respect to the first value at the beginning of each recording. Green: ECs exposed to Bb-GFP (MOI = 200); black: unexposed ECs. Time (h) is represented relative with respect to the time at whichBb was added. Time before exposure is shaded in red. (C) Plot showing the cross-correlation coefficient versus time of the cellular deformation maps obtained via TFM for successive frames separated by different time delays for unexposed or Bb-exposed ECs (MOI = 200) tracked for 24 hpe. An exponential decay function was fitted into the data yielding a rate constant K= 0.01943 for unexposed ECs and K= 0.01504 for Bb-exposed ECs (see STAR Methods). See also Figure S3.

Figure 4

EC monolayer stresses lower during early but not late Bbexposure (A) Sketch of the physical forces present in collectives of cells, and analogue in humans, in 1D. Cells in the monolayer are subjected to tensional and compressive stresses which at any instance balance with the traction stresses on the substrate. Left sketches depict the whole monolayer while right sketches focus on a single cell/human (green). Top. Idealized situation in which all cells are pushed by adjacent cells toward the center of the layer. Cells are subjected to compressive stresses while there are no tensile stresses exerted between cells (${\sigma }_I=0,{\sigma }_{II}$). In the human analogue, humans are subjected to compressive stresses that balance with each other and with the friction exerted in the ground, resulting in a zero net force in each individual when there is no movement. Bottom. Idealized situation in which all the cells in the monolayer are pulled by adjacent cells away from the center of the layer and are thus subjected to tensile stresses only (${\sigma }_I,{\sigma }_{II}=0$). The analogue for humans is also shown. (B) Representative phase contrast image with Bb-GFP fluorescence superimposed (first column), monolayer tensile stresses (${\sigma }_I$, second column) and compressive stresses (${\sigma }_{II}$, third column) for ECs in monolayer at different time points (rows) after exposure to Bb-GFP (MOI =200). Fourth-sixth columns show the corresponding phase contrast image, monolayer tensile stresses and absolute value of compressive stresses for ECs not exposed to Bb. (C) Normalized mean monolayer tensile stresses (${\sigma }_I$) as a function of time after exposure (mean ± SEM, N = 3 independent experiments). Mean ${\sigma }_I$ has been normalized with respect to the first value at the beginning of each recording. Green: ECs exposed to Bb-GFP (MOI = 200); black: unexposed ECs. Time (h) is represented relative to the time at whichBb was added. Time before exposure is shaded in red. See also Figure S4.

Figure 5

EC integrins β1 and αvβ3 colocalize with Bb and β1 shows increased localization compared to unexposed ECs (A) Representative brightfield image of cells superimposed with the Hoechst-stained nuclei image (first column), anti-β1 integrin antibody fluorescence (second column, maximum intensity projection), Bb-GFP fluorescence (third column, maximum intensity projection) and overlay of the last two channels (fourth column) for ECs exposed to Bb-GFP for 8 h. (B) Representative brightfield image of cells superimposed with the Hoechst-stained nuclei image and anti-β1 integrin antibody fluorescence for HMEC-1 not exposed to Bb. (C) Boxplots of normalized mean anti-β1 antibody fluorescence intensity per cell (mean ± SD, dots: individual cells) for ECs exposed to Bb-GFP for 8 h or unexposed ECs. Normalization is done with respect to the mean intensity of unexposed ECs. ∗∗: p<0.01, ns: not significant (Wilcoxon rank-sum test). (D–F) Same as in (A–C) but showing anti-αvβ3 integrin antibody fluorescence. In panels (A) and (D) pink circles denote co-localization of Bb-GFP and the indicated integrins while arrows point to Bb-GFP cells that do not colocalize with integrins. See also Figure S5.

Figure 6

ECs upregulate innate immune signaling pathways at four but not 24 hpe to Bb (A–C) Volcano plots of differentially expressed genes (DE-Gs). The-log₁₀ pvalues are plotted against the average log₂ fold changes in expression. For each pair of compared conditions the upregulated genes of each group are shown in the corresponding color. Each panel refers to a different time after exposure as indicated. (D) PCA of top genes that have ANOVA p value ≤0.05 on FPKM abundance estimations. PC1 versus PC2. (E) Pathway enrichment analysis. Bb-exposed ECs were compared to unexposed ECs based on their enrichment score (-log₁₀p). Resulting barplots for the different times after exposure are shown only for pathways that had-log₁₀p>3. See also Figure S6 and Table S1.

Figure 7

ECs sustainably weaken their force transduction and upregulate NF-κB target genes in response to heat-inactivated Bb (A) Representative phase contrast image overlayed with heat-inactivated Bb-GFP fluorescence (first column), EC traction stress map (second column, Pa), monolayer tensile stresses $({\sigma }_I$, third column, Pa) and absolute value of compressive stresses$({\sigma }_{II}$, fourth column, Pa) for ECs in monolayer at different time points (rows) after exposure to heat-inactivated Bb-GFP (MOI =200). TFM was performed for ECs residing on 3 kPa ECM. (B) Normalized strain energy imparted by ECs during a TFM recording (mean ± SEM, three independent experiments and N= 12 recordings in total). Strain energy has been normalized with respect to the first value at the beginning of each recording. Dark (light) green: ECs exposed to heat-inactivated Bb with an MOI = 200 (MOI= 22); black: unexposed cells. Time (h) is represented relative with respect to the time at whichBb was added. Time before exposure is shaded in red. (C) Same as panel B but showing the normalized mean EC monolayer tensile stresses (${\sigma }_I$) as a function of time after exposure to heat-inactivated Bb-GFP. (D) Relative with respect to GAPDH expression levels of the indicated NF-κB target genes obtained by RT-PCR. N = 3 independent experiments were performed. Three conditions were tested namely ECs exposed to nothing, to heat-inactivated Bb-GFP with an MOI = 200 for 4 h (blue) or for 24h (red). From top to bottom normalized expression of the following genes is shown: CXCL8, ICAM1, NFKBIA. Boxplots show the mean, 25^th and 75^th quartiles, different colors refer to replicates from independent experiments, ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001, ∗∗∗∗: p<0.0001 (Wilcoxon rank-sum test run for each condition’s distribution with respect to control distribution). See also Figure S7.