SARS-CoV-2 induces barrier damage and inflammatory responses in the human iPSC-derived intestinal epithelium

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread and led to global health crises. COVID-19 causes well-known respiratory failure and gastrointestinal symptoms, such as diarrhea, nausea, and vomiting. Thus, human gastrointestinal cell models are urgently needed for COVID-19 research; however, it is difficult to obtain primary human intestinal cells. In this study, we examined whether human induced pluripotent stem cell (iPSC)-derived small intestinal epithelial cells (iPSC-SIECs) could be used as a SARS-CoV-2 infection model. We observed that iPSC-SIECs, such as absorptive and Paneth cells, were infected with SARS-CoV-2, and remdesivir treatment decreased intracellular SARS-CoV-2 replication in iPSC-SIECs. SARS-CoV-2 infection decreased expression levels of tight junction markers, ZO-3 and CLDN1, and transepithelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics. In addition, SARS-CoV-2 infection increased expression levels of proinflammatory genes, which are elevated in patients with COVID-19. These findings suggest iPSC-SIECs as a useful in vitro model for elucidating COVID-19 pathology and drug development.

Keywords: Barrier functions; COVID-19; SARS-CoV-2; Small intestinal epithelial cells; iPSC.

Fig. 1

SARS-CoV-2 infection in human iPSC-SIECs. (A) The expression levels of ACE2 and TMPRSS2 in iPSC-SIECs and adult intestines were examined by RT-qPCR. (B) After the cells were infected with SARS-CoV-2 (MOI = 1, 2.5) for indicated time courses, the intracellular viral copy number was determined by RT-qPCR. (C) The relative value of SARS-CoV-2 copy number was indicated with the infectious condition at MOI = 1 for 24 h as 100%. (D) After SARS-CoV-2 infection (MOI = 1) for 24 h, the cells were stained with antibodies against SARS-CoV-2 nucleocapsid and SARS spike glycoprotein. Nuclei were counterstained with DAPI. Bar = 20 μm. Data are represented as mean ± standard deviation (SD; n = 3).

Fig. 2

Cell-type-specific incorporation of SARS-CoV-2 in human iPSC-SIECs. (A) The expression levels of cell-type-specific markers in SIECs and the adult intestine were examined by RT-qPCR. (B) After SARS-CoV-2 infection (MOI = 1) for 24 h, the cells were stained with antibodies against cell-type specific markers and SARS spike glycoprotein. Nuclei were counterstained with DAPI. Bar = 20 μm. Data are represented as mean ± standard deviation (SD; n = 3).

Fig. 3

Effect of remdesivir on SARS-CoV-2 infection in human iPSC-SIECs. (A) Cells were treated with remdesivir (1 μM) 1 h before SARS-CoV-2 infection (MOI = 1) for 24 h. The intracellular viral copy number was determined by RT-qPCR. Left panel: SARS-CoV-2 RNA copies per well. Right panel: Normalized value of SARS-CoV-2 RNA copies against vehicle control as 100%. (B) After the cells were treated with remdesivir and infected with SARS-CoV-2, viral titers in the culture supernatants were determined by plaque assay in Vero E6 cells. Data are represented as mean ± SD (n = 3). ∗P < 0.05.

Fig. 4

Effect of SARS-CoV-2 infection on the intestinal epithelial barrier. (A) Cells were seeded in Transwell chambers. After the cells were treated with remdesivir and infected with SARS-CoV-2 for 24 h, the TEER values across monolayers were measured. (B) After the cells were infected with SARS-CoV-2 for 24 h, tight junction marker genes (ZO-1, ZO-2, ZO-3, CLDN1, CLDN2) were analyzed by RT-qPCR. (C) After the cells were treated with remdesivir and infected with SARS-CoV-2 for 24 h, tight junction marker genes (ZO-3, CLDN1) were analyzed by RT-qPCR. Data are represented as mean ± SD (n = 3). ∗P < 0.05.

Fig. 5

Effect of SARS-CoV-2 infection on inflammatory responses. Cells were treated with remdesivir (1 μM) 1 h before SARS-CoV-2 infection (MOI = 1) for 24 h. Inflammatory response genes (IL-1β, IL-6, CCL2, CCL3, CCL5, and CXCL10) were analyzed by RT-qPCR. Data are represented as mean ± SD (n = 3). ∗P < 0.05.