Transcriptome Profiling Reveals Pro-Inflammatory Cytokines and Matrix Metalloproteinase Activation in Zika Virus Infected Human Umbilical Vein Endothelial Cells

Abstract

The deformities in the newborns infected with Zika virus (ZIKV) present a new potential public health threat to the worldwide community. Although ZIKV infection is mainly asymptomatic in healthy adults, infection during pregnancy can cause microcephaly and other severe brain defects and potentially death of the fetus. The detailed mechanism of ZIKV-associated damage is still largely unknown; however, it is apparent that the virus crosses the placental barrier to reach the fetus. Endothelial cells are the key structural component of the placental barrier. Endothelium integrity as semi-permeable barrier is essential to control the molecules and leukocytes trafficking across the placenta. Damaged endothelium or disruption of adherens junctions could compromise endothelial barrier integrity causing leakage and inflammation. Endothelial cells are often targeted by viruses, including the members of the Flaviviridae family such as dengue virus (DENV) and West Nile virus (WNV); however, little is known about the effects of ZIKV infection of endothelial cell functions. Our transcriptomic data have demonstrated that the large number of cytokines is affected in ZIKV-infected endothelial cells, where significant changes in 13 and 11 cytokines were identified in cells infected with PRVABC59 and IBH30656 ZIKV strains, respectively. Importantly, these cytokines include chemokines attracting mononuclear leukocytes (monocytes and lymphocytes) as well as neutrophils. Additionally, changes in matrix metalloproteinase (MMPs) were detected in ZIKV-infected cells. Furthermore, we for the first time showed that ZIKV infection of human umbilical vein endothelial cells (HUVECs) increases endothelial permeability. We reason that increased endothelial permeability was due to apoptosis of endothelial cells caused by caspase-8 activation in ZIKV-infected cells.

Keywords: Zika virus; cell permeability; cytokines; inflammation; matrix metalloproteinase.

Figure 1

Zika virus (ZIKV) actively replicates in human umbilical vein endothelial cells (HUVECs). HUVECs were infected with PRVABC59 and IBH30656 ZIKV strains. ZIKV replication was detected by next-generation RNA-seq analysis (panel I), qPCR (panel II), Western blot (panel III), and immunofluorescence assay (panel IV) and plaque assay (panel V). (I) A: Mock-infected HUVECs (12 hpi); B: HUVECs infected with PRVABC59 ZIKV at 12 hpi; C: HUVECs infected with IBH30656 ZIKV at 12 hpi. (II) Total RNA was analyzed by qPCR targeting a region of the viral genome coding for the envelope protein. Relative copies of the viral genomes (ZIKV) were calculated using ∆∆Ct method. (III) Western blot analysis of envelope protein in ZIKV-infected HUVECs. Lane 1: Mock-infected HUVECs; lane 2: HUVECs infected with ZIKV strain PRVABC59; lane 3: HUVECs infected with ZIKV strain IBH30656. (IV) Immunofluorescence analysis of ZIKV envelope protein in the infected HUVECs. Images were captured using Carl Zeiss LSM780 confocal laser-scanning microscope. HUVECs infected with PRVABC59 ZIKV: A: envelope localization, B: nuclei; C: merge of A and B, D-DIC. HUVECs infected with IBH30656 ZIKV strain: E: envelope localization, F: nuclei; G: merge of E and F, H-DIC. Mock-infected HUVECs: I: envelope localization; J: nuclei; K: merge of I and J, L-DIC. Bar represents 10 µm size. (V) Plaque assay analysis of ZIKV replication in HUVECs. Supernatants were collected 72 h post-infection and used to determine infectious virus.

Figure 2

Venn diagram of genes affected in ZIKV-infected HUVECs. Changes in gene transcription were detected as early as 3 hpi, when transcriptional levels of 110 genes were affected in HUVECs infected with PRVABC59 ZIKV strain when compared to the mock-infected controls. Thirty-six genes were found differentially expressed at 3 hpi in HUVECs infected with IBH30656 ZIKV strain as compared to the control cells. Interestingly, 23 genes were affected by both ZIKV strains. At 12 hpi, 610 genes were affected by PRVABC59 ZIKV strain, while expression of only 112 was found changed in IBH30656 ZIKV-infected HUVECs. However, we found a group of genes (137 in PRVABC59 ZIKV and 326 in IBH30656 ZIKV), which were uniquely affected. Expression of 65 genes was affected by both strains.

Figure 3

Pathway analysis of transcriptome data. Pathway analysis was done using the Pathway Studio MammalPlus (Elsevier) tool. Analytes that differ significantly between groups were used for enrichment analysis. Only pathways with p < 0.05 after correction for the multiple comparison were selected. Blue color highlights analytes, which were significantly lower in IBH30656 ZIKV-infected HUVECs. (A) PR vs.CON-12hpi – Extracellular matrix Turnover; (B) NIG vs.CON-12hpi – Extracellular matrix Turnover; (C) - PR vs. CON-12hpi -Neutrophil Activation via FCGR3B.

Figure 4

Analysis of cytokines and MMP activation in ZIKV-infected HUVEC multiplex data. Culture supernatant (50 µl) was used to detect the level of 58 analytes (Bio-Plex human 21-Plex, 27-Plex, 40-Plex; BioRad) and 9 analytes (MMP 9-Plex; BioRad), respectively. Significantly different analytes (between infected and mock-infected groups) were used to generate the diagram. For every analyte, the ratio of cytokine median level in each ZIKV-infected HUVECs to that in mock-infected control was calculated. The binary logarithm of these values was used for sorting of analytes and generation of “lollypop” diagram with “ggplot2” package.

