2020 Feb 27
MAPK Mutations and Cigarette Smoke Promote the Pathogenesis of Pulmonary Langerhans Cell Histiocytosis
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MAPK Mutations and Cigarette Smoke Promote the Pathogenesis of Pulmonary Langerhans Cell Histiocytosis
Pulmonary Langerhans cell histiocytosis (PLCH) is a rare smoking-related lung disease characterized by dendritic cell (DC) accumulation, bronchiolocentric nodule formation, and cystic lung remodeling. Approximately 50% of patients with PLCH harbor somatic BRAF-V600E mutations in cells of the myeloid/monocyte lineage. However, the rarity of the disease and lack of animal models have impeded the study of PLCH pathogenesis. Here, we establish a cigarette smoke-exposed (CS-exposed) BRAF-V600E-mutant mouse model that recapitulates many hallmark characteristics of PLCH. We show that CD11c-targeted expression of BRAF-V600E increases DC responsiveness to stimuli, including the chemokine CCL20, and that mutant cell accumulation in the lungs of CS-exposed mice is due to both increased cellular viability and enhanced recruitment. Moreover, we report that the chemokine CCL7 is secreted from DCs and human peripheral blood monocytes in a BRAF-V600E-dependent manner, suggesting a possible mechanism for recruitment of cells known to dominate PLCH lesions. Inflammatory lesions and airspace dilation in BRAF-V600E mice in response to CS are attenuated by transitioning animals to filtered air and treatment with a BRAF-V600E inhibitor, PLX4720. Collectively, this model provides mechanistic insights into the role of myelomonocytic cells and the BRAF-V600E mutation and CS exposure in PLCH pathogenesis and provides a platform to develop biomarkers and therapeutic targets.
Chemokines; Dendritic cells; Immunology; Molecular pathology; Pulmonology.
Conflict of interest statement
Conflict of interest: The authors have declared that no conflict of interest exists.
Figure 1. Exposure of mice with BRAF-V600E expression in CD11c
+ DCs to CVS results in PLCH-like lesions.
A) Representative H&E-stained sections showing lung pathology in BRAF VE mice exposed to FA or CS for 4 months. Red arrowheads indicate parenchymal nodule formation and red arrows indicate perivascular and peribronchiolar cell infiltration. Black arrows indicate cyst-like lesions and black arrowheads indicate areas of heterogeneous alveolar tissue destruction. Scale bar: 100 μm. Representative image of n = 8 mice per group. ( B) Number and volume of pulmonary nodules. Nodules were identified as large inflammatory cell infiltrates not surrounding an airway or vessel. The volume of each individual nodule was estimated as described in Methods. n = 8 mice per group. ( C) Enumeration of pulmonary cystic structures more than 200 μm in diameter. Representative lesions from n = 8 mice per group. ( D) Representative IHC staining of nodular inflammatory lesions in lungs of BRAF VE mice exposed to CS for 4 months. Scale bar: 250 μm. Representative lesions from n = 8 mice per group. For experiments shown, 1-way ANOVA with Tukey’s multiple-comparisons analysis was performed. * P < 0.05.
Figure 2. Increased inflammatory cells and disrupted DC homeostasis in the lungs of BRAF
A) Absolute cell numbers of leukocytes, DCs, macrophages, and T cells in the dissociated lungs of WT ( n = 5–8 mice per group) and BRAF VE ( n = 5–7 mice per group) mice were determined by flow cytometry. Leukocytes were identified as CD45 +; DCs were identified as CD11c +, MHC II +, and autofluorescence mid/low cells; macrophages were identified as CD11c + and autofluorescence hi cells; and T cells were identified as CD11c – and CD3 +cells ( n = 5 mice per group). ( B) The absolute numbers of CD11b + DCs and CD103 + DCs were determined by flow cytometry. Both subsets were gated from the DC population. ( C) The number of DCs expressing maturation marker CD86 was determined by flow cytometry ( n = 5 mice per group). ( D and E) DCs were isolated from lungs and treated with or without 20 ng/mL IFN-γ for 2 hours before being treated with 1 μg/mL poly(I:C) or 1 μg/mL LPS for 16 hours. The supernatant was collected and IL-6 and IL-12 p40 were measured by ELISA ( n = 5–7 mice per group). For experiments shown, ANOVA (1 way in A and B, 2 way in D and E) with Tukey’s multiple-comparisons analysis was performed. * P < 0.05. Data represent mean ± SEM.
Figure 3. BRAF-V600E mutation is associated with increased cell viability and expression of the antiapoptotic protein B cell lymphoma leukemia-x molecule.
A) Phospho-ERK (p-ERK) expression in BMDCs from WT and BRAFV-600E mice following the administration of Cre Recombinase Adenovirus mCherry (Ad-Cre) to induce BRAFV-600E expression was determined by flow cytometry. In indicated groups, 1 μM PLX4720 (BRAF inhibitor) was added to the BMDC culture at day 6. Data shown are representative of 4 independent experiments. ( B) Absolute number of BMDCs ( n = 4 WT/day; n = 4 BRAF VE/day) determined by flow cytometry at the indicated times. ( C) BRAFV-600E expression increases BMDC viability in vitro. GM-CSF was removed from the culture medium at day 7, and apoptosis was quantified by annexin V and PI staining after 16 hours using flow cytometry. Live cells are annexin V – and PI –. Data shown are representative of 4 independent experiments. ( D) BRAFV-600E expression increases antiapoptotic protein Bcl-xL expression in vitro. The expression of the antiapoptotic marker Bcl-xL in BMDCs was measured by flow cytometry. Data shown are representative of 4 independent experiments. ( E) BRAFV-600E expression increases CD11c + cell viability in vivo. CD11c cells were isolated from WT and BRAF VE mice, and the viability was assessed by flow cytometry as described above. Data are shown as the mean ± SEM; n = 4 per each group. For the experiments shown, B and E used 1-way ANOVA with Tukey’s multiple-comparisons test. Data shown are mean ± SEM. * P < 0.05.
