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. 2011 May;17(5):596-603.
doi: 10.1038/nm.2356. Epub 2011 Apr 17.

The tumor necrosis factor family member LIGHT is a target for asthmatic airway remodeling

Taylor A Doherty  1 , Pejman Soroosh, Naseem Khorram, Satoshi Fukuyama, Peter Rosenthal, Jae Youn Cho, Paula S Norris, Heonsik Choi, Stefanie Scheu, Klaus Pfeffer, Bruce L Zuraw, Carl F Ware, David H Broide, Michael Croft
Affiliations

The tumor necrosis factor family member LIGHT is a target for asthmatic airway remodeling

Taylor A Doherty et al. Nat Med. 2011 May.

Abstract

Individuals with chronic asthma show a progressive decline in lung function that is thought to be due to structural remodeling of the airways characterized by subepithelial fibrosis and smooth muscle hyperplasia. Here we show that the tumor necrosis factor (TNF) family member LIGHT is expressed on lung inflammatory cells after allergen exposure. Pharmacological inhibition of LIGHT using a fusion protein between the IgG Fc domain and lymphotoxin β receptor (LTβR) reduces lung fibrosis, smooth muscle hyperplasia and airway hyperresponsiveness in mouse models of chronic asthma, despite having little effect on airway eosinophilia. LIGHT-deficient mice also show a similar impairment in fibrosis and smooth muscle accumulation. Blockade of LIGHT suppresses expression of lung transforming growth factor-β (TGF-β) and interleukin-13 (IL-13), cytokines implicated in remodeling in humans, whereas exogenous administration of LIGHT to the airways induces fibrosis and smooth muscle hyperplasia, Thus, LIGHT may be targeted to prevent asthma-related airway remodeling.

