A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV-2 at the immune–epithelial interface

Significance By modeling in vitro the cross-talk between epithelial and immune cells, this work provides possible origins for the profound inflammatory perturbations that are a hallmark of COVID-19, and the relative protection of children from severe disease. The initial interaction between immune cells and epithelial cells infected with SARS-CoV-2, or transduced to express the proteins the virus encodes, elicits a specific response, not observed with other pathogenic viruses, that presages perturbations seen in patients with severe COVID-19. Thus, the severe manifestations of COVID-19 may be rooted in the very first response that it elicits from immunocytes.

cultured in DMEM supplemented with 2% FBS, penicillin (50 U/ml), and streptomycin (50 mg/ml). To remove confounding cytokines and other factors, viral stocks were purified by ultracentrifugation through a 20% sucrose cushion at 80,000 x g for 2 h at 4°C (1). Viral titers were determined in Vero E6 cells by tissue culture infectious dose 50 (TCID50) assay and calculated using the Spearman-Kärber algorithm.
All work with EBOV and CoV2 was performed in the biosafety level 4 (BSL-4) facility of the National Emerging Infectious Diseases Laboratories at Boston University, Boston, MA following approved SOPs and inactivation procedures.

Influenza A virus propagation and titration
Influenza A PR8-GFP virus (A/Puerto Rico/8/1934(H1N1), hereafter IAV) and Madin-Darby canine kidney (MDCK) cells were provided by D. Lingwood (Ragon Institute). IAV stock was made using MDCK cells and titer was determined by plaque assay as described previously (2).

Viral infection of Caco-2 cells for co-culture
One day prior to infection, Caco-2 cells were seeded at a density of 10 5 cells per well of a 24-well tissue culture plate or 25.10 3 cells per well of a 96-well tissue-culture plate. Twenty-four hours later, cells were infected with CoV2 or EBOV at a nominal MOI of 10, with IAV at nominal MOI ranging from 0.1-10. After an adsorption period (2h for CoV2 and EBOV, 1h for IAV), the inoculum was removed, replaced with fresh media (2% FBS supplemented DMEM), and cells were incubated at 37˚C for 35 h prior to coculturing with PBMC.

Plasmids and transfections
Expression plasmids were kindly provided by D. Gordon

Coculture of PBMCs with infected or transfected epithelial cells
The same experimental design was used for viral-co-cultures and transfectant-co-cultures. 35 h post-infection or 24-48 h post-transfection, adherent epithelial cells were washed gently twice with standard culture media (DMEM, 10% FBS, penicillin (50 U/ml), streptomycin (50 mg/ml), and 1% MEM non-essential amino acids) to remove cell debris. Frozen PBMCs were thawed in a 37°C water bath for 90-120 sec then added dropwise to 9 mL of pre-warmed culture medium and centrifuged at 300 x g for 7 min. After removing supernatant, the PBMC pellet was resuspended in pre-warmed culture medium at a concentration of 1.5.10 6 cells/ml. This PBMC suspension was slowly added to the epithelial cells at a final concentration of 7.5.10 5 PBMCs/well (24-well plate, infections) or 2.10 5 PBMCs/well (96-well plate, transfections). PBMCs and epithelial cells were then co-cultured for 14 h in a 37°C incubator (5% CO2).
For Transwell transfectant experiments, HEK cells were transfected in standard flat-bottom plates, dissociated into single cell suspension, and re-plated at 30,000 cells per well onto 24-well Transwell polyester 0.4 μm pore membrane inserts (Corning) with media filling both top and bottom chambers. Twenty-four hours after seeding, media from both chambers was replaced and 2.10 5 freshly thawed PBMCs were added to the bottom chamber for 14 h co-culture.
CoV2 and IAV co-cultures were performed in three independent experiments (one pilot, one main experiment and one replication experiment) with at least 3 biological replicates per condition. EBOV co-culture was performed for one experiment with 3 independent replicates.
Transfectant co-culture were performed in two independent experiments for each cell line (each experiment used a different preparation of plasmid DNA), and each experiment included 2 biological replicates for each transfected protein.

Cell treatments
To determine the response to LPS, PBMCs were cultured for 14 h, in parallel to HEK cocultures, with or without 1 ng/mL of LPS (LPS from E. coli O55:B5, Sigma, Cat# L2880) for 14 h. To assess the effects of co-culture with lysed cells, a freshly passaged single cell suspension of HEK cells at 2.5.10 5 cells/ml was frozen on dry ice, then thawed rapidly at 37°C. The freeze-thawed suspension was centrifuged at 500 x g to eliminate debris and added at a 10-fold dilution in PBMC cultures for 14 h.

Magnetic isolation of PBMCs in viral co-cultures
Due to lack of flow cytometry sorting in the BSL-4 containment laboratory, different In parallel, Caco-2 cells seeded in a 96-well plate were infected with one of the three viruses and cultured alone. At 48 h post infection (similar harvesting timepoint as for co-cultures), the cells were washed and directly lysed in 125 µL of TCL/ME lysis buffer, and heat-treated as above.

Population Low-input RNA-seq
Low-input RNA-seq was performed following the standard ImmGen low-input protocol (www.immgen.org), from the 5μl of collected lysis buffer. For the viral co-cultures, magnetically isolated samples were centrifuged at maximum speed (17,000 x g) for 3 min at 4°C before loading, in order to pellet the cellular debris and magnetic particles, the cleaned RNA lysate remaining in the supernatant. Smart-seq2 libraries were prepared as described previously (4) with slight modifications. Briefly, total RNA was captured and purified on RNAClean XP beads (Beckman Coulter). Polyadenylated mRNA was then selected using an anchored oligo(dT) primer (50 -AAGCAGTGGTATCAACGCAGAGTACT30VN-30) and converted to cDNA via reverse transcription. First strand cDNA was subjected to limited PCR amplification followed by Tn5 transposon-based fragmentation using the Nextera XT DNA Library Preparation Kit (Illumina).
Samples were then PCR amplified for 12 cycles using barcoded primers such that each sample carries a specific combination of eight base Illumina P5 and P7 barcodes for subsequent pooling and sequencing. Paired-end sequencing was performed on an Illumina NextSeq 500 using 2 x 38bp reads with no further trimming.

RNA-seq data processing and QC
Reads were aligned to the human genome (GENCODE GRCh38 primary assembly and Quality control: Samples with less than 1 million uniquely mapped reads were automatically excluded from normalization to mitigate the effect of poor-quality samples on normalized counts. Samples having fewer than 8,000 genes with over ten reads were also removed from the data. We screened for contamination by using known cell type specific transcripts (per ImmGen ULI RNAseq and microarray data). Finally, the RNA integrity for all samples was

Statistical analysis
Unless specified otherwise, the data are presented as mean ± SD and tests of associations