Journal: bioRxiv

Article Title: Infant RSV infection desensitizes β2-adrenergic receptor via CXCL11-CXCR7 signaling in airway smooth muscle

doi: 10.1101/2025.01.13.632772

Figure Lengend Snippet: ( A ) Representative fluorescence images of CXCR7 staining in infant ASMCs following 48-hr treatment with Mock- or RSV-CM collected from neonatal ALI cultures. Scale bar, 100 μm. ( B ) Representative Western blot for CXCR7 in infant ASMCs after 48-hr treatment with Mock- or RSV-CM of neonatal ALI cultures. β-actin was the loading control in densitometry. N=4 donors. ( C ) Summary plots of 10μM His-precontracted infant airway in response to Form at indicated concentrations in infant PCLSs that were incubated with Mock-CM, RSV-CM, and RSV-CM+CXCR7 antagonist (10 µM) for 48 hours. N=4-6 slices from 4 donors. ( D ) Representative Western blot for β2AR in primary infant ASMCs following 48-hr treatment with Mock- or RSV-CM in the presence of CXCR7 antagonist (10 µM) or vehicle control. β-actin was the loading control in densitometry. N=4 donors with 2 independent repeats. ( E ) Representative confocal fluorescence images showing colocalization of CXCR7 and β2AR on the cell membrane of infant ASMCs. Scale bar,10 μm. ( F ) Representative co-immunoprecipitation (IP) assay for β2AR and mAChR3 by the CXCR7 antibody. β-actin was the loading control for input cell lysates. ( G ) Densitometry to measure the enrichment of CXCR7, β2AR, and mAChR3 by the CXCR7 antibody normalized to IgG in the co-IP assay in (F). Statistical significance was calculated by the Kruskal-Wallis test with a post-hoc test with BYK procedure in (C) and the Wilcoxon test in (B) and (D) *p<0.05. ns, not significant.

Article Snippet: Treatment with CXCL11 (200 ng/ml, R&D, 672-IT-025) or CXCR7 antagonist (10 μM, MCE, HY-139643) was performed by mixing RSV and the reagent in a volume of 10 µL following by intranasal inoculation.

Techniques: Fluorescence, Staining, Western Blot, Control, Incubation, Membrane, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Infant RSV infection desensitizes β2-adrenergic receptor via CXCL11-CXCR7 signaling in airway smooth muscle

doi: 10.1101/2025.01.13.632772

Figure Lengend Snippet: ( A ) Schematic of RSV A2-line19F infection of BALB/c mouse pups at postnatal day 7 (P7) and P8 (1×10 6 PFU/10 µl) with and without the CXCR7 antagonist (10µM, 10 µl). At day 3 post-infection, mice were analyzed for RSV infection and airway contraction using prepared PCLSs in (B-E). ( B ) Cxcl11 gene expression in the lungs from Mock- and RSV-infected BALb/c pups by qRT-PCR. Data were normalized to 18s rRNA. Each mark represents one mouse. ( C ) Summary plots of airway contraction in response to methacholine (MCh) and relaxation in response to Form at indicated concentrations. N=8 PCLSs in 3 Mock-infected pups. N=18 PCLSs from 5 RSV-infected pups. ( D ) Summary plots of airway contraction by 0.5 µM MCh and relaxation in response to Form at indicated concentrations. N=12 PCLSs from 4 Mock-infected pups. N=21 PCLSs from 5 RSV-infected pups. N=11 PCLSs from 4 RSV+CXCR7 antagonist pups. ( E ) Airway relaxation in response to 10 and 100 nM Form in (D) was replotted for comparison between the three groups. ( F ) Schematic of RSV A2-line19F infection (1×10 6 PFU/10 µL) of C57BL/6 mouse pups at P7 and P8 with and without CXCL11 (200 ng/mL, 10 µL). At day 3 post-infection, pups were analyzed for airway contraction using prepared PCLSs ( G , H ). ( G ) Summary plots of 0.5 µM MCh-induced contraction followed by 10 and 100 nM Form treatment to induce relaxation. N=11 PCLSs from 3 Mock-infected pups. N=14 PCLSs from 4 RSV-infected pups. N=13 PCLSs from 4 RSV+CXCL11 pups. ( H ) Replotted 10 and 100 nM Form-induced airway relaxation in the three groups. Statistical significance was performed with a two-way ANOVA test with a post-hoc Sidak test in (C), a Mann-Whitney test in (B), and a Kruskal-Wallis test with a post-hoc test with BYK procedure in (E) and (H). *p<0.05, and ns, not significant.

Article Snippet: Treatment with CXCL11 (200 ng/ml, R&D, 672-IT-025) or CXCR7 antagonist (10 μM, MCE, HY-139643) was performed by mixing RSV and the reagent in a volume of 10 µL following by intranasal inoculation.

Techniques: Infection, Expressing, Quantitative RT-PCR, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Infant RSV infection desensitizes β2-adrenergic receptor via CXCL11-CXCR7 signaling in airway smooth muscle

doi: 10.1101/2025.01.13.632772

Figure Lengend Snippet: ( A ) Schematic of nasopharyngeal aspirate (NPA) collection from infants with bronchiolitis caused by RSV (RSV-NPA) or by rhinovirus or metapneumovirus (nonRSV-NPA). ( B ) ELISA of CXCL11 levels in RSV-NPA and nonRSV-NPA samples. Each mark represents one patient. ( C ) Summary plots of airway contraction in infant PCLSs in response to His and relaxation in response to Form at indicated concentrations. Infant PCLSs were pretreated with saline or RSV-NPA reconstituted medium for 48 hours. RSV-NPA was pooled from 6 different infant patients. ( D ) Form-induced airway relaxation in saline (N=11 PCLSs from 4 donors) and RSV-NPA (N=14 PCLSs from 4 donors) groups in (C) was replotted for comparison. ( E ) Summary plots of airway contraction in infant PCLSs in response to His and relaxation in response to Form at indicated concentrations. Infant PCLSs were pretreated with RSV-NPA medium or RSV-NPA medium+10µM CXCR7 antagonist for 48 hours. ( F ) Form-induced airway relaxation in RSV-NPA (N=4 PCLSs from 4 donors) or RSV-NPA+CXCR7 antagonist groups (N=7 PCLSs from 4 donors) in (E) was replotted for comparison. ( G ) Summary plots of Form-induced relaxation of His (10 µM)-precontracted airways in infant PCLSs. The infant PCLSs were treated with vehicle (N=7 PCLSs from 3 donors) or TNFα (100 ng/ml) + IL1β (10 ng/ml) (N=6 PCLSs from 3 donors). Statistical significance was performed with a Mann-Whitney test in (B, D, F and G), a two-way ANOVA test with a post-hoc Sidak test in (C) and (E). *p<0.05, and ns, not significant.

Article Snippet: Treatment with CXCL11 (200 ng/ml, R&D, 672-IT-025) or CXCR7 antagonist (10 μM, MCE, HY-139643) was performed by mixing RSV and the reagent in a volume of 10 µL following by intranasal inoculation.

Techniques: Enzyme-linked Immunosorbent Assay, Saline, Comparison, MANN-WHITNEY