Figure 4. BRAF-V600E mutation increases the recruitment of DCs to the lung in a CCL20-dependent manner.
A) Representative IHC staining for CCL20 in lung sections from healthy controls (HC) and patients with PLCH ( n = 3/group). Scale bar: 100 μm. ( B) CCL20 concentration in the BAL of mice ( n = 5/group) exposed to FA or CS for 4 months was measured by ELISA. Data shown are mean ± SEM of 5 independent experiments. ( C) CCL20 secretion by WT or BRAF VE donor BMDCs ( n = 6/group) treated with/without LPS plus IFN-γ was measured by ELISA. Data are representative of 5 independent experiments. ( D) Intracellular cAMP levels in WT or BRAF VE BMDCs ( n = 5/group) were measured after treatment with forskolin followed by CCL20 stimulation (300 ng/mL). Data are representative of 3 independent experiments. ( E) The number of WT or BRAF VE BMDCs that migrated from the upper to lower chamber of a Transwell plate toward CCL20 (300 ng/mL) was determined by flow cytometry after 3 hours ( n = 6/group). Data are representative of 3 independent experiments. ( F) Two million WT or BRAF VE CD45.2 donor BMDCs were intravenously injected into recipient CD45.1 mice that had been previously exposed to FA/CS for 6 months. The lungs were harvested 2 days later, and the numbers of donor cells in the lung and mLNs of the recipient mice were determined by gating on CD45.2 expression. Data shown are mean ± SEM of 3 independent experiments. ( B– F) * P < 0.05, ANOVA (1 way in B and C, 2 way in D– F) with Tukey’s multiple-comparisons test. Data shown are mean ± SEM.
Figure 5. BRAF-V600E mutation induces DCs production of CCL7.
A) Peripheral blood was collected from WT or BRAF VE mice exposed to FA/CS for indicated periods, and the CCL7 level in the serum was measured by ELISA ( n = 4/group). ( B) WT or BRAF VE pulmonary DCs were treated with/without 1 μg/mL LPS or 1 μM BRAF-specific inhibitor PLX4720 or both, and CCL7 in the supernatant was measured by ELISA ( n = 5/group). ( C) Representative CCL7 staining of BMDCs ( n = 5/group) from WT or BRAF VE mice treated with 1 μg/mL LPS overnight. Blue, DAPI; green, CCL7. Data are representative of 4 independent experiments. ( D and E) The secretion of ( D) CCL7 and ( E) CCL2 from WT or BRAF VE BMDCs treated with/without Ad-Cre at 150 multiplicity of infection (MOI), 1 μg/mL LPS and 1 μM PLX4720, was determined by ELISA ( n = 5/group). Data are representative of 5 independent experiments. ( F) CCL7 and CCL2 mRNA expression level in WT or BRAF VE BMDCs treated with/without 1 μg/mL LPS and 1 μM PLX4720 was determined by real-time PCR. Data are representative of 3 independent experiments. * P < 0.05, 2-way ANOVA with Tukey’s multiple-comparisons test. Data shown are mean ± SEM.
Figure 6. Increased CCL7 in the serum and CD11c
+ PBMCs of PLCH patients.
A) CCL7 levels were measured in the serum of never smokers ( n = 10), current smokers ( n = 10), and PLCH patients ( n = 22) by ELISA. Groups were compared using Kruskal-Wallis 1-way analysis of variance on ranks with Dunn’s multiple-comparisons test. * P < 0.05. ( B) PBMCs were isolated from healthy controls and PLCH patients and treated with or without LPS and IFN-γ overnight. The CCL7 + cells were identified exclusively in the CD11c + population of PBMCs by flow cytometry. Representative plots from 3 patients and controls are shown.
Figure 7. CS and TAM withdrawal ameliorates PLCH phenotypes in BRAF
A) Scheme of withdrawal experiments. Mice were treated with TAM chow and exposed to CS and then switched to normal chow and FA for the indicated periods. ( B) CCL7 levels in the serum and CCL20 levels in the BAL of mice ( n = 5 mice/group) at 5 months on TAM chow, after exposure to FA or CS for 4 months, followed by continued treatment or cessation of TAM plus CS cessation (-Cess). ( C) The absolute number of inflammatory cells in dissociated lungs of WT or BRAF VE mice ( n = 4/group) exposed to FA or CS for 4 months was determined by flow cytometry. ( B and C) * P < 0.05, 2-way ANOVA with Tukey’s multiple-comparisons test. Data shown are mean ± SEM.
All figures (7)
Cyclin D1 and BRAF V600E Immunohistochemical Staining in Pulmonary Langerhans Cell Histiocytosis.
Histopathology. 2020 Jan 21. doi: 10.1111/his.14068. Online ahead of print.
Genetic landscape of adult Langerhans cell histiocytosis with lung involvement.
Eur Respir J. 2020 Feb 27;55(2):1901190. doi: 10.1183/13993003.01190-2019. Print 2020 Feb.
Eur Respir J. 2020.
Pulmonary Langerhans Cell Histiocytosis: An Update From the Pathologists' Perspective.
Arch Pathol Lab Med. 2016 Mar;140(3):230-40. doi: 10.5858/arpa.2015-0246-RA.
Arch Pathol Lab Med. 2016.
Current understanding and management of pulmonary Langerhans cell histiocytosis.
Thorax. 2017 Oct;72(10):937-945. doi: 10.1136/thoraxjnl-2017-210125. Epub 2017 Jul 8.
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