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Figures

Figure 1
Figure 1
Blockade of LIGHT or LTαβ inhibits airway remodeling and AHR induced by HDM. (a) Protocol for HDM-induced remodeling. WT mice were given three intranasal (i.n.) challenges with HDM extract, once per week. LTβR-Fc or IgG was given 24 h before each additional intranasal HDM challenge over the next 4 weeks. i.p., intraperitoneal. (b) Lung sections were stained for Masson's trichrome (top left and middle) and collagen-1 (bottom left and middle) and scored for the extent of fibrosis (top right, n = 54–75 airways per group). Induced total lung collagen was measured (bottom right, pooled from four mice per group, two experiments shown). (c) Lung sections stained for α-smooth muscle actin (left) and scored for extent of induced peribronchial smooth muscle (right, n = 49–70 airways per group). Induced reflects levels above those detected in mice receiving three intranasal challenges before LTβR-Fc treatment. (d) Peak airway resistance with increasing doses of methacholine and baseline resistance without methacholine exposure (six or seven mice per group). *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001, means ± s.e.m., Mann-Whitney test. Data are from two or three independent experiments. Scale bars, 100 μm.
Figure 2
Figure 2
LIGHT-deficient mice are resistant to airway remodeling induced by HDM. WT and Tnfsf14−/− mice received HDM intranasally once per week for 3 weeks, then twice per week for 4 weeks. Mice were killed 1 day after the last challenge. (a) Lung sections stained with Masson's trichrome (top) and collagen-1 (middle) and scoring for fibrosis (bottom left, n = 35–36 airways per group, means ± s.e.m., Mann-Whitney test). Total lung collagen was also measured (bottom right, eight mice per group, means ± s.e.m., Mann-Whitney test). (b) Peribronchial smooth muscle area (left, n = 34–35 airways per group, means ± s.e.m., Mann-Whitney test) and lung sections stained for α-smooth muscle actin (right). Levels reflect those above lung measurements from naive mice (a and b). (c) Invasive lung function test and resistance after challenge with 48 mg ml−1 methacholine (means ± s.e.m., Mann-Whitney test from six or seven mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 100 μm.
Figure 3
Figure 3
LIGHT controls lung TGF-β1 production and accumulation of LAP+ macrophages. Mice were chronically challenged with HDM or OVA. (a) Free TGF-β1 concentrations assessed in lung homogenates from WT mice treated as in Figure 1 (data from six to eight mice per group; acute signifies levels before immunoglobulin treatment; levels of two naive mice also shown, means ± s.e.m., Mann-Whitney, *P < 0.03); WT and Tnfsf14−/− mice treated as in Figure 2 (data from three or four mice per group, means ± s.e.m., t test, *P < 0.02); WT mice treated as in Supplementary Figure 6 (data from four pooled mice per group run in duplicate, mean ± s.e.m., except single IgG group); WT and Tnfsf14−/− mice treated as in Supplementary Figure 6 (data from four pooled mice per group run in quadruplicate, means ± s.e.m., t test, *P < 0.01). (b) Lung sections from WT mice in Supplementary Figure 6 stained for LTβR expression. Scale bar, 50 μm. (c) Lung cells from WT mice in Supplementary Figure 6 analyzed for Mac-3 and CD11c (left), and the gated population evaluated for LTβR expression (right). Filled histogram indicates isotype staining. (d) Lung cells from WT and Tnfsf14−/− mice in Supplementary Figure 6 analyzed for Mac-3 and CD11c (top and bottom) and absolute numbers of Mac-3+CD11c+ cells (right, pooled lung cells from four mice per group). (e) Immunofluorescent staining of lung sections from a representative WT mouse from Figure 1 stained for Mac-3 (red), LAP (green) and DAPI (blue). Scale bar, 50 μm. Image zoom also depicted (bottom). Scale bar, 25 μm. (f) Gated Mac-3+CD11c+ cells from lungs of mice in Figure 1 and Supplementary Figure 6 analyzed for LAP expression (top), enumeration of total LAP+ macrophages per lung (middle, n = 4 mice per group, means ± s.e.m., two experiments shown for OVA and one for HDM, *P < 0.05, t test) and flow analysis for LAP expression gating on Siglec-F+CD11c+ macrophages (bottom).
Figure 4
Figure 4
LTβR stimulation promotes fibrosis and TGF-β production by lung macrophages. (a) Lung sections stained with Masson's trichrome (left) and extent of induced peribronchial fibrosis (top right; 44–68 airways per group IgG and anti-LTβR, means ± s.e.m., Mann-Whitney test, *P < 0.05). Scale bar, 100 μm. Mac-3+ CD11c+LAP+ cells per lung were enumerated (bottom right, pooled from six mice per group). WT mice were immunized and acutely challenged with OVA over 28 d and then injected with LTβR agonist antibody (anti-LTβR) or rat IgG every 3–4 d for 2 weeks. (b,c) Analysis of Siglec-F+CD11c+ lung macrophages (b, top) from naive mice after stimulation with rat IgG or anti-LTβR and analyzed for surface LAP expression after 2 d (b, bottom), TGF-β1 mRNA (c, left, ***P < 0.0005) or TGF-β1 protein after HDM was added in the last 8 h of culture (c, right, **P < 0.005). Results are triplicates from each group. (d) Flow cytometry analysis of LAP−Siglec-F+CD11c+Mac-3+ lung macrophages sorted (left) and stimulated with rat IgG or anti-LTβR and analyzed for LAP expression (right). (e) Flow cytometry analysis of intracellular TGF-β in purified lung macrophages stimulated with recombinant LIGHT, in the presence or absence of ERK inhibitor. Data are representative of at least two experiments.
Figure 5
Figure 5
LIGHT-induced airway remodeling is in part dependent on TGF-β. (a,b) Lung sections were stained for trichrome (top row, scale bar, 100 μm), collagen-1 (second row, scale bar, 100 μm), α-smooth muscle actin (third row, scale bar, 50 μm) and scored for fibrosis and smooth muscle area (bottom row, 40 airways per group, means ± s.e.m., Mann-Whitney, *P < 0.05, **P < 0.005). WT mice primed with HDM over 3 weeks were treated with intranasal rLIGHT or PBS given eight times over 2 weeks (a) or pCDNA3 mouse LIGHT plasmid or control plasmid given four times over 2 weeks (b). Antibody to TGF-β (anti-TGF) or isotype control IgG was also injected as indicated. Induced reflects levels above those detected in lungs of mice receiving PBS (a) or control plasmid (b). A, airway; BV, blood vessel.
Figure 6
Figure 6
LIGHT promotes IL-13 production by lung eosinophils. (a) IL-13 content in mice primed and challenged with HDM or OVA. Lung homogenates from mice treated as in Figures 1 and 2 and Supplementary Figure 6 were analyzed (five to seven mice per group from Figure 1, three or four mice per group from Figure 2 and triplicates of pooled samples from four to seven mice per group from Supplementary Figure 6, means ± s.e.m., t test, *P < 0.05, **P < 0.01, ***P < 0.005). (b) Flow cytometry analysis of sorted granulocytes (>95% eosinophils by cytospin, bottom; scale bar, 50 μm) from WT mice after acute intranasal OVA challenge for LTβR (top middle) and HVEM expression (top right). Isotype control in gray. (c) Flow cytometry analysis of CD45+CD11c− granulocyte-gated lung eosinophils (top and bottom left) from mice in Supplementary Figure 6 for intracellular IL-13 directly ex vivo (middle right). Siglec-F+CD11c− eosinophils from mice in Figure 1 (top right) and Figure 2 (bottom right) were stained for IL-13 expression. (d) Flow cytometry analysis of BALF (bottom left) and lung cells (top left) and intracellular IL-13 (middle and right) was analyzed in cells gated on forward and side scatter (left, >95% eosinophils). Cells from WT mice immunized and challenged with OVA over 8 days were cultured for 48 h with rLIGHT or medium added during the last 24 h. Data are representative of two independent experiments.
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