Journal: Frontiers in Immunology

Article Title: RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response

doi: 10.3389/fimmu.2023.1197356

Figure Lengend Snippet: RSV activates XBP1s. hSAECs were pre-treated with solvent (DMSO) or selective IRE1α kinase inhibitor RNase attenuator (KIRA8, 10 μM) for 2 h, followed by 3 h RSV adsorption in continued presence of DMSO or KIRA8 (MOI = 1.0). Separately, hSAECs were treated with solvent (DMSO) or 50 nM Tg to directly activate the UPR during the 3h RSV adsorption as above. After the 3 h RSV adsorption, cells were washed and either harvested (0 h) or cultured for additional 24 h in continued presence of solvent, KIRA8 or Tg. Q-RT-PCR was used to quantitate changes in XBP1s (A, D, G) and unspliced XBP1 (XBP1u) mRNAs (B, E, H) . The ratio of fold induction XBP1s to XBP1u was calculated (C, F, I) . Error bars are mean ± SD with four independent replicates. n.s., not signifiicant; *, P<0.05; **, P<0.01; ***, P<0.001; post-hoc analysis.

Article Snippet: Immortalized primary human small airway epithelial cells hSAECs and type II transformed alveolar carcinoma (A549) cells were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). hSAECs were grown in SAGM (Lonza) and A549 in DMEM/F12 (Gibco supplemented with 10% FBS) in a humidified 5% CO 2 environment ( , ).

Techniques: Solvent, Adsorption, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response

doi: 10.3389/fimmu.2023.1197356

Figure Lengend Snippet: The IRE1α-XBP1s pathway has complex effects on RSV replication and the innate immune response. hSAECs, treated with solvent (DMSO) or selective IRE1α kinase inhibitor RNase attenuator (KIRA8, 10 μM) for 2 h, were infected by RSV at an MOI of 1.0 for 3 h with continued presence of DMSO or KIRA8. Separately, hSAECs were infected by RSV (MOI=1.0) for 3 h in the presence of DMSO or 50 nM Tg activating the UPR. After 3 h viral adsorption, cells were washed and at 0 h or cultured for an additional 4, 8, 16 or 24 h in the presence of DMSO, KIRA8 or Tg as indicated. Q-RT-PCR was carried out for the mRNA levels of XBP1s (A) , RSV N (B) , IFNβ (C) , IFNL2 (D) and MX1 (E) . Error bars are mean ± SD with four independent replicates. **, P<0.01, post-hoc analysis.

Article Snippet: Immortalized primary human small airway epithelial cells hSAECs and type II transformed alveolar carcinoma (A549) cells were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). hSAECs were grown in SAGM (Lonza) and A549 in DMEM/F12 (Gibco supplemented with 10% FBS) in a humidified 5% CO 2 environment ( , ).

Techniques: Solvent, Infection, Adsorption, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response

doi: 10.3389/fimmu.2023.1197356

Figure Lengend Snippet: Expression of functionally active FLAG epitope-tagged XBP1s (FXBP1s). (A) FXBP1s expression. Western immunoblot of hSAECs transduced with empty (pCT) or FXBP1s-expressing lentiviral vector at an MOI of 2.0 for 48 h, or mock-treated or treated with tunicamycin (TM, 0.5 μg/ml for 8 h) or thapsigargin (Tg, 50 nM for 6 h) as indicated. Nuclear extracts were prepared and stained with anti-FLAG M2 Ab (top) or anti-XBP1s antibodies (middle panel). TATA box binding protein (TBP) was used as loading control (bottom panel). Molecular weight (MW) markers (in kDa) are shown. Ratio, the relative intensities of XBP1s immunoblotting signals normalized to TBP. Note the selective anti-FLAG M2 staining of ~60 kDa protein encoded by the FXBP1s expression vector and the significantly higher reactivity of the anti-FLAG M2 antibody (Sigma) than the anti-XBP1s antibody (BioLegend). Note TM and Tg induce significantly higher levels of XBP1s than that produced by FXBP1s transduction. (B) Immunofluorescence microscopy was performed to assess FXBP1s transduction efficiency and nuclear translation. Cells were infected with empty (pCT) or FXBP1s-expressing lentivirus (MOI=2). 24 h later, cells were mock or RSV infected (MOI=1) for an additional 24 h before fixation and staining with anti-FLAG M2 (red). Nuclei were counter-stained with DAPI (blue). Top panel, anti-FLAG M2 staining; Bottom panel, anti-FLAG M2 and DAPI staining are merged. Scale bar of 50 μm is shown. Note the >50% transduction of the cell population and nuclear localization.

