Timothy B. Oriss

Activation of c-Kit in dendritic cells regulates T helper cell differentiation and allergic asthma.

Dendritic cells (DCs) are integral to the differentiation of T helper cells into T helper type 1 TH1, TH2 and TH17 subsets. Interleukin-6 (IL-6) plays an important part in regulating these three arms of the immune response by limiting the TH1 response and promoting the TH2 and TH17 responses. In this study, we investigated pathways in DCs that promote IL-6 production. We show that the allergen house dust mite (HDM) or the mucosal adjuvant cholera toxin promotes cell surface expression of c-Kit and its ligand, stem cell factor (SCF), on DCs. This dual upregulation of c-Kit and SCF results in sustained signaling downstream of c-Kit, promoting IL-6 secretion. Intranasal administration of antigen into c-Kit–mutant mice or neutralization of IL-6 in cultures established from the lung-draining lymph nodes of immunized wild-type mice blunted the TH2 and TH17 responses. DCs lacking functional c-Kit or those unable to express membrane-bound SCF secreted lower amounts of IL-6 in response to HDM or cholera toxin. DCs expressing nonfunctional c-Kit were unable to induce a robust TH2 or TH17 response and elicited diminished allergic airway inflammation when adoptively transferred into mice. Expression of the Notch ligand Jagged-2, which has been associated with TH2 differentiation, was blunted in DCs from c-Kit–mutant mice. c-Kit upregulation was specifically induced by TH2- and TH17-skewing stimuli, as the TH1-inducing adjuvant, CpG oligodeoxynucleotide, did not promote either c-Kit or Jagged-2 expression. DCs generated from mice expressing a catalytically inactive form of the p110δ subunit of phosphatidylinositol-3 (PI3) kinase (p110D910A) secreted lower amounts of IL-6 upon stimulation with cholera toxin. Collectively, these results highlight the importance of the c-Kit–PI3 kinase–IL-6 signaling axis in DCs in regulating T cell responses.

Treg-mediated immunosuppression involves activation of the Notch-HES1 axis by membrane-bound TGF-β

Studies in humans and mice show an important role for Tregs in the control of immunological disorders. The mechanisms underlying the immunosuppressive functions of Tregs are not well understood. Here, we show that CD4+ T cells expressing Foxp3 and membrane-bound TGF-beta (TGF-beta(m+)Foxp3+), previously shown to be immunosuppressive in both allergic and autoimmune diseases, activate the Notch1-hairy and enhancer of split 1 (Notch1-HES1) axis in target cells. Soluble TGF-beta and cells secreting similar levels of soluble TGF-beta were unable to trigger Notch1 activation. Inhibition of Notch1 activation in vivo reversed the immunosuppressive functions of TGF-beta(m+)Foxp3+ cells, resulting in severe allergic airway inflammation. Integration of the TGF-beta and Notch1 pathways may be an important mechanism for the maintenance of immune homeostasis in the periphery.

WNT5A Is a Regulator of Fibroblast Proliferation and Resistance to Apoptosis

Usual interstitial pneumonia (UIP) is a specific histopathologic pattern of interstitial lung fibrosis that may be idiopathic or secondary to autoimmune diseases and environmental exposures. In this study, we compared gene expression patterns in primary fibroblasts isolated from lung tissues with UIP histology and fibroblasts isolated from lung tissues with normal histology using expression microarrays. We found that WNT5A was significantly increased in fibroblasts obtained from UIP lung tissues compared with normal lung fibroblasts, an observation verified by quantitative real-time RT-PCR and Western blot. Because the role of WNT5A in UIP is unknown, we treated normal lung fibroblasts or UIP lung fibroblasts with WNT5A, and found that WNT5A increased proliferation as well as relative resistance to H2O2-induced apoptosis. This effect was not mediated through the canonical WNT/beta-catenin pathway, as WNT5A induced a decrease in beta-catenin levels in the same cells. In addition, WNT5A induced increases in fibronectin and alpha(5)-integrin in normal lung fibroblasts. Collectively, our data suggest that WNT5A may play a role in fibroblast expansion and survival characteristics of idiopathic pulmonary fibrosis and other fibrotic interstitial lung diseases that exhibit UIP histological patterns.

Early infection with respiratory syncytial virus impairs regulatory T cell function and increases susceptibility to allergic asthma.

