Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 1. Generation of iPSC-derived alveolar epithelial type II cells (iAECIIs) as an HCoV-229E infection experimental model. (A) Outline of experimental design. (B) Bright-field images showing the morphology of iPSCs 3 and 5 days after seeding. (C) Immunofluorescent staining showing the expression of NANOG marker in iPSCs. Nuclei are stained with DAPI. (D) Schematic diagram showing a stepwise protocol of differentiation of iPSCs into iAECIIs. (E) Bright-field images showing cell morphology at the indicated time points of differentiation of iPSCs into iAECIIs.

Article Snippet: The following primary antibodies were used: SFTPB, HCoV-229E spike protein (PAB21477–100/ PAB21477–500; The Native Antigen Company, Kidlington, UK), HCoV229E nucleocapsid protein (#40,640-T62; Sino Biological, Beijing, China), GAPDH (10,494–1-AP; Rosemont, IL, USA).

Techniques: Derivative Assay, Infection, Staining, Expressing, Marker

Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 3. iAECIIs as a model of infection with HCoV-229E. (A) Immunofluorescent staining of iAECII-containing spheroids showing the co-expression of EpCAM iAECII marker and CD13 HCoV-229E receptor. (B) Bright field images of iAECIIs cultures at 24 and 48 h post-infection (hpi) with HCoV-229E. (C) Immunofluorescent staining of viral dsRNA in iAECII culture after 48 h of infection with HCoV-229E. White arrows show the dsRNA signal. In (A) and (C) – nuclei are stained with DAPI. (D) qRT-PCR analysis of the expression of HCoV-229E S protein-encoding mRNA in iAECIIs infected with HCoV-229E for the indicated time periods. Data are expressed relative to 0 h time point. Means are shown, n = 3, SD error bars, p < 0.005 (Student’s test) (E) Western blot showing the expression of HCoV-229E S and N proteins in iAECIIs infected with HCoV-229E for the indicated time periods. GAPDH – loading control.

Article Snippet: The following primary antibodies were used: SFTPB, HCoV-229E spike protein (PAB21477–100/ PAB21477–500; The Native Antigen Company, Kidlington, UK), HCoV229E nucleocapsid protein (#40,640-T62; Sino Biological, Beijing, China), GAPDH (10,494–1-AP; Rosemont, IL, USA).

Techniques: Infection, Staining, Expressing, Marker, Quantitative RT-PCR, Western Blot, Control

Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 4. Infected iAEClls enhance HL-60 migration and adhesion behavior. (A) Bright-field images of HL-60 neutrophils co-cultured with the medium conditioned by uninfected iAECIIs (Ctrl) and conditioned by iAECIIs infected with HCoV-229E for 48 h (48 hpi). White arrows point at granules. (B) Schematic outline of transwell migration assay. (C) Transwell migration assay showing the migration capacity of HL-60 neutrophils in response to a medium conditioned by iAECIIs infected with HCoV-229E for 24 or 48 h (24 hpi or 48 hpi). Ctrl – uninfected control. Data shown as means with SD error bars, n = 3, *p < 0.005 (ANOVA). (D) Schematic outline of adhesion assay. (E) Representative fluorescence microscopy images of adherent HL-60 neutrophils after incubation with infected iAECIIs (24 hpi and 48 hpi) for the indicated time periods. (F) Cell count of adherent HL-60 neutrophils. Mean values are shown with SD error bars, n = 3, *p < 0.005 (ANOVA). In (E) and (F), Ctrl – virus-free control.

Article Snippet: The following primary antibodies were used: SFTPB, HCoV-229E spike protein (PAB21477–100/ PAB21477–500; The Native Antigen Company, Kidlington, UK), HCoV229E nucleocapsid protein (#40,640-T62; Sino Biological, Beijing, China), GAPDH (10,494–1-AP; Rosemont, IL, USA).

