Identification of SARS-CoV-2 inhibitors using lung and colonic organoids. Wynn, T. A., Chawla, A. In addition, the expression of ACE2 and TMPRSS2 was undetectable in the bulk RNA-seq of iMs (Supplementary Fig. In summary, these results demonstrated a differential immune response of iM2s versus iM1s or iM0s upon SARS-CoV-2 infection. The yellow-orange color in the merged images indicates the colocalization of SGK1 and CD68 + macrophages in decidual tissue, n = 3, at 630 . Data are presented as mean values SD. This was followed with differentiation into functional macrophages by treatment with monocyte inducing cytokines, IL-3, and Macrophage-colony stimulating factor (M-CSF) (Supplementary Fig. Nature Communications thanks Lesley Forrester, Antonio Sica and the other anonymous reviewers for their contribution to the peer review of this work. The analysis showed decreased expression of proliferation-associated genes MKI67 and TOP2A and increased expression of apoptosis-related genes TP53, CASP3, BAX, MCL1, in the iLungs co-cultured with iM1s, but not in that co-cultured with iM2s (Supplementary Figs. PubMed hESC differentiation into endoderm was performed in serum-free differentiation (SFD) medium of IMDM/F12 (3:1) (Life Technologies) supplemented with N2 (Life Technologies), B27 (Life Technologies), 50g/ml ascorbic acid, 2mM Glutamax, 0.4M monothioglycerol, and 0.05% BSA at 37C in a 5% CO2/5% O2/95% N2 environment. We detected SARS-N protein in all types of cells except A549 cells, indicating the viruses could enter Calu3 cells and iMs, and had different ratios of overlapped regions (Supplementary Fig. performed the scRNA- and bulk RNA sequencing, and raw datasets conversion to matrices. Negative controls stained with control IgG instead of primary antibodies were always performed with sample measurements. Article After 24h, Y27632 was withdrawn from the medium and cells were cultured for another 24h. At day 0, cells were firstly induced by macrophage differentiation basal medium (SFD-M) which is RPMI 1640 medium supplemented with 2% B27 (Thermo Fisher Scientific), 1% L-GlutaMAX-I and 50g/ml ascorbic acid (Sigma Aldrich) and 10ng/ml BMP4 (R&D Systems) for 24h. Afterward, the medium was changed to SFD-M medium containing 10ng/ml BMP4 and 2M GSK3 inhibitor CHIR99021 (Cayman Chemical) for another 48h. On day 3, cells were replated onto Matrigel-coated dishes at a density of 4104 cells/ cm2 in SFD-M medium with 50ng/ml VEGF (R&D Systems) and 10ngng/ml FGF2 (R&D Systems) for 48h. On day 5, the medium was replaced with basal medium with 50ng/ml VEGF, 10ngng/ml FGF2 and 10uM TGF inhibitor SB431542 (R&D Systems) for another 72h. On day 810, floating cells were collected and medium was changed and supplemented with 50ng/ml M-CSF and 10ng/ml IL-3 (R&D Systems) for another 3-5 days. CD68 and CD163 are used to identify macrophages in tissue sections. 4c). . Differential gene expression analysis was performed by DESeq249. Slider with three articles shown per slide. indicates a non-significant difference. The hESC line-RUES2 and H1 are both purchased from WiCell and were cultured on irradiated mouse embryonic fibroblasts (Global Stem) at a density of 20,00025,000 cells/cm2 in a medium of DMEM/F12, 20% knockout serum replacement (Life Technologies), 0.1mM -mercaptoethanol (Sigma Aldrich) and 20ng/ml bFGF (R&D Systems), and medium was changed daily. Proc. 28, 511515 (2010). 6d, e) and were readily polarized to CD68+CD206+ macrophages, or CD68+FCN1+STAT1hi macrophages (Supplementary Fig. Philos. 8, 1352 (2017). 1b), in agreement with the above-cited report. Deciphering human macrophage development at single-cell resolution. SARS-CoV-2, isolate nCoV/Washington/1/2020 (kindly provided by the National Biocontainment Laboratory, Galveston, TX), was propagated in Vero E6 cells in DMEM supplemented with 2% FBS, 4.5g/L D-glucose, 4mM L-glutamine, 10mM Non-Essential Amino Acids, 1mM Sodium Pyruvate and 10mM HEPES44. positive cells in lungs were randomly counted from different visions of slides by confocal microscopy. 