CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung

Journal name:
Nature Medicine
Volume:
16,
Pages:
1305–1312
Year published:
DOI:
doi:10.1038/nm.2253
Received
Accepted
Published online

Abstract

Allergic asthma is a T helper type 2 (TH2)-dominated disease of the lung. In people with asthma, a fraction of CD4+ T cells express the CX3CL1 receptor, CX3CR1, and CX3CL1 expression is increased in airway smooth muscle, lung endothelium and epithelium upon allergen challenge. Here we found that untreated CX3CR1-deficient mice or wild-type (WT) mice treated with CX3CR1-blocking reagents show reduced lung disease upon allergen sensitization and challenge. Transfer of WT CD4+ T cells into CX3CR1-deficient mice restored the cardinal features of asthma, and CX3CR1-blocking reagents prevented airway inflammation in CX3CR1-deficient recipients injected with WT TH2 cells. We found that CX3CR1 signaling promoted TH2 survival in the inflamed lungs, and injection of B cell leukemia/lymphoma-2 protein (BCl-2)-transduced CX3CR1-deficient TH2 cells into CX3CR1-deficient mice restored asthma. CX3CR1-induced survival was also observed for TH1 cells upon airway inflammation but not under homeostatic conditions or upon peripheral inflammation. Therefore, CX3CR1 and CX3CL1 may represent attractive therapeutic targets in asthma.

At a glance

Figures

  1. Airway disease in CX3CR1-deficient mice.
    Figure 1: Airway disease in CX3CR1-deficient mice.

    (af) Cx3cr1GFP/GFP (GFP/GFP) and Cx3cr1+/+ (+/+) littermates were sensitized with LACK, challenged with five consecutive aerosols, and analyzed 1 d (a) or 2 d (bf) later. (a) Dynamic lung resistance and compliance, as monitored by plethysmography in Cx3cr1GFP/GFP and Cx3cr1+/+ mice after LACK or PBS challenge. Data show means ± s.e.m. in one representative experiment out of three. n = 6–8 mice per group. P values were obtained by comparing Cx3cr1GFP/GFP and Cx3cr1+/+ mice upon LACK sensitization and challenge. (b) Quantification of a FACS analysis of BALF cells from Cx3cr1GFP/GFP and Cx3cr1+/+ mice. E, eosinophils; N, neutrophils; L, lymphocytes. Data show means ± s.e.m. of three pooled experiments. n = 18–24 mice per group. (c) Representative microscopic images of lung sections at a tenfold magnification after MSB staining. (d) Quantification of mucus after periodic acid–Schiff staining. Data show means ± s.e.m. of two experiments. n = 6 mice per group. (e) IL-4, IL-5, IL-13 and IFN-γ contents in lung extracts of LACK-sensitized mice challenged with LACK or PBS. Data show one representative experiment out of three, with bars indicating the means. n = 6–8 mice per group. (f) IL-4 and IL-5 intracellular staining of LACK-stimulated, pooled lung cells from Cx3cr1GFP/GFP and Cx3cr1+/+ mice. Data show means ± s.e.m. of two experiments. *P < 0.05, **P < 0.01.

  2. AHR, airway inflammation and cytokine response in mice treated with CX3CR1 blocking reagents.
    Figure 2: AHR, airway inflammation and cytokine response in mice treated with CX3CR1 blocking reagents.

    (a) Experimental protocol. BALB/c mice were sensitized on days 0 and 7 with two intraperitoneal injections of 10 μg of LACK in alum and challenged on day 17 with 40 μg of LACK intranasally (i.n.). For prophylactic treatments, mice were treated on days 16, 18 and 21 with either 50 μg of polyclonal antibodies to CX3CR1 intravenously, or 50 μg of FKN-AT or PBS i.n. For curative treatments, mice were treated on days 18, 20 and 21 with 50 μg of FKN-AT i.n. (b) AHR, as measured by whole-body plethysmography. Data show means ± s.e.m. of one representative experiment out of five for the prophylactic treatments and out of three for the curative treatments. n = 6 mice per group. (c) Dynamic lung resistance and compliance, as monitored by plethysmography. Data show means ± s.e.m. in one representative experiment out of two. n = 6–8 mice per group. (d) Quantification of a FACS analysis of BALF cells. Data show means ± s.e.m. of three pooled experiments. n = 8–12 mice per group. (e) IL-4, IL-5 and IL-13 concentrations in the supernatants of lung cells from individual mice incubated with or without LACK for 72 h. Data show means ± s.e.m. in one representative experiment out of two. n = 4–6 mice per group. NS, non significant; *P < 0.05; **P < 0.01; ***P < 0.001.