Techniques: Infection, Migration, Cell Culture, Transwell Migration Assay, Control, Cell Adhesion Assay, Fluorescence, Microscopy, Incubation, Cell Counting, Virus

Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 5. Identification of cytokines secreted by infected iAECIIs and their effect on HL-60 cells. (A) Cytokine array analysis showing the levels of the indicated cy tokines in the medium conditioned by iAECIIs infected with HCoV-229E for 24 or 48 h (24 hpi and 48 hpi). Ctrl – uninfected control. Right panel: cytokine label legend. (B) Western blot analysis of the expression of IL-8 and ICAM-1 in HCoV-229E-infected iAECIIs. (C) Transwell migration assay showing the migration capacity of HL-60 neutrophils in response to a medium conditioned by iAECIIs infected with HCoV-229E for 48 h (48 hpi) in the absence or presence of an IL-8-neutralizing antibody (IL-8 nAb). Ctrl – uninfected control conditioned medium. Data shown as means with SD error bars, n = 3, *p < 0.005, (ANOVA). (D) Representative fluorescence microscopy images of adherent HL-60 neutrophils after incubation with infected iAECIIs in the absence or presence of an ICAM-1-neutralizing antibody (ICAM-1 nAb). Ctrl – uninfected control. (E) Cell count of adherent HL-60 neutrophils. Mean values are shown with SD error bars, n = 3, *p < 0.005.

Article Snippet: The following primary antibodies were used: SFTPB, HCoV-229E spike protein (PAB21477–100/ PAB21477–500; The Native Antigen Company, Kidlington, UK), HCoV229E nucleocapsid protein (#40,640-T62; Sino Biological, Beijing, China), GAPDH (10,494–1-AP; Rosemont, IL, USA).

Techniques: Infection, Control, Western Blot, Expressing, Transwell Migration Assay, Migration, Fluorescence, Microscopy, Incubation, Cell Counting

Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 6. RNA-seq analysis identifies upstream cytokine pathways in the immune responses by neutrophils. (A and B) Cytoscape ClueGO networks of upregulated genes by infection of iAECIIs with HCoV-229E (A) or triggered by infected HCoV-229 conditioned medium in HL-60 cells (B). (C and D) Bubble plots showing the most enriched GO-BP terms among the genes upregulated by the infection of iAECIIs with HCoV-229E (C) or the genes upregulated in HL-60 cells by cultivation in the infected HCoV-229-conditioned medium (D). (E and F) Hierarchical clustering heatmaps showing the signatures of genes differentially regulated in infected iAECIIs (E) and conditioned medium-stimulated HL-60 cells (F). (G and H) X2K network analysis showing the kinases and transcription factors predicted to regulate differentially expressed genes in infected iAECIIs (G) and conditioned medium-stimulated HL-60 cells (H).

Article Snippet: The following primary antibodies were used: SFTPB, HCoV-229E spike protein (PAB21477–100/ PAB21477–500; The Native Antigen Company, Kidlington, UK), HCoV229E nucleocapsid protein (#40,640-T62; Sino Biological, Beijing, China), GAPDH (10,494–1-AP; Rosemont, IL, USA).

Techniques: RNA Sequencing, Infection

Journal: medRxiv

Article Title: Neurologic sequalae of COVID-19 are determined by immunologic imprinting from previous Coronaviruses