5e, f). Although primary macrophages are more functionally, or phenotypically, representative of the native macrophages of the tissue from which they are derived, they proliferate slowly, are difficult to obtain, and are often poorly characterized14. Nature 560, 377381 (2018). For induction of AFE, the endoderm cells were cultured in SFD medium supplemented with 1.5M Dorsomorphin dihydrochloride (R&D Systems) and 10M SB431542 (R&D Systems) for 48h, and then switched to 24h of 10M SB431542 and 1M IWP2 (R&D Systems) treatment. In agreement with this phenotype, the RNA-seq analysis detected higher viral loads in the iM2s than in iM1s or iM0s, after incubating iMs with SARS-CoV-2 for 24h (Supplementary Fig. Human primary macrophages derived in vitro from circulating monocytes comprise adherent and non-adherent subsets with differential expression of Siglec-1 and CD4 and permissiveness to HIV-1 infection. 5h). 117, 1728 (2015). PubMed Han, Y. et al. 9, 205 (2018). ADS After 24h of incubation with the SARS-CoV-2 virus (USA-WA1/2020, MOI=0.1), a significantly greater decrease in the amount of viral proteins (SARS-CoV-2 nucleocapsid (N) proteins) was observed in the co-culture of iLungs and iMs compared to the co-culture of iLungs and 293T cells (293T cells were used as a co-culture control, based on our preliminary data and previous report that the permissiveness of 293T to SARS virus is low32). These results aligned with the phenotype of pro-inflammatory activities of iM1s, as both bulk RNA-seq and scRNA-seq data detected a set of pro-inflammatory factors, IL-1B, IL-18, STAT1, FCN1, CXCL9, CXCL10, CXCL11, CXCL16, and CCL2 highly expressed in iM1s (Supplementary Figs. Liao, M. et al. However, M1 and non-activated (M0) macrophages, but not M2 macrophages, significantly up-regulate inflammatory factors upon viral infection. Circ. Most COVID-19 patients show mild to moderate symptoms of fever, dry cough, fatigue, and diarrhea. We thank the other members in Huanhuan Joyce Chen lab (University of Chicago), Qizhou Lian Lab (University of Hong Kong), Shuibing Chen (Weill Cornell Medicine), Todd Evans (Weill Cornell Medicine), Sarina Zhao (Lab school, University of Chicago), the Varmus Laboratory (Weill Cornell Medicine), and Oebiotech. Xu, C. et al. Iba1 (+) and Gal-3 (+) macrophages showed both M1- and M2-immunophenotypes. Plasschaert, L. W. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Thus, we examined if macrophages were dominantly presented in the diseased patients lungs. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Rev. 4 ). b Heatmap analysis of cytokines, chemokines, receptors, phagocytosis and cell death related genes in iM0, iM1s and iM2 cells at 24hpi or 48 hpi. To make sense of macrophages polarization in different distribution patterns, studies could be conducted around . J. Exp. M1 macrophages secrete proinflammatory cytokines (TNF-, IL-6, and IL-12) and chemokines (MCP-1), and induce inflammation and tissue destruction [25]. They reported the pathological changes of lungs in critical COVID-19 infection41 (a 66 year-old male, a 62 year-old female, and a 31 year-old male; all patients are Asian Chinese). In contrast, no significant alteration of the aforementioned cell death-related genes or signaling pathways was observed in iM2s following viral infection (Fig. Principal component analysis was performed using Log2 FPKM values with R ggplot2 package. 1a). A Broader Perspective in the Third Year of a Worldwide Pandemic, Evaluating the transmission feasibility of SARS-CoV-2 Omicron (B.1.1.529) variant to 143 mammalian hosts: insights from S protein RBD and host ACE2 interaction studies. Subramanian, A. et al. Although the traditional M1/M2 model can be applied to in vitro cultured and stimulated cells to describe different types of macrophage activation, new insights into the intricate network of tissue macrophages clearly reveal that it fails to adequately describe the functional and phenotypic diversity of these cells in vivo . Several effective methods have recently been described16,17,18 for generating major cell types found in human lung tissues by directed differentiation of hPSCs using growth factors and chemicals to alter cell fate-determining signaling pathways. However, characterisation of polarised macrophages in situ has remained difficult. The average ratios of CD68 + iNOS + M1 macrophages to CD68 + CD163 + M2 macrophages (M1/M2 ratios) were greater than 2.5 for the wounds aged 2-5 days. McCray, P. B. Jr et al. Immunohistologic methods were used to identify macrophage surface markers CD68 (pan macrophages), CD80 and CCR7 (M1 profile), and CD163 (M2 profile) during the remodeling process. 