  3. Airway disease in T cell-injected mice.
    Figure 3: Airway disease in T cell–injected mice.

    (a,b) CD4+ T cells (5 × 106) from Cx3cr1GFP/GFP or Cx3cr1+/+ mice were injected into the indicated recipients. LACK-sensitized mice were challenged with LACK or PBS. Data show means ± s.e.m. in one representative experiment out of two. n = 6 mice per group. (a) AHR, as measured by whole-body plethysmography. (b) Quantification of a FACS analysis of BALF cells. (cf) LACK-specific Thy1.1+ polyclonal TH2 cells (2 × 106) were injected into Cx3cr1GFP/GFP recipients. Recipients were treated with FKN-AT or PBS, challenged with LACK or PBS, and analyzed 5 d (c) or 6 d (df) later. (c) AHR, as monitored by whole-body plethysmography in mice treated with FKN-AT or PBS. LACK-sensitized, PBS-challenged mice were used as controls. Data show means ± s.e.m. in one experiment out of two. n = 6 mice per group. The black circles are obscured by the gray ones. (d) Quantification of a FACS analysis of BALF cells from FKN-AT–treated or PBS-treated mice. Data show means ± s.e.m. of two experiments. n = 12 mice per group. (e) Quantification of a FACS analysis of MLN, lung, spleen and BALF cells from FKN-AT–treated and PBS-treated mice. Data show means ± s.e.m. n = 6 mice per group. (f) IL-4, IL-5 and IL-13 concentrations in the supernatants of lung cells incubated with or without LACK. Data show means ± s.d. of triplicate wells. *P < 0.05; **P < 0.01; ***P < 0.001.

  4. Number and phenotype of CX3CR1-expressing CD4+ T cells.
    Figure 4: Number and phenotype of CX3CR1-expressing CD4+ T cells.

    (ad) Cx3cr1+/GFP (a,c,d) and WT (b) mice were sensitized and challenged with LACK and killed 1 d after the last aerosol. (a) GFP expression, after gating on live CD3+CD4+ cells, in the indicated organs in which blood cells were removed by cardiac perfusion with PBS. Data show representative FACS profiles. Numbers indicate the mean frequency ± s.e.m. of GFP+ cells among CD4+ T cells. n = 12 mice per group pooled from three experiments. (b) Cx3cr1 mRNA levels, as measured by quantitative PCR, in CD4+ T cells purified from spleen, MLN, lung and BAL. Data are expressed as Cx3cr1 / 18s rRNA ratio. (c) FACS analysis of T1/ST2 expression. Data show mean frequencies after gating on live CD3+CD4+ cells. n = 6 mice per group. (d) T cell cytokine secretion assessed by ELISA and intracellular staining. Top, 2 × 104 sorted GFP+ or GFP cells were incubated with LACK and 5 × 105 T cell–depleted spleen cells for 72 h. Supernatants were analyzed for IL-4, IL-5, IL-13 and IFN-γ contents. Data show means ± s.d. of three experiments. Bottom, quantification of intracellular staining of sorted GFP+ or GFP cells. Data show means ± s.e.m. of three experiments. *P < 0.05; **P < 0.01.

  5. Frequency and phenotype of injected TH2 cells.
    Figure 5: Frequency and phenotype of injected TH2 cells.

    (ad) Thy1.1+Thy1.2 Cx3cr1GFP/GFP and Thy1.1+Thy1.2+ Cx3cr1+/GFP TCR-transgenic TH2 cells were co-injected into naive (a,b) or OVA-sensitized (c,d) BALB/c mice (1.5 × 106 cells of each per mouse). Recipients were exposed to LACK aerosols, and cells were analyzed by FACS. (a) The frequency of Cx3cr1GFP/GFP and Cx3cr1+/GFP donor cells in individual mice in two experiments. n = 8 mice per group, except for MLN before aerosol and BALF after the third aerosol, for which each dot represents pools of four and two mice, respectively. (b) Representative profiles after gating on CX3CR1-proficient (black histogram) and CX3CR1-deficient (empty histogram) donor or endogenous CD4+ T cells (gray histogram). Numbers indicate mean fluorescence intensities (MFI) ± s.e.m. of eight mice from two experiments. (c,d) Mice were exposed to five aerosols of OVA only or LACK and OVA, and lung and BALF cells were analyzed by FACS 1 d later. (c) Representative FACS profiles after gating on live CD4+ T cells. Numbers indicate mean frequencies ± s.e.m. of donor cells in one representative experiment out of two. n = 4 mice per group. (d) GFP expression by donor and endogenous CD4+ T cells in OVA-sensitized, challenged mice. Data show representative FACS profiles after gating on CX3CR1-proficient (black histogram), CX3CR1-deficient (empty histogram) and endogenous CD4+ T cells (gray histogram). Numbers indicate mean fluorescence intensities ± s.e.m. of eight mice from two experiments.

