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. 2023 Apr;151(4):976-990.e5.
doi: 10.1016/j.jaci.2022.11.016. Epub 2022 Dec 5.

Lymphotoxin beta receptor signaling directly controls airway smooth muscle deregulation and asthmatic lung dysfunction

Haruka Miki  1 , William B Kiosses  2 , Mario C Manresa  1 , Rinkesh K Gupta  1 , Gurupreet S Sethi  1 , Rana Herro  1 , Ricardo Da Silva Antunes  1 , Paramita Dutta  1 , Marina Miller  3 , Kai Fung  4 , Ashu Chawla  4 , Katarzyna Dobaczewska  2 , Ferhat Ay  1 , David H Broide  3 , Alexei V Tumanov  5 , Michael Croft  6
Affiliations

Lymphotoxin beta receptor signaling directly controls airway smooth muscle deregulation and asthmatic lung dysfunction

Haruka Miki et al. J Allergy Clin Immunol. 2023 Apr.

Abstract

Background: Dysregulation of airway smooth muscle cells (ASM) is central to the severity of asthma. Which molecules dominantly control ASM in asthma is unclear. High levels of the cytokine LIGHT (aka TNFSF14) have been linked to asthma severity and lower baseline predicted FEV1 percentage, implying that signals through its receptors might directly control ASM dysfunction.

Objective: Our study sought to determine whether signaling via lymphotoxin beta receptor (LTβR) or herpesvirus entry mediator from LIGHT dominantly drives ASM hyperreactivity induced by allergen.

Methods: Conditional knockout mice deficient for LTβR or herpesvirus entry mediator in smooth muscle cells were used to determine their role in ASM deregulation and airway hyperresponsiveness (AHR) in vivo. Human ASM were used to study signals induced by LTβR.

Results: LTβR was strongly expressed in ASM from normal and asthmatic subjects compared to several other receptors implicated in smooth muscle deregulation. Correspondingly, conditional deletion of LTβR only in smooth muscle cells in smMHCCreLTβRfl/fl mice minimized changes in their numbers and mass as well as AHR induced by house dust mite allergen in a model of severe asthma. Intratracheal LIGHT administration independently induced ASM hypertrophy and AHR in vivo dependent on direct LTβR signals to ASM. LIGHT promoted contractility, hypertrophy, and hyperplasia of human ASM in vitro. Distinguishing LTβR from the receptors for IL-13, TNF, and IL-17, which have also been implicated in smooth muscle dysregulation, LIGHT promoted NF-κB-inducing kinase-dependent noncanonical nuclear factor kappa-light-chain enhancer of activated B cells in ASM in vitro, leading to sustained accumulation of F-actin, phosphorylation of myosin light chain kinase, and contractile activity.

Conclusions: LTβR signals directly and dominantly drive airway smooth muscle hyperresponsiveness relevant for pathogenesis of airway remodeling in severe asthma.

Keywords: AHR; LIGHT; LTβR; TNF superfamily; TNFSF14; airway smooth muscle; asthma; contractility; noncanonical NF-κB.

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Conflict of interest statement

Conflict of interest statement: M.C has patents related to LIGHT and lung inflammation.

