Andrew C. Melton

IL-17A produced by αβ T cells drives airway hyper-responsiveness in mice and enhances mouse and human airway smooth muscle contraction

Emerging evidence suggests that the T helper 17 (TH17) subset of αβ T cells contributes to the development of allergic asthma. In this study, we found that mice lacking the αvβ8 integrin on dendritic cells did not generate TH17 cells in the lung and were protected from airway hyper-responsiveness in response to house dust mite and ovalbumin sensitization and challenge. Because loss of TH17 cells inhibited airway narrowing without any obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of TH17 cytokines on mouse and human airway smooth muscle function. Interleukin-17A (IL-17A), but not IL-17F or IL-22, enhanced contractile force generation of airway smooth muscle through an IL-17 receptor A (IL-17RA)–IL-17RC, nuclear factor κ light-chain enhancer of activated B cells (NF-κB)–ras homolog gene family, member A (RhoA)–Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling cascade. Mice lacking integrin αvβ8 on dendritic cells showed impaired activation of this pathway after ovalbumin sensitization and challenge, and the diminished contraction of the tracheal rings in these mice was reversed by IL-17A. These data indicate that the IL-17A produced by TH17 cells contributes to allergen-induced airway hyper-responsiveness through direct effects on airway smooth muscle.

Mfge8 diminishes the severity of tissue fibrosis in mice by binding and targeting collagen for uptake by macrophages

Milk fat globule epidermal growth factor 8 (Mfge8) is a soluble glycoprotein known to regulate inflammation and immunity by mediating apoptotic cell clearance. Since fibrosis can occur as a result of exaggerated apoptosis and inflammation, we set out to investigate the hypothesis that Mfge8 might negatively regulate tissue fibrosis. We report here that Mfge8 does decrease the severity of tissue fibrosis in a mouse model of pulmonary fibrosis; however, it does so not through effects on inflammation and apoptotic cell clearance, but by binding and targeting collagen for cellular uptake through its discoidin domains. Initial analysis revealed that Mfge8-/- mice exhibited enhanced pulmonary fibrosis after bleomycin-induced lung injury. However, they did not have increased inflammation or impaired apoptotic cell clearance after lung injury compared with Mfge8+/+ mice; rather, they had a defect in collagen turnover. Further experiments indicated that Mfge8 directly bound collagen and that Mfge8-/- macrophages exhibited defective collagen uptake that could be rescued by recombinant Mfge8 containing at least one discoidin domain. These data demonstrate a critical role for Mfge8 in decreasing the severity of murine tissue fibrosis by facilitating the removal of accumulated collagen.

Expression of αvβ8 integrin on dendritic cells regulates Th17 cell development and experimental autoimmune encephalomyelitis in mice

Th17 cells promote a variety of autoimmune diseases, including psoriasis, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. TGF-β is required for conversion of naive T cells to Th17 cells, but the mechanisms regulating this process are unknown. Integrin αvβ8 on DCs can activate TGF-β, and this process contributes to the development of induced Tregs. Here, we have now shown that integrin αvβ8 expression on DCs plays a critical role in the differentiation of Th17 cells. Th17 cells were nearly absent in the colons of mice lacking αvβ8 expression on DCs. In addition, these mice and the DCs harvested from them had an impaired ability to convert naive T cells into Th17 cells in vivo and in vitro, respectively. Importantly, mice lacking αvβ8 on DCs showed near-complete protection from experimental autoimmune encephalomyelitis. Our results therefore suggest that the integrin αvβ8 pathway is biologically important and that αvβ8 expression on DCs could be a therapeutic target for the treatment of Th17-driven autoimmune disease.

Intestinal dendritic cells specialize to activate transforming growth factor-β and induce Foxp3 + regulatory T cells via integrin αvβ8

Background & Aims The intestinal immune system is tightly regulated to prevent responses against the many nonpathogenic antigens in the gut. Transforming growth factor (TGF)-β is a cytokine that maintains intestinal homeostasis, in part by inducing Foxp3+ regulatory T cells (Tregs) that suppress immune responses. TGF-β is expressed at high levels in the gastrointestinal tract as a latent complex that must be activated. However, the pathways that control TGF-β activation in the intestine are poorly defined. We investigated the cellular and molecular pathways that control activation of TGF-β and induction of Foxp3+ Tregs in the intestines of mice to maintain immune homeostasis. Methods Subsets of intestinal dendritic cells (DCs) were examined for their capacity to activate TGF-β and induce Foxp3+ Tregs in vitro. Mice were fed oral antigen, and induction of Foxp3+ Tregs was measured. Results A tolerogenic subset of intestinal DCs that express CD103 were specialized to activate latent TGF-β, and induced Foxp3+ Tregs independently of the vitamin A metabolite retinoic acid. The integrin αvβ8, which activates TGF-β, was significantly up-regulated on CD103+ intestinal DCs. DCs that lack expression of integrin αvβ8 had reduced ability to activate latent TGF-β and induce Foxp3+ Tregs in vitro and in vivo. Conclusions CD103+ intestinal DCs promote a tolerogenic environment in the intestines of mice via integrin αvβ8-mediated activation of TGF-β.