2d). The . Cluster of differentiation (CD)68 may be used as a pan-macrophage or M1 marker, whereas CD163 may be used as an M2 marker. We discovered that pro- and anti-inflammatory macrophages both have similar capacities to eliminate the virus in the context of a moderate viral load. Next, bulk RNA-seq was performed to thoroughly examine the immune response in iMs after SARS-CoV-2 infection (Fig. Article To obtain 4g, and Supplementary Fig. 7c) upon stimulation by IL-4 or IFN and LPS, respectively (Supplementary Figs. CD68+ TAMs correlated with favorable prognosis in several organs, such as prostate [ 20 ], lung [ 21 ], and brain tumors [ 22 ]. In contrast, iM2s mainly expressed anti-inflammatory factors or immunoregulatory genes such as TGM2, APOE, A2M, CCL13, CCL26, and TREM2 (Supplementary Figs. We found that SARS-CoV-2 infection induced or strengthened inflammatory reaction in M0 or M1 macrophages, leading to expression of a cohort of inflammatory factors with similarity to those observed in patients with severe COVID-19 and CRS39. Huang, C. et al. Induction of pro-inflammatory cytokines (IL-1 and IL-6) and lung inflammation by COVID-19: anti-inflammatory strategies. The protocol details are summarized in Supplementary Fig. ADS In vitro generation of human pluripotent stem cell derived lung organoids. Hoffmann, M. et al. N. Engl. Nico et al. Therefore, we conducted a series of experiments to investigate the immune response of M1, M2, or non-activated macrophages (M0) to SARS-CoV-2 infection, which could reflect the differential responses seen in patients at different stages of macrophage activation. f Representative bright-field and fluorescence images of the co-culture of lung cells and macrophages derived from hPSC line RUES2. c Immunofluorescence (IF) staining on Non-COVID-19 or COVID-19 distal lung tissues using antibodies against CD80 (M1 marker) or CD163 (M2 marker). The role of M1 macrophages is to secrete pro-inflammatory cytokines and chemokines, present antigens, and thus participate in the positive immune response and function as an immune monitor. 8, 420422 (2020). Stem Cell Rep. 11, 348362 (2018). The normalized libraries were pooled and sequenced on Illumina Novaseq 6000 sequencer with pair-end 50 cycles.The sequencing libraries sequenced with paired-end 50bps on NovaSeq6000 sequencer. Macrophage polarization is increasingly recognised as an important pathogenetic factor in inflammatory and neoplastic diseases. helped with FACS on macrophage phenotype and cytokine expression analysis. Polarized or non-polarized iMs were incubated with SARS-CoV-2 for 4h followed by the collection of the supernatant and extraction of the RNA from infected cells at 6, 24, and 48h-post infection for downstream analysis (Fig. Clinical characteristics of coronavirus disease 2019 in China. Heatmaps were generated using R gplots package. Bar graph showing the GO enrichments of the overlap genes. The number of CD68+ macrophages was obviously up-regulated in the DGalN-treated group (Figure (Figure4). Our results demonstrate different immune reaction patterns among distinct phenotypes of macrophages; SARS-CoV-2 infection tends to trigger a cascade of inflammatory pathways and augment inflammation in M1 and M0 macrophages, but not in M2 macrophages. 5ad). CAS p values were calculated by one-way ANOVA with Tukeys multiple comparison test. conceived the project and recruited the collaborating partners. provided and characterized the clinical human lung tissues. 8a, b). Abnormal M1/M2 polarization of decidual macrophages might predispose immune maladaptation in recurrent pregnancy loss (RPL). Med. ADS 74 Such CD68-positive macrophages are called tumor-associated . 