doi: 10.1101/2022.11.07.22282030

Figure Lengend Snippet: (A) Bar dot plots show the level of IgG1 antibodies against non-coronaviruses (EBV, Flu, HSV1) in individuals who did not develop (no PASC, in blue) or developed neurological PASC (neuroPASC, in purple). MFI=Median Fluorescence Intensity. Mann-Whitney, ns= statistically not significant. (B) Heatmap indicates the average difference (%) of SARS-CoV-2 -specific (Spike, S1, S2, RBD and NC) and common Coronaviruses-specific (229E, OC43, NL63, HKU1) IgG subclasses (IgG1, IgG2, IgG3, IgG4), IgM and IgA1, IgA2, binding capacity to FcγRs (2A, 2B, 3A, 3B), FcαR, between individuals with no PASC and with neuroPASC. Each column represents an antibody feature. Positive values (purple) indicate features enriched in no PASC, negative values (blue) indicate features enriched in neuroPASC, null values (white) indicate features equally present in both no PASC and neuroPASC. Common CoVs = other non-SARS-CoV-2 common Coronaviruses; NC= SARS-CoV-2 nucleocapsid; RBD= SARS-CoV-2 receptor binding domain. Mann-Whitney was used to define statistically significant differences at a univariate level between no PASC and neuroPASC, *p<0.01 or less. ( C-D ) Multivariate analysis of antibody signatures in individuals with neuroPASC and no PASC. Partial least square discriminant analysis (PLSDA) on LASSO-selected features was used to resolve antibody profiles in neuroPASC and no PASC. Dots represent individual samples (no PASC, blue; neuroPASC, red) across SARS-CoV-2 specific (Spike, S1, S2, RBD, NC), and common Coronaviruses (229E, OC43, NL63, HKU1) specific antibody features. Bar graph shows LV1 loadings of LASSO-selected features ranked by their Variable Importance in Projection (VIP). Features enriched in no PASC are in blue, features enriched in neuroPASC are in red. Ten-fold cross validation was performed, resulting in 84% cross validation accuracy (p<0.01). (E) Chord plots indicate the Spearman correlation coefficients between the LASSO-selected features enriched in no PASC (left) and in neuroPASC (right), and the non-LASSO selected antibody features across SARS-CoV-2 antigens (Spike, S1, S2, RBD, NC), and other common Coronaviruses antigens (229E, OC43, NL63, HKU1). Only coefficients >0.5 are plotted, p<0.01 after Benjamini-Hochberg correction for multiple comparisons. Positive values represent direct correlations, negative values represent inverse correlations (none of the features were inversely correlated with any other feature).

Article Snippet: The antibody responses were assessed against 5 different SARS-CoV-2 antigens, including Spike WT (Sino Biological 40589-V08H4), RBD WT (Sino Biological 40592-V08H), S1 WT (Sino Biological 40591-V08H), S2 WT (Sino Biological 40590-V08B) and Nucleocapsid (Sino Biological 40588-V08B), as well as 4 non-SARS-CoV-2 coronaviruses, including HCoV-OC43 (Sino Biological 40607-V08B), -HKU1 (Sino Biological 40606-V08B), -NL63 (Sino Biological 40604-V08B), -229E (Sino Biological 40605-V08B).

Techniques: Fluorescence, MANN-WHITNEY, Binding Assay

Journal: medRxiv

Article Title: Neurologic sequalae of COVID-19 are determined by immunologic imprinting from previous Coronaviruses

doi: 10.1101/2022.11.07.22282030

Figure Lengend Snippet: (A) Heatmap indicating the average difference (%) of antibody responses to SARS-CoV-2 (Spike, S1, S2, RBD and NC), other common Coronaviruses (229E, OC43, NL63, HKU1) and non-coronaviruses (EBV, Flu, HHV1), including IgG subclasses (IgG1, IgG2, IgG3, IgG4), IgM and IgA, binding capacity to FcγR (2A, 2B, 3A, 3B) and FcαR, between serum and CSF from individuals with neuroPASC. Positive values (purple) indicate features enriched in serum, negative values (blue) indicate features enriched in CSF, null values (white) indicate features equally present in both serum and CSF. There were no features enriched in CSF. Common CoVs = other non-SARS-CoV-2 common Coronaviruses (229E, OC43, NL63, HKU1); NC= nucleocapsid; RBD= receptor binding domain (B-C) Multivariate analyses of antibody signatures in serum and CSF from individuals with neuroPASC. Multilevel partial least square discriminant analysis (M-PLSDA) on LASSO-selected features was used to resolve antibody profiles in serum:CSF pairs. Dots represent individual samples (serum, blue; CSF, purple) across SARS-CoV-2 antigens (Spike, S1, S2, RBD, NC), and common Coronaviruses (229E, OC43, NL63, HKU1). Bar graph shows LV1 loadings of LASSO-selected features ranked by their Variable Importance in Projection (VIP). Features enriched in serum are in purple, no features were enriched in CSF. Ten-fold cross validation was performed, resulting in 100% cross validation accuracy (p<0.01). (D-E) Bar dot plots indicate CSF to serum ratios of IgG1, FcγR (2A, 2B, 3A) and FcαR binding capacity for SARS-CoV-2 (Spike, S1, S2, RBD, NC antigens combined) and other common Coronaviruses (229E, OC43, NL63, HKU1 combined) antibodies in individuals with neuroPASC. Mann-Whitney, *p<0.05, ***p<0.001 ****p<0.0001. CoV=common Coronaviruses; NC= nucleocapsid; RBD= receptor binding domain (F) Bar dot plots show the binding capacity of antibodies to the neonatal Fc receptor (FcRn) of serum and CSF SARS-CoV-2 antibodies targeting different antigens (Spike, S1, S2, RDB, NC) in individuals with neuroPASC. Mann-Whitney, *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001. MFI=Median Fluorescence Intensity; NC=nucleocapsid; RBD=receptor binding domain (G) Bar dot plots indicate the FcRn binding capacity of serum and CSF SARS-CoV-2 antibodies (Spike, S1, S2, RBD, NC, combined), compared to common Coronaviruses (229E, OC43, NL63, HKU1 combined) and non-coronaviruses (EBV, Flu, HSV1 combined). Kruskal Wallis test, Dunn’s correction for multiple comparisons. **p<0.01, ****p<0.0001. MFI=Median Fluorescence Intensity; NC= nucleocapsid; RBD= receptor binding domain