Article Snippet: Immortalized primary human small airway epithelial cells hSAECs and type II transformed alveolar carcinoma (A549) cells were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). hSAECs were grown in SAGM (Lonza) and A549 in DMEM/F12 (Gibco supplemented with 10% FBS) in a humidified 5% CO 2 environment ( , ).

Techniques: Expressing, FLAG-tag, Western Blot, Transduction, Plasmid Preparation, Staining, Binding Assay, Control, Molecular Weight, Produced, Immunofluorescence, Microscopy, Infection

Journal: Frontiers in Immunology

Article Title: RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response

doi: 10.3389/fimmu.2023.1197356

Figure Lengend Snippet: CUT&RUN Analysis of FLAG-XBP1s Binding. (A) CUT&RUN analysis was applied to pCT (C1-C3) or FXBP1-transduced cells in the absence (M1-M3) or presence of RSV infection (R1-R3; MOI =1, 24h). Shown are violin plots of fragment length distribution of cleavage fragments after removal of adapters. Note the distinct 70 and 180 nt pattern in the M-series and R-series for FXBP1s fragments that are absent in the nonspecific cleavage pattern of the control series. (B) Linear plot of fragment length of CUT&RUN data illustrating the 180 nt cut pattern, consistent with nucleosomal distribution. (C) Correlation plots of individual CUT&RUN binding. Each individual replicate from empty vector pCT (CON1-CON3) or FXBP1 transduced cells in the absence (MM1-MM3) or presence of RSV infection (RM1-RM3; MOI =1, 24h). Note the high cross-correlation of the MM1-MM3 and RM1-RM3 with each other in the same replicate treatment that are distinct from that of control. (D) Volcano plot of FXBP1s peaks in uninfected vs RSV infected hSAECs. Differential analysis of FXBP1s binding was determined after normalization to library depth and differential analysis using DIFFBIND. X axis, Fold Change of binding occupancy. Y axis, -log10(adjusted p value using benjamini-hochberg, padj). Green symbols, FXBP1s peaks reduced by RSV infection; Red symbols, FXBP1s peaks upregulated by RSV infection. (E) Motif enrichment of FXBP1s binding sites. Motifs are rank ordered by the frequency of binding sites. On the left is the sequence logo followed by the JASPAR matrix name. For each, the p value of enrichment is also shown (symbol). Note enrichment of Fos-related protein 1 (Fra1) and GC sequences (POL-GC, CREB and NFY).

Article Snippet: Immortalized primary human small airway epithelial cells hSAECs and type II transformed alveolar carcinoma (A549) cells were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). hSAECs were grown in SAGM (Lonza) and A549 in DMEM/F12 (Gibco supplemented with 10% FBS) in a humidified 5% CO 2 environment ( , ).