1c, d and Supplementary Fig. d Quantification of CD80+ and CD163+ macrophages in Non-COVID-19 or COVID-19 distal lung tissues. Ziegler, C. G. et al. World Allergy Organ 13, 100126 (2020). and Y.C. An hPSC-derived tissue-resident macrophage model reveals differential responses of macrophages to ZIKV and DENV infection. Med. The next day, GFP-labeled latex beads (1.0m mean particle size, Sigma) were added at a ratio of iMACs: beads = 1:10. On days 15 and 16, the lung progenitor cells were replaced after 1-min trypsinization onto fibronectin-coated plates, in the presence of SFD containing 3M CHIR99021, 10ng/ml human FGF10, 10ng/ml human FGF7, in a 5% CO2/air environment. Scale bar = 100m. The sequencing data were primarily analyzed by CellRanger pipeline v3.0.2 (10 Genomics). Article Finally, inhibiting viral entry into target cells using an ACE2 blocking antibody diminished viral infection and enhanced the elimination of viruses. Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus. For co-culture analysis, Macrophages and lung cells were re-clustered and re-analyzed, respectively. Recently, a transgenic mouse strain37 has been made with human ACE2 expression regulated by a human cytokeratin-18 promoter, but the ACE2 expression in humans is more complex than its expression in the mice. Immunostaining results showed that iM2s had the most SARS-CoV-2- N proteins, not in the iLungs, while the N protein was clearly found in iLungs co-cultured with iM1s, iM0s, and 293T cells (Fig. N=3 independent biological replicates were used for all experiments unless otherwise indicated. The abundance of transcripts was measured with Cufflinks in Fragments Per Kilobase of exon model per Million mapped reads (FPKM)46,47. RNA concentrations were measured using the NanoDrop system (Thermo Fisher Scientific). EPO treatment significantly attenuated pro-inflammatory genes (IL1, iNOS, and CD68) and augmented anti-inflammatory genes (IL10 and CD163) and the cell-surface marker CD206. Google Scholar. Gene Ontology (GO) or Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis comparing iM1s, iM2s, and iM0s validated the activation of classical signaling pathways such as JAK-STAT and Toll-like receptor signaling pathways in iM1s; and cell-cell adhesion, osteoclast differentiation, and phagosome related pathways in iM2s (Supplementary Fig. These results indicated that cell death or decrease in cell viability may occur in iM1s or iM0s, especially 48h after infection; this phenotype could attribute to viral damage or hyper-inflammatory reaction of macrophages upon infection. Nat. Statistically significant differences are calculated using an unpaired two-tailed unpaired Students t-test. 4c and Supplementary Fig. Zhang, J. et al. Moreover, Gene Set Enrichment Analysis (GSEA) of KEGG signaling pathways underlined that Toll-like receptor and chemokine signaling pathways were highly enriched in iM1s, indicating a pro-inflammatory state, while adhesion and extracellular matrix receptor-related genes were upregulated in iM2s, indicating a state of alternative activation (Fig. The authors declare no competing interests. Ther. . To model the immune response of macrophages to SARS-CoV-2 infection in lung cells, the virus was introduced to the co-culture system (Fig. The diagnosis of COVID-19 pneumonia was based on the Coronavirus Pneumonia Prevention and Control Plan (7th edition) newly issued by the National Health Commission, China42. Dye, B. R. et al. 1gi), which represented alveolar and interstitial macrophages, respectively. Lian, Q., Zhang, K., Zhang, Z. et al. PubMed . CD68 + cells are a functionally active subset of macrophages that are associated with increased iNOS and arginase staining and altered gene expression. H.J.C., Q.Z.L., and F.D. Google Scholar. The differentially expressed genes were found by vst method and the top 3000 differentially expressed genes were selected for PCA analysis. It is urgent to understand the interactions among permissive cells, macrophages, and the SARS-CoV-2 virus, thereby offering important insights into effective therapeutic strategies. c Venn plot comparing the overlap of upregulated and downregulated genes upon SARS-CoV-2 infection in iM1 and iM0 cells at 24hpi. In general, M1 cells destroy pathogens by producing a large amount of pro-inflammatory cytokines. The pan marker for macrophages in rat is CD68. USA 102, 1554515550 (2005). Nuclei were counterstained by Hoechst 33342 (Sigma-Aldrich). PVAT surrounding mesenteric arteries was fixed and immunolabeled for total macrophages (MT, F4/80), M1-like macrophages (M1, CD68) and M2-like macrophages (M2, CD206).