Endothelin-1 stimulates human colonic myofibroblast contraction and migration

Background: Although the contractile, migratory, and proliferative responses of subepithelial myofibroblasts to injury have been postulated to be important events in intestinal wound healing, contractile force generation and migration by these cells has not been investigated previously, and the signals that regulate proliferation by these cells are poorly understood. Aims: The primary aim of this study was to test the hypothesis that the inflammatory mediator endothelin-1 modulates contraction, migration, and proliferation of intestinal myofibroblasts. We also sought to examine the signal transduction pathways which might underlie these putative effects. Methods: Contraction, migration, proliferation, cytosolic [Ca2+], and myosin phosphorylation were measured in human colonic subepithelial myofibroblasts in the absence and presence of endothelin receptor agonists and antagonists. Results: Endothelin-1, but not interleukin 1α, interleukin 6, interleukin 8, interleukin 10, or tumour necrosis factor α, induced a rapid and robust generation of contractile force, which was associated with an increase in cytosolic [Ca2+] and myosin phosphorylation. Inhibition of rho associated kinase reduced endothelin-1 stimulated myosin phosphorylation and contractile force development. Endothelin-1 stimulated migration with a dose-response relationship similar to that observed for contraction. Endothelin A and B receptors mediated contraction while migration was mediated predominantly through endothelin B receptors. Platelet derived growth factor and serum, but not endothelin-1, induced proliferation. Conclusions: Endothelin-1 stimulates colonic subepithelial myofibroblast contraction and migration via endothelin receptor mediated myosin phosphorylation. These results support an important role for subepithelial myofibroblasts in the injury response of the gut and consequently intestinal wound repair.

p38 MAP kinase mediates platelet-derived growth factor-stimulated migration of hepatic myofibroblasts

Although the migration of hepatic myofibroblasts (HMFs) contributes to the development of fibrosis, the signals regulating migration of these cells are poorly understood. In this study, we tested the hypothesis that HMF migration is stimulated by platelet‐derived growth factor‐BB (PDGF‐BB) through p38 mitogen‐activated protein (MAP) kinase and extracellular signal‐regulated kinase (ERK) signaling pathways. This hypothesis was addressed by directly visualizing the migration of cultured human HMFs into a wound. PDGF‐BB stimulated membrane ruffling, migration, and proliferation. PDGF‐BB also induced activation of p38 MAP kinase, its downstream effector, heat shock protein (HSP) 27, ERK 1 and ERK 2, and p125 focal adhesion kinase (FAK). Selective antagonism of p38 MAP kinase blocked PDGF‐BB‐stimulated HSP 27 phosphorylation, membrane ruffling, and migration, but did not alter PDGF‐BB‐induced proliferation. Selective antagonism of ERK kinase inhibited PDGF‐BB‐induced ERK phosphorylation and proliferation, but did not affect PDGF‐BB‐stimulated migration. Concentrations of PDGF‐BB that stimulated migration and proliferation did not influence myosin‐dependent contractility. Neither selective inhibition of p38 MAP kinase nor ERKs altered PDGF‐BB‐induced activation of FAK. In conclusion, these results provide novel evidence indicating that (1) HMF migration is stimulated by PDGF‐BB through the regulation of membrane ruffling by a p38 MAP kinase signaling pathway, (2) whereas p38 MAP kinase mediates PDGF‐BB‐stimulated migration, but not proliferation, ERKs mediate PDGF‐induced proliferation, but not migration, and (3) increases in myosin‐dependent contractility are not required for PDGF‐BB‐stimulated migration.