Techniques: Binding Assay, Infection, Control, Plasmid Preparation, Sequencing

Journal: Frontiers in Immunology

Article Title: RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response

doi: 10.3389/fimmu.2023.1197356

Figure Lengend Snippet: mRNA expression changes in XBP1s-bound cytokine regulators. (A) Hierarchical cluster of IL10 signaling and cytokine genes in RSV-infected hSAECs (from the GSEA classification in Figure 1F ). Genes are organized by row, treatment conditions by column. Color indicates row-wise Z-score. Dendrogram is Euclidian distance. Scale indicates z-score statistic for each condition. Note the major cluster are type I and III IFN and IFN stimulated genes (ISGs) whose expression are increased by RSV infection. (B-G) Expression changes for genes directly regulated by XBP1s. For each gene shown, plotted are transcripts per million (TPM) from mock infected, RSV infected or RSV + IRE1α inhibitor (KIRA8) treated cells. Genes plotted are: (B) Interferon Regulatory Factor 1 (IRF1); (C) Colony Stimulating Factor 2 (CSF2), (D) Inhibin Subunit Beta A (INHBA); (E) NFKB Inhibitor Alpha (NFKBIA); (F) Dual Specificity Phosphatase 10 (DUSP10); and (G) Interleukin 15 (IL15). Box plots are 10-90% interquartile range with mean indicated by horizontal line. Each symbol is an independent replicate. Error bars are ± SD. ***, P<0.001, post-hoc analysis.

Article Snippet: Immortalized primary human small airway epithelial cells hSAECs and type II transformed alveolar carcinoma (A549) cells were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). hSAECs were grown in SAGM (Lonza) and A549 in DMEM/F12 (Gibco supplemented with 10% FBS) in a humidified 5% CO 2 environment ( , ).

Techniques: Expressing, Infection

Journal: Frontiers in Immunology

Article Title: RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response

doi: 10.3389/fimmu.2023.1197356

Figure Lengend Snippet: IRF1 is a regulator of XBP1s-dependent IFN gene regulatory network: effects of IRF1 silencing. (A) Western immunoblot of IRF1 in hSAECs transfected with scrambled (Scr) or IRF1-targeting siRNA. Cells were extracted after mock (-) or RSV infection (MOI=1, 24 h). Top panel, IRF1 staining. Specific IRF1 staining is seen at 50 kDa (arrow). Bottom, TATA-binding protein (TBP) staining is internal control. Left, molecular weight markers (in kDa). (B-I) Q-RT-PCR analysis of hSAECs after IRF1 silencing. hSAECs were siRNA transfected and RSV infected for 10 or 24 h and gene expression determined by Q-RT-PCR. Shown are 10-90% interquartile ranges of mRNA fold change relative to mock infected hSAECs. Gene expression is plotted for: (B) IRF1 mRNA; (C) IFNL2 mRNA; (D) IL29 mRNA; (E) MX1 mRNA; (F) OAS1 mRNA; (G) TLR2 mRNA; (H) IFIT1 mRNA; (I) IFITM1 mRNA. **, P<0.01, ***, P<0.001, post-hoc analysis.

Article Snippet: Immortalized primary human small airway epithelial cells hSAECs and type II transformed alveolar carcinoma (A549) cells were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). hSAECs were grown in SAGM (Lonza) and A549 in DMEM/F12 (Gibco supplemented with 10% FBS) in a humidified 5% CO 2 environment ( , ).

Techniques: Western Blot, Transfection, Infection, Staining, Binding Assay, Control, Molecular Weight, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: Frontiers in Immunology

Article Title: RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response

doi: 10.3389/fimmu.2023.1197356

Figure Lengend Snippet: IRF1 is a regulator of XBP1s-dependent IFN gene regulatory network: effects of IRF1 expression. To examine the effects of physiological IRF1 expression, IRF1 expression was conducted in IRF1 -/- A549 cells generated by CRISPR/Cas9 site-directed recombination. Shown are 10-90% interquartile ranges of mRNA fold change relative to mock infected hSAECs. Gene expression is plotted for: (A) IRF1 mRNA; (B) IFNB mRNA; (C) IFNL2 mRNA; (D) IFITM1 mRNA; and (E) MX1 mRNA; **, P<0.01, ****, P<0.0001, post-hoc analysis.

Article Snippet: Immortalized primary human small airway epithelial cells hSAECs and type II transformed alveolar carcinoma (A549) cells were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). hSAECs were grown in SAGM (Lonza) and A549 in DMEM/F12 (Gibco supplemented with 10% FBS) in a humidified 5% CO 2 environment ( , ).