Article Snippet: The antibody responses were assessed against 5 different SARS-CoV-2 antigens, including Spike WT (Sino Biological 40589-V08H4), RBD WT (Sino Biological 40592-V08H), S1 WT (Sino Biological 40591-V08H), S2 WT (Sino Biological 40590-V08B) and Nucleocapsid (Sino Biological 40588-V08B), as well as 4 non-SARS-CoV-2 coronaviruses, including HCoV-OC43 (Sino Biological 40607-V08B), -HKU1 (Sino Biological 40606-V08B), -NL63 (Sino Biological 40604-V08B), -229E (Sino Biological 40605-V08B).

Techniques: Binding Assay, MANN-WHITNEY, Fluorescence

Journal: medRxiv

Article Title: Neurologic sequalae of COVID-19 are determined by immunologic imprinting from previous Coronaviruses

doi: 10.1101/2022.11.07.22282030

Figure Lengend Snippet: (A) The radial plots indicate the levels of IgG1 and FcγR2A binding capacity of SARS-CoV-2 antibodies (Spike, S1, S2, RBD, upper half circle) and common Coronaviruses (229E, OC43, NL63, lower half circle) in individuals with neuroPASC, divided into those with good versus poor outcome. Each sector represents a z-scored antibody feature. RBD=receptor binding domain (B-C) Volcano plots showing the correlation of each SARS-CoV-2 and common Coronaviruses (229E, OC43, NL63, HKU1)-antibody feature with modified Rankin Scale (mRS). Spearman correlation coefficients are indicated in the x-axis (positive correlation on the right, red; negative correlation on the left, blue), and the statistical significance is indicated in the y-axis (-log10[p-values]). Values above black dashed line indicate statistically significant correlations (p adjusted value<0.01, Benjamini-Hochberg correction for multiple comparisons). Negative correlations, indicating good outcome, were identified with serum antibodies (left graph, in blue), whereas negative correlations, indicating poor outcome, were identified with CSF antibody profiles (right graph, in red).

Article Snippet: The antibody responses were assessed against 5 different SARS-CoV-2 antigens, including Spike WT (Sino Biological 40589-V08H4), RBD WT (Sino Biological 40592-V08H), S1 WT (Sino Biological 40591-V08H), S2 WT (Sino Biological 40590-V08B) and Nucleocapsid (Sino Biological 40588-V08B), as well as 4 non-SARS-CoV-2 coronaviruses, including HCoV-OC43 (Sino Biological 40607-V08B), -HKU1 (Sino Biological 40606-V08B), -NL63 (Sino Biological 40604-V08B), -229E (Sino Biological 40605-V08B).

Techniques: Binding Assay, Modification