Hepatic stellate cell protrusions couple platelet-derived growth factor-BB to chemotaxis†

Hepatic stellate cells play an essential role in the livers injury response. Although stellate cells are defined by the presence of cytoplasmic protrusions, the function of these characteristic structures has been obscure. We hypothesized that stellate cell protrusions act by coupling injury‐associated stimuli to chemotaxis. To test this hypothesis, we developed an assay for directly visualizing the response of living stellate cells in early primary culture to local stimulation of the tips of protrusions with platelet‐derived growth factor‐BB (PDGF). Stellate cells exhibited elongate protrusions containing actin, myosin, and tubulin. PDGF, but not cytochrome C, localized at a protrusion tip induced a coordinated series of morphological events—cell spreading at the tip, movement of the cell body toward the PDGF, and retraction of trailing protrusions— that resulted in chemotaxis. Soluble PDGF and AG 1296, a receptor tyrosine kinase inhibitor, both reduced stellate cell chemotaxis. PDGF‐induced chemotaxis was associated with an early and transient increase in myosin phosphorylation within the spreading lamella. We observed that blebbistatin, a myosin II inhibitor, completely and reversibly blocked protrusion‐mediated lamella formation and chemotaxis. Moreover, blockade of MRLC phosphorylation with the myosin light chain kinase inhibitor, ML‐7, or the rho kinase inhibitor, Y‐27632, blocked lamella formation, myosin phosphorylation within the protrusion, and chemotaxis. Conclusion: These results support a model in which protrusions permit stellate cells to promptly detect PDGF distant from their cell bodies and transduce this signal into mechanical forces that propel the cell toward the site of injury. (HEPATOLOGY 2007.)

Rho and p38 MAP Kinase Signaling Pathways Mediate LPA-Stimulated Hepatic Myofibroblast Migration

Although hepatic myofibroblast migration plays a key role in the livers injury response, the signal transduction pathways mediating the migration of this cell type are uncertain. Recently, we reported that lysophosphatidic acid (LPA) stimulates the migration of hepatic myofibroblasts. The goal of this study was to test the hypothesis that rho and p38 MAP kinase signaling pathways mediate LPA-stimulated hepatic myofibroblast migration. We measured migration, myosin regulatory light chain and p38 MAP kinase phosphorylation, and contractile force generation by human hepatic myofibroblasts. LPA stimulated migration in a dose-dependent and saturable manner that was partially blocked by Y-27632, a rho-associated kinase inhibitor, as well as by SB-202190, a p38 MAP kinase inhibitor. LPA also induced myosin regulatory light chain phosphorylation and contractile force generation in a Y-27632 dependent, and SB-202190 independent fashion. Moreover, LPA stimulated a dose-dependent and saturable phosphorylation of p38 MAP kinase, which was not altered by Y-27632 or C3 transferase, a rho inactivator. These novel results suggest that LPA stimulates hepatic myofibroblast migration via distinct pathways that signal through rho and p38 MAP kinase.

Adaptive Immune Regulation of Mammary Postnatal Organogenesis

Postnatal organogenesis occurs in an immune competent environment and is tightly controlled by interplay between positive and negative regulators. Innate immune cells have beneficial roles in postnatal tissue remodeling, but roles for the adaptive immune system are currently unexplored. Here we show that adaptive immune responses participate in the normal postnatal development of a non-lymphoid epithelial tissue. Since the mammary gland (MG) is the only organ developing predominantly after birth, we utilized it as a powerful system to study adaptive immune regulation of organogenesis. We found that antigen-mediated interactions between mammary antigen-presenting cells and interferon-γ (IFNγ)-producing CD4+ T helper 1 cells participate in MG postnatal organogenesis as negative regulators, locally orchestrating epithelial rearrangement. IFNγ then affects luminal lineage differentiation. This function of adaptive immune responses, regulating normal development, changes the paradigm for studying players of postnatal organogenesis and provides insights into immune surveillance and cancer transformation.

Unexpected Role for Adaptive αβTh17 Cells in Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a devastating disorder characterized by increased alveolar permeability with no effective treatment beyond supportive care. Current mechanisms underlying ARDS focus on alveolar endothelial and epithelial injury caused by products of innate immune cells and platelets. However, the role of adaptive immune cells in ARDS remains largely unknown. In this study, we report that expansion of Ag-specific αβTh17 cells contributes to ARDS by local secretion of IL-17A, which in turn directly increases alveolar epithelial permeability. Mice with a highly restrictive defect in Ag-specific αβTh17 cells were protected from experimental ARDS induced by a single dose of endotracheal LPS. Loss of IL-17 receptor C or Ab blockade of IL-17A was similarly protective, further suggesting that IL-17A released by these cells was responsible for this effect. LPS induced a rapid and specific clonal expansion of αβTh17 cells in the lung, as determined by deep sequencing of the hypervariable CD3RβVJ region of the TCR. Our findings could be relevant to ARDS in humans, because we found significant elevation of IL-17A in bronchoalveolar lavage fluid from patients with ARDS, and rIL-17A directly increased permeability across cultured human alveolar epithelial monolayers. These results reveal a previously unexpected role for adaptive immune responses that increase alveolar permeability in ARDS and suggest that αβTh17 cells and IL-17A could be novel therapeutic targets for this currently untreatable disease.