Techniques: Expressing, Generated, CRISPR, Infection, Gene Expression

Journal: Frontiers in Immunology

Article Title: RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response

doi: 10.3389/fimmu.2023.1197356

Figure Lengend Snippet: IRF3 mediates type I, but not type III IFNs in RSV-induced anti-viral response. hSAECs were transfected overnight by control or human IRF3-specific siRNA (Dharmacon SMARTpool, 25 nM) using DharmaFECT 1 (2.0 μl per 24-well), followed by 48 h culture in fresh medium. The siRNA transfected cells were then mock- or RSV-infected for 10 or 24 h. Q-RT-PCR was performed for the mRNA levels of IRF3 (A) , IFNβ (B) , IFNL1 (C) , IFNL2 (D) , IFNL3 (E) , BST2 (F) , IFITM1 (G) , MX1 (H) and RSV N (I) . Error bars are ± SD with four independent replicates. **, P<0.01; ***, P<0.001, post-hoc analysis. Note the significant reduction in RSV-induced IFNβ in IRF3-silenced cells, the lack of effect on IFL2, 3 mRNAs and the promoting effect of IRF3 knockdown on RSV replication in 24 h. n.s., not significant; *, P<0.05, post-hoc analysis.

Article Snippet: Immortalized primary human small airway epithelial cells hSAECs and type II transformed alveolar carcinoma (A549) cells were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). hSAECs were grown in SAGM (Lonza) and A549 in DMEM/F12 (Gibco supplemented with 10% FBS) in a humidified 5% CO 2 environment ( , ).

Techniques: Transfection, Control, Infection, Reverse Transcription Polymerase Chain Reaction, Knockdown

Journal: Frontiers in Immunology

Article Title: RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response

doi: 10.3389/fimmu.2023.1197356

Figure Lengend Snippet: IRE1-XBP1s requirement in RSV-induced IRF1 expression. The requirement for IRE1α-XBP1s in IRF1 mRNA expression was examined in hSAECs after lentiviral silencing. (A) Experimental schematic. (B) Effect of IRE1α silencing. Nontargeting shRNA and IRE1α-targeted shRNA expressing hSAECs were RSV infected for 24 h (MOI=1) and IRF1 gene expression determined by Q-RT-PCR. Shown are 10-90% interquartile ranges of mRNA fold change relative to mock infected hSAECs. (C) Effect of XBP1 silencing. Nontargeting and XBP1-targeting lentivirus shRNA transduced cells were infected with RSV and IRF1 mRNA measured as above. ***, P<0.001, post-hoc analysis. (D) Role of IRF1 enhancer in RSV-induced IRF1 expression. Experimental schematic. hSAECs expressing NH2-terminal fusion of Krox KRAB repressor domain with enzymatically inactive Cas9 (dCas9) were transduced with nontargeting or IRF1 5’-enhancer-targeting single guide RNA (sgRNAs). The IRF1 -specific sgRNA guides KRAB-dCas9 to the IRF1 5’-enhancer for transcriptional repression. (E) Effect of KRAB-dCas9 on RSV-induced IRF1 mRNA expression. KRAB-dCas9 expressing hSAECs were transduced with non-targeting or IRF1 5’-enhancer-targeting sgRNAs. Cells were RSV infected and IRF1 mRNA measured at 0, 18 and 24 h after infection. ***, P<0.001, post-hoc analysis. (F, G) XBP1s primes phospho-Ser2 Pol II binding to the IRF1 enhancer. XChIP for XBP1s and p-Ser2 Pol II binding on the IRF1 5’-enhancer. hSAECs were mock- or RSV infected (MOI=1, 24 h) in the absence or presence of KIRA8, or transduced with empty lentiviral vector or FXBP1s for 48 h. For each XChIP, Q-gPCR for IRF1 5’-enhancer abundance was performed. Data are presented as fold change over mock-infected cells. Individual symbols are biological replicates. (F) XBP1s binding; (G) p-Ser2 Pol II binding. Error bars are ± SD. **, P<0.01, post-hoc analysis.

