SARS-CoV-2 infection of human brain microvascular endothelial cells leads to inflammatory activation through NF-κB non-canonical pathway and mitochondrial remodeling

Abstract

Neurological effects of COVID-19 and long-COVID-19 as well as neuroinvasion by SARS-CoV-2 still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro infection by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the Blood-Brain Barrier. Despite the low to non-productive viral replication, SARS-CoV-2-infected cultures displayed increased apoptotic cell death and tight junction protein expression and immunolocalization. Transcriptomic profiling of infected cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression, and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

Figure 1.. Characterization of infectivity profile of HBMECs by SARS-CoV-2.

(A) HBMECs were infected with different MOIs of SARS-CoV-2 (variant D614G) and viral production and release to supernatant was analyzed by RT-qPCR for Envelope (E) gene from 0 to 72 hours post infection (hpi). As compared to Vero cells, HBMECs showed a non-productive infection. (B) At desired time points (6 and 24 hpi) total RNA from cultures and expression of Spike1 and E genes were analyzed by RT-qPCR. Infection with MOI 0.1 showed an increase in expression of these two transcripts at 24 hpi (p>0.05). (C) Evaluation of SARS-CoV-2 receptors expression in infected HBMECs. ACE2 mRNA had a significant decrease at 24 hpi with the MOI 0.1, which did not translate to protein levels (right panel). TMPRSS2 had a slight increase in protein content at 24 hpi. (D) SARS-CoV-2 induces apoptotic cell death in Vero and HBME cells at 24 hpi as measured by cleaved caspase-3 immunostaining. *: p<0.05; ****: p<0.0001, Two-Way ANOVA with Bonferroni post-test of at least five independent experiments.

Figure 2.

Effects of SARS-CoV-2 on tight junctional proteins in Vero and HBMECs. A: Cells were stained for tight junction adaptor protein ZO-1 (red) and SARS-CoV-2 Spike1 (in green). ZO-1 was affected in infected cultures at 24 hpi as shown in higher magnification in the insets. B: Morphometrical analyses of ZO-1 fluorescence intensity and TiJOR (D) showed increased ZO-1 signal and TiJOR index at 6 hpi with the MOI 0.01. TJ proteins ZO-1 and Claudin-5 mRNA levels remained unaffected by infection (C), but were increased at 24 hpi by MOI 0.1 (D). *: p<0.05 One-way ANOVA with Bonferroni post-test (in D) or Two-Way ANOVA with Bonferroni post-test (in B and C); ****: p<0.0001, Two-Way ANOVA with Bonferroni post-test. Each point in C and D correspond to independent cultures and in B correspond to microscopic field from four independent cultures. Representative blots in D from three independent experiments.

Figure 3.. Transcriptomic profiling of SARS-CoV-2 infection on BBB-forming cells.

HBMECs were infected with MOIs 0.01 and 0.1 and analyzed by RNA-Seq. (A) Volcano plot depicting the overall profile of differentially expressed genes in cultures infected with MOI 0.1 at 24 hpi, with up-regulated genes shown in purple and down-regulated in green. (B) Heatmap diagram depicting expression levels of the most significantly altered genes by MOI 0.1 (3 right columns) as compared to uninfected controls (3 left columns). (C) Cnetplot visualization of functional enrichment results with up-regulated genes, depicting the functional correlation of genes with the most significant GO terms. (D) Enrichment functional analysis of GO terms most affected by SARS-CoV-2 in HBMECs indicate inflammatory endothelial activation, as well as mitochondrial dysfunction and ribosomal-related gene expression. (E) RT-qPCR validation of most significantly altered genes detected in the RNA-Seq indicate activation of non-canonical NF-κB pathway, with massive increase in TNF-α, lymphotoxin B (LTB, or TNF-C) and downstream target genes such as IL-6, CXCL1, −2 and −8. NFKB1 (p105/p50) and NFKB2 (p100/p52) as well as JUNB did not show alteration in infected cultures. *: p<0.05; **: p<0.01; ****: p<0.0001, Two-Way ANOVA with Bonferroni post-test of at least 5 independent experiments. MOI: multiplicity of infection; GO: gene ontology.

Figure 4.. Production of angiogenic-related molecules is modulated by SARS-CoV-2 in HBMECs.

(A) Conditioned medium from Mock and SARS-CoV-2-infected HBMEC cultures (both with MOI 0.01 and 0.1) were analyzed with Proteome Profiler Human Angiogenic Antibody Array and detected by chemoluminescence, each protein detected in duplicated spots. (B) Densitometric analysis of membranes in A revealed the analytes with strongest signal and which were affected by infection. (C) RT-qPCR analysis of angiogenesis-related genes in HBMECs revealed that PTX3 and HIF-1α were increased in infected cultures. *: p<0.05; **: p<0.01, Two-Way ANOVA with Bonferroni post-test of at least five independent experiments.

Figure 5:. SARS-CoV-2-induced mitochondrial remodeling in HBMECs.

Mitochondrial networks were detected by TOMM20 immunostaining (A) and TEM (B). MiNA analysis of TOMM20 revealed that SARS-CoV-2 induced an increase in mitochondrial footprint, branch length mean and summed branch length (C). Mitochondrial density was calculated by TEM images (D), which also revealed increased fusion and association to multivesicular bodies (B). E: RT-qPCR (upper panel) and western blotting (lower panel) analyses revealed that despite fission-related genes (Fis1, Drp1 and MFF) were up-regulated in MOI 0.01-infected cultures, only Mfn2 protein levels were increased in MOI 0.1-infected cultures. TOMM20 protein levels also remained unaltered. *: p<0.05; **: p<0.01; ***:p<0.001; ****: p<0.0001, Two-way ANOVA with Bonferroni post-test. Each dot in graphs represents one cell (in C), one mitochondrion (in D) or one independent experiment (in E). Bottom right panels depict blots from E. Scale bars: 50 μm for A and 500 nm for B.