Immune tolerance is instituted early in life, during which time regulatory T (Treg) cells have an important role. Recurrent infections with respiratory syncytial virus (RSV) in early life increase the risk for asthma in adult life. Repeated infection of infant mice tolerized to ovalbumin (OVA) through their mothers milk with RSV induced allergic airway disease in response to OVA sensitization and challenge, including airway inflammation, hyper-reactivity and higher OVA-specific IgE, as compared to uninfected tolerized control mice. Virus infection induced GATA-3 expression and T helper type 2 (TH2) cytokine production in forkhead box P3 (FOXP3)+ Treg cells and compromised the suppressive function of pulmonary Treg cells in a manner that was dependent on interleukin-4 receptor α (IL-4Rα) expression in the host. Thus, by promoting a TH2-type inflammatory response in the lung, RSV induced a TH2-like effector phenotype in Treg cells and attenuated tolerance to an unrelated antigen (allergen). Our findings highlight a mechanism by which viral infection targets a host-protective mechanism in early life and increases susceptibility to allergic disease.

Transforming Growth Factor β Blocks Tec Kinase Phosphorylation, Ca2+ Influx, and NFATc Translocation Causing Inhibition of T Cell Differentiation

Transforming growth factor (TGF)-β inhibits T cell proliferation and differentiation. TGF-β has been shown to inhibit the expression of transcription factors such as GATA-3 and T-bet that play important roles in T cell differentiation. Here we show that TGF-β inhibits T cell differentiation at a more proximal step. An early event during T cell activation is increased intracellular calcium levels. Calcium influx in activated T cells and the subsequent activation of transcription factors such as NFATc, events essential for T cell differentiation, are modulated by the Tec kinases that are downstream of the T cell receptor and CD28. We show that in stimulated CD4+ T cells, TGF-β inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation. Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-β limits T cell differentiation along both the Th1 and Th2 lineages.

Dynamics of Dendritic Cell Phenotype and Interactions with CD4+ T Cells in Airway Inflammation and Tolerance

An emerging concept is that different types of dendritic cells (DCs) initiate different immune outcomes, such as tolerance vs inflammation. In this study, we have characterized the DCs from the lung draining lymph nodes of mice immunized for allergic airway inflammation or tolerance and examined their interactions with CD4+ T cells. The DC population derived from tolerized mice was predominantly CD11c+, B220+, Gr-1+, CD11b−, and MHC class IIlow, which resembled plasmacytoid-type DCs whereas DCs from the inflammatory condition were largely CD11c+, B220−, Gr-1−, CD11b+, and MHC class IIhigh resembling myeloid-type DCs. The DCs from the tolerogenic condition were poor inducers of T cell proliferation. DCs from both conditions induced T cell IL-4 production but the T cells cultured with tolerogenic DCs were unresponsive to IL-4 as indicated by inhibition of STAT6 activation and expression of growth factor-independent 1, which has been recently shown to be important for STAT6-activated Th2 cell expansion. Our data suggest that airway tolerance vs inflammation is determined by the DC phenotype in lung draining lymph nodes.

Indoleamine 2,3-dioxygenase in lung dendritic cells promotes Th2 responses and allergic inflammation

Indoleamine 2,3 dioxygenase (IDO) has emerged as an important mediator of immune tolerance via inhibition of Th1 responses. However, the role of IDO in antigen-induced tolerance or allergic inflammation in the airways that is regulated by Th2 responses has not been elucidated. By using IDO−/− mice, we found no impairment of airway tolerance, but, surprisingly, absence of IDO provided significant relief from establishment of allergic airways disease, as evident from attenuated Th2 cytokine production, airway inflammation, mucus secretion, airway hyperresponsiveness, and serum ovalbumin-specific IgE. Myeloid dendritic cells isolated from lung-draining lymph nodes of mice immunized for either Th1 or Th2 response revealed fewer mature dendritic cells in the lymph nodes of IDO−/− mice. However, the net functional impact of IDO deficiency on antigen-induced responses was more remarkable in the Th2 model than in the Th1 model. Collectively, these data suggest that IDO is not required for the induction of immune tolerance in the airways but plays a role in promoting Th2-mediated allergic airway inflammation via unique effects on lung dendritic cells.

