CD103+ dendritic cell–fibroblast crosstalk via TLR9, TDO2, and AHR signaling drives lung fibrogenesis
Hannah Carter, Rita Medina Costa, Taylor S. Adams, Talon M. Gilchrist, Claire E. Emch, Monica Bame, Justin M. Oldham, Steven K. Huang, Angela L. Linderholm, Imre Noth, Naftali Kaminski, Bethany B. Moore, Stephen J. Gurczynski
Hannah Carter, Rita Medina Costa, Taylor S. Adams, Talon M. Gilchrist, Claire E. Emch, Monica Bame, Justin M. Oldham, Steven K. Huang, Angela L. Linderholm, Imre Noth, Naftali Kaminski, Bethany B. Moore, Stephen J. Gurczynski
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Research Article Immunology Pulmonology

CD103+ dendritic cell–fibroblast crosstalk via TLR9, TDO2, and AHR signaling drives lung fibrogenesis

Abstract

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring and loss of lung function. With limited treatment options, patients die from the disease within 2–5 years. The molecular pathogenesis underlying the immunologic changes that occur in IPF is poorly understood. We characterize noncanonical aryl-hydrocarbon receptor (ncAHR) signaling in DCs as playing a role in the production of IL-6 and increased IL-17+ cells, promoting fibrosis. TLR9 signaling in myofibroblasts is shown to regulate production of TDO2, which converts tryptophan into the endogenous AHR ligand kynurenine. Mice with augmented ncAHR signaling were created by crossing mice harboring a floxed AHR exon 2 deletion (AHRΔex2) with mice harboring a CD11c-Cre. Bleomycin (blm) was used to study fibrotic pathogenesis. Isolated CD11c+ cells and primary fibroblasts were treated ex vivo with relevant TLR agonists and AHR-modulating compounds to study how AHR signaling influenced inflammatory cytokine production. Human datasets were also interrogated. Inhibition of all AHR signaling rescued fibrosis; however, AHRΔex2 mice treated with blm developed more fibrosis, and DCs from these mice were hyperinflammatory and profibrotic upon adoptive transfer. Treatment of fibrotic fibroblasts with TLR9 agonist increased expression of TDO2, and fibrotic fibroblasts activated IL-6 production in CD103+ DCs. Study of human samples corroborated the relevance of these findings in patients with IPF. We also show, for the first time to our knowledge, that AHR exon 2 floxed mice retain the capacity for ncAHR signaling.

Authors

Hannah Carter, Rita Medina Costa, Taylor S. Adams, Talon M. Gilchrist, Claire E. Emch, Monica Bame, Justin M. Oldham, Steven K. Huang, Angela L. Linderholm, Imre Noth, Naftali Kaminski, Bethany B. Moore, Stephen J. Gurczynski

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Figure 1

CD103+ DCs accumulate in the fibrotic lung and show evidence of proinflammatory AHR signaling.

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CD103+ DCs accumulate in the fibrotic lung and show evidence of proinfla...
(A and B) Mice (n = 6–10 mice per group (A) and 5 per group (B) were treated with 0.75 U/kg blm for 0, 7, or 14 days (A) or 14 days (B). Lungs were harvested, and single-cell suspensions were analyzed by flow cytometry for the presence of CD103+ DCs (CD45+, CD11c+, MHC II+, CD24+, CD64–, CD103+) and lung leukocytes shown as a t-distributed stochastic neighbor embedding (tSNE) multidimensional reduction plot (full gating strategy in Supplemental Figure 1). Alv. Macs, alveolar macrophages; Mo. Alv. Macs, monocyte-derived alveolar macrophages; Int. Macs, interstitial macrophages; Inf. Monocytes, inflammatory monocytes. (C–E) Groups of mice (n = 5–9 mice per group [C] or n = 5–8 per group [D and E]) were treated with 0.75 U/kg blm for 14–21 days. Lungs were harvested, and collagen was quantified by hydroxyproline assay or qRT-PCR. (F) Blm-treated mice (n = 20 mice per group, pooled and analyzed in triplicate) were harvested 21 days following instillation. DCs were isolated from whole lung via a 2-step magnetic bead purification strategy using CD11c- and CD103-specific antibodies (CD11c+, CD103– cells were retained and analyzed separately). After culturing for an additional 24 hours, expression of IL-6 in the supernatant was determined via ELISA. (G) Mice (n = 4 per group) were treated with 0.75 U/kg blm for 7 days, after which lungs were harvested and single-cell suspensions were analyzed via flow cytometry for AHR expression in the indicated cell subsets. (H–J) Mice (n = 5 per group) were treated with 0.75 U/kg blm or saline control (WT). Single-cell suspensions were prepared from pooled collagenase-digested lung tissue at 7 days after blm, and CD11c+ cells were purified via magnetic bead isolation. CD103+ DCs and AMs were purified via FACS from bulk CD11c+ magnetic sorted cells. Expression of the indicated transcripts was analyzed via qRT-PCR in each population. All experiments shown are representative of at least 3 independent experiments; statistical significance was determined via ANOVA or Student’s t test in F (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).