Figures

Figure 1.
Figure 1.. LTβR and HVEM are expressed by human healthy and asthmatic airway smooth muscle cells.
(A) Expression of transcripts of the receptors for LIGHT, TNF, IL-13/IL-4, and IL-17 from published RNA-seq data of human healthy and asthmatic ASM (GSE119578/9; GSE58434; GSE94335). (B) Flow analysis of LTβR and HVEM expression on healthy and asthma donor-derived ASM.
Figure 2.
Figure 2.. ASM-specific LTβR-deficient mice exhibit reduced ASM mass and AHR in allergen-induced experimental severe asthma.
(A) HVEM and LTβR expression on ASM (GFP+) from smMHCCre(eGFP), smMHCCreHVEMfl/fl or smMHCCreLTβRfl/fl mice. Data from 3 experiments. (B-G) smMHCCre, smMHCCreHVEMfl/fl, or smMHCCreLTβRfl/fl mice were given intranasal challenges of HDM extract over 6 weeks (n = 4–6/group). (B) Confocal analysis of lung bronchi (scale 20 μm). F-actin (Phalloidin, yellow), alpha smooth muscle actin (red), DAPI (blue). Data representative of 3 experiments. (C) Quantitation of ASM volume based on phalloidin staining around the bronchi from (B) (n = 6–9/group, 10–15 tertiary bronchi per mouse). Data representative of 3 experiments. (D) Airway resistance (AHR) after methacholine challenge, measured by Flexivent (n = 5–8/group). Data representative of two experiments. *P < 0.05, smMHCCre vs. LTβRfl/fl. (E) Flow quantitation of total lung tissue smooth muscle cells (ASM). Data points, individual mice. (F) Flow quantitation of total lung inflammatory/immune cells. Data points, individual mice. (G) Representative H&E stained lung sections (scale 100 μm). n =4–6 mice/group. Data representative of 3 experiments. Means ± SEM. *P < 0.05, **P < 0.01.
Figure 3.
Figure 3.. LIGHT-LTβR signals increase airway smooth muscle mass and induce AHR in mice.
Mice received intratracheal recombinant LIGHT or PBS over 3 days. (A-B) Confocal microscopy of lung bronchial sections for expression of F-actin (Phalloidin, yellow) and α-smooth muscle actin (red). (A) Representative images of bronchi (scale 20 μm). (B) Imaris 3D imaging analysis quantification of ASM volume (phalloidin) around individual bronchi (n = 4–5 mice/group, 10–15 tertiary bronchi per mouse). Data means ± SEM and representative of 3 experiments. *P < 0.05, **P < 0.01. (C) Airway resistance (AHR) after methacholine challenge, measured by Flexivent (n = 4 mice/group). *P < 0.05, smMHCCre vs. LTβRfl/fl. Data means ± SEM and representative of 2 experiments.
Figure 4.
Figure 4.. LIGHT induces human ASM proliferation and contractile activity.
(A) HVEM and LTβR expression on human ASM assessed by flow after stimulation with LIGHT for 24 or 48hr. Similar data in 3 experiments. (B) Images of ASM in collagen 3D gels. Left: Bright field, Right: Phalloidin (red), DAPI (blue). (C) ASM collagen gel contraction at 24–72hr after stimulation with LIGHT. Data combined means from triplicates from 3 experiments. (D) Percentages of BrdU+ ASM stimulated with LIGHT for 48hr. Cells either first incubated in serum free media for 16 hours for proliferative ASM, or for 7 days for contractile ASM. Data means of triplicates and representative of 3 experiments. (E) ASM volume after stimulation with LIGHT, measured by confocal microscopy. 300–400 ASM, in two replicate experiments were analyzed. Data means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 5.
Figure 5.. LIGHT induces actin polymerization and migratory ability in human ASM.
(A) RNA-seq analysis of contractile molecules in human ASM treated with LIGHT. Log2 fold change after 4hr compared to PBS treated cells. (B) GSEA of cell structure-related genes from RNA-seq of ASM stimulated with LIGHT compared to PBS for 4hr. (C) Confocal images of ASM treated with LIGHT for 6hr and stained with Phalloidin (red), Vinculin (green), DAPI (blue). Graphs show volume of F-actin (phalloidin) and focal adhesions (vinculin) normalized to individual cell sizes (scale 15 μm). 300–400 ASM, in two replicate experiments were analyzed. (D) Images of wounded monolayers of ASM stimulated with LIGHT for 12hr; left, fluorescence intensity of F-actin shown (yellow to red); right, remaining wound highlighted in yellow outline. Percentage of wound area remaining after LIGHT stimulation compared to PBS (scale 200 μm). Data combined from 3 independent experiments. Data means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6.
Figure 6.. LIGHT-dependent non-canonical NF-κB signaling induces actin polymerization and ASM contractility.
(A) Activation of canonical NF-κB (pp65) and non-canonical NF-κB (processing of p100 to p52) assessed in human ASM stimulated with LIGHT over 120 mins or 24hr. Data representative of 3 experiments. (B) Confocal analysis of ASM treated with LIGHT with or without NIK-SMI, an inhibitor of non-canonical NF-κB. Volume of phalloidin and vinculin normalized to cell size from triplicates (scale 10 μm). 300–400 ASM, in two replicate experiments were analyzed. (C) Gel contraction assay of LIGHT-stimulated ASM with or without inhibitors of canonical (BAY11–7082) and non-canonical (NIK-SMI) NF-κB. Percentage gel contraction compared to PBS in triplicates. Data representative of 2 experiments. Data means ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 7.
Figure 7.. LIGHT induces non-canonical NF-κB-dependent activation of Rac1/PAK1 and phosphorylation of MLC in ASM.
(A-C) pMLC2 (A), pMYPT and pPAK1 (B), and active Rac1-GTP immunoprecipitated with PAK1 (C) in human ASM stimulated with LIGHT for the indicated times. (D-E) Processing of p52, and pPAK1 (D) and pMLC2 (E) in ASM stimulated with LIGHT for 12hr, with or without inhibitors of canonical (BAY) and non-canonical NF-κB (NIK-SMI). (F) Active Rac1-GTP immunoprecipitated with PAK1 in ASM stimulated with LIGHT for 4hr with or without NIK-SMI. All data in A-F representative of 3 experiments. (G) Gel contraction of ASM stimulated with LIGHT, with or without an inhibitor of Rac1. Percentage gel contraction compared to PBS in triplicates. (H) Confocal analysis of ASM treated with LIGHT with or without a Rac1 inhibitor. Volume of phalloidin and vinculin normalized to cell size from triplicates (scale 10 μm). 300–400 ASM, in two replicate experiments were analyzed. Data representative of 2 experiments. Data means ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001.
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