Figure 5

Interaction between cytokines in ZIKV-infected HUVECs: green, activation; blue, binding; black, reaction; thicker the line = stronger interaction; String 9.0 (http://string-db.org) high confidence 0.7 (circled are clusters k means 3). (A) 1BH30656 ZIKV strain, (B) PRVABC59 ZIKV strain.

Figure 6

Monocyte migration across ZIKV-infected endothelial monolayer. HUVECs were seeded in the upper compartment of the Boyden chamber and infected with PRVBC59 or IBH30656 ZIKV (MOI 0.1). Three days later, monocytes (10⁵ cells/inset) were added in the upper compartment. Migrated monocytes were analyzed 24 h later in the lower compartment. Experiments were repeated four times. (A) Monocyte count in the lower chamber. Monocytes were counted in five separate fields; *p < 0.03, **p < 0.0001 by paired t test. Data shown are the mean ± Sterr of four independent experiments. (B) Immunofluorescence analysis of monocytes in the lower chamber: A: mock-infected control; B: PRVBC59 ZIKV infected; C: IBH30656 ZIKV infected.

Figure 7

Transwell permeability assay. HUVECs were seeded onto Transwell inserts and infected with PRVABC59 or IBH30656 ZIKV (MOI 0.1) or mock. FITC-dextran was added into the upper compartment of the Transwell system. Aliquots of culture medium from the lower chamber were collected at selected time points to determine the presence of dextran molecule. Data are presented as a percent change in permeability as compared to mock-infected control. MMPs were inhibited using GM6001 (10 µM). Also, GM6001 negative control (10 µM) was used. (A) FITC-dextran permeability of HUVECs infected with PRVABC59 or IBH30656 ZIKV; (B) FITC-dextran permeability of HUVECs infected with PRVABC59 or IBH30656 ZIKV and treated with GM6001; (C) FITC-dextran permeability of HUVECs infected with PRVABC59 or IBH30656 ZIKV and treated with GM6001, negative control. ***p < 0.0001 by paired t test. Data shown are the mean ± Sterr of three independent experiments.

Figure 8

Immunofluorescence analysis of VE cadherin expression in ZIKV-infected HUVECs. HUVECs were infected with PRVABC59 and IBH30656 ZIKV strains for 72 h; monolayers were fixed and probed with rat anti-VE cadherin antibody. Nuclei were stained with TO-PRO-3. Images were captured using Carl Zeiss LSM 780 confocal laser-scanning microscope. (A) mock-infected control HUVECs; (B) HUVECs infected with PRVABC59 ZIKV; (C) HUVECs infected with IBH30656 ZIKV. White arrow: VE cadherin expression in control HUVECs; yellow arrow: decreased VE cadherin expression in ZIKV-infected HUVECs; red arrow: free of VE cadherin spaces between ZIKV-infected HUVECs. Bar represents 10 µm size.

Figure 9

Caspase-8 and annexinV activation in ZIKV-infected HUVECs. ZIKV replication was analyzed using Western blot (panel A) and qPCR (panel B). (A) Western blot analysis of envelop protein, caspase-8, and annexinV in ZIKV-infected HUVECs. HUVECs were infected with PRVABC59 and IBH30656 ZIKV. At 72 hpi, total proteins were collected and used for the detection of envelop protein, caspase-8, and annexinV. GAPDH was detected as the loading control. Lane 1: mock-infected HUVECs, lane 2: HUVECs infected with ZIKV strain PRVABC59; lane 3: HUVECs infected with ZIKV strain IBH30656. (B) qPCR analysis of virus transcript accumulation and caspase-8 transcription activation in ZIKV-infected HUVECs. Relative copies of each transcript were calculated using ∆∆Ct method. (C) IFA analysis of annexinV expression in ZIKV-infected HUVECs. A: mock-infected control; B: ZIKV strain PRVABC59; C: ZIKV strain IBH30656. Bar represents 10 µm size. (D) Effect of ZIKV replication on caspase-8 expression. UV-inactivated and replication-competent ZIKV PRVABC59 and IBH30656 were used to infect HUVECs. Total RNA was collected at 72 hpi and analyzed using qPCR. Relative copies of the viral genomes (ZIKV) were calculated using ∆∆Ct method. (E) Effect of ZIKV infection on cell vitality: mock-infected and ZIKV-infected HUVECs (IBH30656 and PRVABC59 strains) were counted using trypan blue. Experiments were performed in triplicate for three independent times.

Figure 10

Effect of caspase-8 inhibition on transwell permeability of ZIKV-infected HUVECs. HUVECs were seeded onto Transwell inserts and infected with IBH30656, PRVABC59 ZIKV (MOI 0.1), or mock. FITC-dextran was added into the upper compartment of the Transwell system. Aliquots of culture medium from the lower chamber were collected at selected time points to determine the presence of dextran molecule. Data are presented as a percent change in permeability as compared to the mock-infected control. Caspase-8 was inhibited using Ac-IETD-CHO (50 µM).

Figure 11

Activation of p-p38 MAPK, p38 MAPK, and NF-κB kinases in ZIKV-infected HUVECs. HUVECs were infected with PRVABC59 and IBH30656 strains of ZIKV. At 72 hpi, total proteins were collected and used to determine the level of p-p38 MAPK, p38 MAPK, and NF-κB kinases. GAPDH was detected as loading control.