  6. T cell proliferation and apoptosis.
    Figure 6: T cell proliferation and apoptosis.

    (af) Equal numbers of LACK-specific Cx3cr1GFP/GFP and Cx3cr1+/GFP TCR-transgenic TH2 (ae) or TH1 (f) cells were injected into naive mice. (a) Apoptosis of transferred cells, as assessed by TUNEL. Recipients were challenged with LACK aerosols and analyzed 24 h after the 3rd aerosol. Data show representative FACS profiles after gating on CX3CR1-proficient or CX3CR1-deficient cells. Data show mean frequencies ± s.e.m. of TUNEL+ cells among CX3CR1-proficient (black histograms) and CX3CR1-deficient (gray histograms) cells. One representative experiment out of two is shown. n = 6 mice per group. (b) Frequencies of infected T cells following aerosol challenges. CX3CR1-proficient and CX3CR1-deficient (TH2 cells) were infected with a BCL-2–encoding or a control retroviral vector. Equal numbers of transduced cells were injected into naive recipients. Data show donor cell frequency in individual mice, with bars indicating the mean in one representative experiment out of two. (c) Quantification of a FACS analysis of BALF cells 1 d after Cx3cr1GFP/GFP recipients were injected with transduced TH2 cells and exposed to LACK aerosols. Data show means ± s.e.m. of two pooled experiments. n = 6 mice per group. (d) Quantification of a FACS analysis of donor cells 1 or 3 weeks after injection. Data show mean frequency ± s.e.m. of CX3CR1-proficient and CX3CR1-deficient donor cells in a representative experiment out of two. n = 6 mice per group. (e) Quantification of a FACS analysis of lymph node cells 4 d after the mice were immunized with LACK in CFA or CFA only 1 d after cell injection. Data show mean frequency ± s.e.m. of CX3CR1-proficient and CX3CR1-deficient donor cells in a representative experiment out of two. n = 3 mice per group. (f) Quantification of a FACS analysis of CD4+ donor TH1 cells from mice exposed to LACK aerosols. Data show the frequency of CX3CR1-proficient or CX3CR1-deficient donor cells in individual mice in one representative experiment out of two. n = 5, except for BALF before and after the first aerosol, for which each dot represents a pool of three mice. *P < 0.05; **P < 0.01; ***P < 0.001.

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Author information

Affiliations

  1. University of Nice-Sophia Antipolis, U924, Valbonne, France.

    • Cyrille Mionnet,
    • Vanessa Buatois,
    • Valerie Milcent,
    • Nicolas Glaichenhaus &
    • Valerie Julia
  2. Institut National de la Santé et de la Recherche Médicale, Paris, France.

    • Cyrille Mionnet,
    • Vanessa Buatois,
    • Akira Kanda,
    • Valerie Milcent,
    • Sebastien Fleury,
    • David Lair,
    • Marie Langelot,
    • Yannick Lacoeuille,
    • Antoine Magnan,
    • David Dombrowicz,
    • Nicolas Glaichenhaus &
    • Valerie Julia
  3. University of Lille 2, U1011, Lille, France.

    • Akira Kanda,
    • Sebastien Fleury &
    • David Dombrowicz
  4. Institut Pasteur de Lille, Lille, France.

    • Akira Kanda,
    • Sebastien Fleury &
    • David Dombrowicz
  5. University of Nantes, UMR915, Institut du Thorax, Nantes, France.

    • David Lair,
    • Marie Langelot,
    • Yannick Lacoeuille &
    • Antoine Magnan
  6. Dynavax Technologies, Berkeley, California, USA.

    • Edith Hessel &
    • Robert Coffman

Contributions

C.M. and V.B. conducted most of the experiments and contributed to data analysis. A.K., S.F. and D.D. measured lung resistance and compliance and performed lung histology. V.M. provided technical help. D.L., M.L., Y.L. and A.M. performed experiments with HDM. E.H. and R.C. contributed to early experiments aimed at monitoring chemokine receptor expression in lung T cells. N.G. and V.J. wrote the manuscript. V.J. conceived of and directed the project.

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The authors declare no competing financial interests.

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