Article Snippet: Immortalized primary human small airway epithelial cells hSAECs and type II transformed alveolar carcinoma (A549) cells were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). hSAECs were grown in SAGM (Lonza) and A549 in DMEM/F12 (Gibco supplemented with 10% FBS) in a humidified 5% CO 2 environment ( , ).

Techniques: Expressing, shRNA, Infection, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Transduction, Binding Assay, Plasmid Preparation

Journal: Respiratory Research

Article Title: ABCB1 and ABCG2 drug transporters are differentially expressed in non-small cell lung cancers (NSCLC) and expression is modified by cisplatin treatment via altered Wnt signaling

doi: 10.1186/s12931-017-0537-6

Figure Lengend Snippet: Wnt dependent differential regulation of drug transporters. Relative mRNA expression of a ) ABCB1 and ABCG2 expression in 3D HMVEC-L-NHLF-SAEC aggregates treated with canonical Wnt pathway inhibitor, IWR and inducer, LiCl for 3 h. mRNA of treated samples is compared to untreated controls, n = 3 b ) ABCB1 and ABCG2 expression in 3D HMVEC-L-NHLF-SAEC co-culture aggregates following recombinant human Wnt5a treatment shows similar pattern that SCC primary samples. mRNA expression of treated samples is compared to untreated controls, n = 3, mRNA expression of primary SCC samples is compared to normal lung tissue

Article Snippet: Primary human small airway epithelial cells (SAEC), normal human lung fibroblasts cells (NHLF) and normal human lung microvascular endothelial cells (HMVEC-L) were cultured according to the manufacturers’ recommendations (Lonza, Basel, Switzerland).

Techniques: Expressing, Co-Culture Assay, Recombinant

Journal: Respiratory Research

Article Title: ABCB1 and ABCG2 drug transporters are differentially expressed in non-small cell lung cancers (NSCLC) and expression is modified by cisplatin treatment via altered Wnt signaling

doi: 10.1186/s12931-017-0537-6

Figure Lengend Snippet: Effects of cisplatin treatment. Relative mRNA expression of a ) Wnt5a and Wnt7b in cisplatin treated 3D HMVEC-L-NHLF-SAEC co-culture aggregates. mRNA expression of treated samples is compared to untreated controls, n = 3 b ) ABCB1 and ABCG2 in cisplatin treated (29.7 μM, 3 h) 3D HMVEC-L-NHLF-SAEC co-culture aggregates. mRNA expression is normalized to beta-actin, n = 3 c ) Representative images of Ser675 phosphorylated beta-catenin immune-fluorescent staining in control and cisplatin treated 3D HMVEC-L-NHLF-SAEC aggregates. Fluorescence intensity are representations of three different experiments as mean ± SEM. Scale bar 50 μm, magnification 63×. d ) Western-blot analysis and densitometric quantification of Ser675 phosphorylated beta-catenin and beta-catenin proteins in different cell fractions of control and cisplatin treated A549 lung adenocarcinoma cell cultures, n = 3 e ) functional activity of ABCB1 and ABCG2 drug transporters in 3D co-culture aggregates following canonical Wnt pathway inhibitor, IWR and inducer, LiCl treatment. Data are presented as mean ± SEM of multidrug resistance activity factor values (MAF), n = 3. MAF values ≤ 20 are considered as MDR negative

Article Snippet: Primary human small airway epithelial cells (SAEC), normal human lung fibroblasts cells (NHLF) and normal human lung microvascular endothelial cells (HMVEC-L) were cultured according to the manufacturers’ recommendations (Lonza, Basel, Switzerland).

Techniques: Expressing, Co-Culture Assay, Staining, Control, Fluorescence, Western Blot, Functional Assay, Activity Assay