The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe

The intensity of hemolytic anemia has been proposed as an independent risk factor for the development of certain clinical complications of sickle cell disease, such as pulmonary hypertension, hypoxemia and cutaneous leg ulceration. A composite variable derived from several individual markers of hemolysis could facilitate studies of the underlying mechanisms of hemolysis. In this study, we assessed the association of hemolysis with outcomes in sickle cell anemia. A hemolytic component was calculated by principal component analysis from reticulocyte count, serum lactate dehydrogenase, aspartate aminotransferase and total bilirubin concentrations in 415 hemoglobin SS patients. Association of this component with direct markers of hemolysis and clinical outcomes was assessed. As primary validation, both plasma red blood cell microparticles and cell-free hemoglobin concentration were higher in the highest hemolytic component quartile compared to the lowest quartile (P≤0.0001 for both analyses). The hemolytic component was lower with hydroxyurea therapy, higher hemoglobin F, and alpha-thalassemia (P≤0.0005); it was higher with higher systemic pulse pressure, lower oxygen saturation, and greater values for tricuspid regurgitation velocity, left ventricular diastolic dimension and left ventricular mass (all P<0.0001). Two-year follow-up analysis showed that a high hemolytic component was associated with an increased risk of death (hazard ratio, HR 3.44; 95% confidence interval, CI: 1.2–9.5; P=0.02). The hemolytic component reflects direct markers of intravascular hemolysis in patients with sickle cell disease and allows for adjusted analysis of associations between hemolytic severity and clinical outcomes. These results confirm associations between hemolytic rate and pulse pressure, oxygen saturation, increases in Doppler-estimated pulmonary systolic pressures and mortality (Clinicaltrials.gov identifier: NCT00492531).

Macrophages Regulators of Sex Differences in Asthma

Females are more susceptible to development of asthma than are males. In a mouse model of ovalbumin-induced airway inflammation, with aggravated disease in females compared with males, we studied interactions between immune and resident lung cells during asthma development to elucidate which processes are affected by sex. We studied numbers of regulatory T cells (Tregs), effector T cells, myeloid dendritic cells (mDCs), and alternatively activated macrophages (AAMPhi), and their functional capabilities. Male and female mice had comparable Treg numbers in lung tissue and comparable Treg function, but effector T cells had expanded to a greater extent in lungs of females after ovalbumin exposure. This difference in T cell expansion was therefore not the result of lack of Treg control, but appeared to be driven by a greater number of inflammatory mDCs migrating from the lungs to lymph nodes in females. Resident lung cells can influence mDC migration, and AAMPhi in lung tissue were found to be involved. Artificially elevating the number of AAMPhi in lung tissue increased the migration of mDCs and airway inflammation. We found greater numbers of AAMPhi in female lungs than in males; we therefore postulate that AAMPhi are involved in increased airway inflammation found in female mice.

TNF-alpha from inflammatory dendritic cells (DCs) regulates lung IL-17A/IL-5 levels and neutrophilia versus eosinophilia during persistent fungal infection.

Aspergillus fumigatus is commonly associated with allergic bronchopulmonary aspergillosis in patients with severe asthma in which chronic airway neutrophilia predicts a poor outcome. We were able to recapitulate fungus-induced neutrophilic airway inflammation in a mouse model in our efforts to understand the underlying mechanisms. However, neutrophilia occurred in a mouse strain-selective fashion, providing us with an opportunity to perform a comparative study to elucidate the mechanisms involved. Here we show that TNF-α, largely produced by Ly6c+CD11b+ dendritic cells (DCs), plays a central role in promoting IL-17A from CD4+ T cells and collaborating with it to induce airway neutrophilia. Compared with C57BL/6 mice, BALB/c mice displayed significantly more TNF-α–producing DCs and macrophages in the lung. Lung TNF-α levels were drastically reduced in CD11c-DTR BALB/c mice depleted of CD11c+ cells, and TNF-α–producing Ly6c+CD11b+ cells were abolished in Dectin-1−/− and MyD88−/− BALB/c mice. TNF-α deficiency itself blunted accumulation of inflammatory Ly6c+CD11b+ DCs. Also, lack of TNF-α decreased IL-17A but promoted IL-5 levels, switching inflammation from a neutrophil to eosinophil bias resembling that in C57BL/6 mice. The TNF-αlow DCs in C57BL/6 mice contained more NF-κB p50 homodimers, which are strong repressors of TNF-α transcription. Functionally, collaboration between TNF-α and IL-17A triggered significantly higher levels of the neutrophil chemoattractants keratinocyte cytokine and macrophage inflammatory protein 2 in BALB/c mice. Our study identifies TNF-α as a molecular switch that orchestrates a sequence of events in DCs and CD4 T cells that promote neutrophilic airway inflammation.