Alison J. Logar

TH17 Cells Mediate Steroid-Resistant Airway Inflammation and Airway Hyperresponsiveness in Mice

Steroid-resistant asthma comprises an important source of morbidity in patient populations. TH17 cells represent a distinct population of CD4+ Th cells that mediate neutrophilic inflammation and are characterized by the production of IL-17, IL-22, and IL-6. To investigate the function of TH17 cells in the context of Ag-induced airway inflammation, we polarized naive CD4+ T cells from DO11.10 OVA-specific TCR-transgenic mice to a TH2 or TH17 phenotype by culturing in conditioned medium. In addition, we also tested the steroid responsiveness of TH2 and TH17 cells. In vitro, TH17 cytokine responses were not sensitive to dexamethasone (DEX) treatment despite immunocytochemistry confirming glucocorticoid receptor translocation to the nucleus following treatment. Transfer of TH2 cells to mice challenged with OVA protein resulted in lymphocyte and eosinophil emigration into the lung that was markedly reduced by DEX treatment, whereas TH17 transfer resulted in increased CXC chemokine secretion and neutrophil influx that was not attenuated by DEX. Transfer of TH17 or TH2 cells was sufficient to induce airway hyperresponsiveness (AHR) to methacholine. Interestingly, AHR was not attenuated by DEX in the TH17 group. These data demonstrate that polarized Ag-specific T cells result in specific lung pathologies. Both TH2 and TH17 cells are able to induce AHR, whereas TH17 cell-mediated airway inflammation and AHR are steroid resistant, indicating a potential role for TH17 cells in steroid-resistant asthma.

Prospective identification of myogenic endothelial cells in human skeletal muscle

We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.

Interleukin-17 Is Required for T Helper 1 Cell Immunity and Host Resistance to the Intracellular Pathogen Francisella tularensis

The importance of T helper type 1 (Th1) cell immunity in host resistance to the intracellular bacterium Francisella tularensis is well established. However, the relative roles of interleukin (IL)-12-Th1 and IL-23-Th17 cell responses in immunity to F. tularensis have not been studied. The IL-23-Th17 cell pathway is critical for protective immunity against extracellular bacterial infections. In contrast, the IL-23-Th17 cell pathway is dispensable for protection against intracellular pathogens such as Mycobacteria. Here we show that the IL-23-Th17 pathway regulates the IL-12-Th1 cell pathway and was required for protective immunity against F.tularensis live vaccine strain. We show that IL-17A, but not IL-17F or IL-22, induced IL-12 production in dendritic cells and mediated Th1 responses. Furthermore, we show that IL-17A also induced IL-12 and interferon-gamma production in macrophages and mediated bacterial killing. Together, these findings illustrate a biological function for IL-17A in regulating IL-12-Th1 cell immunity and host responses to an intracellular pathogen.

Recombinant Adenovirus Induces Maturation of Dendritic Cells via an NF-κB-Dependent Pathway

ABSTRACT Recombinant adenovirus (rAd) infection is one of the most effective and frequently employed methods to transduce dendritic cells (DC). Contradictory results have been reported recently concerning the influence of rAd on the differentiation and activation of DC. In this report, we show that, as a result of rAd infection, mouse bone marrow-derived immature DC upregulate expression of major histocompatibility complex class I and II antigens, costimulatory molecules (CD40, CD80, and CD86), and the adhesion molecule CD54 (ICAM-1). rAd-transduced DC exhibited increased allostimulatory capacity and levels of interleukin-6 (IL-6), IL-12p40, IL-15, gamma interferon, and tumor necrosis factor alpha mRNAs, without effects on other immunoregulatory cytokine transcripts such as IL-10 or IL-12p35. These effects were not related to specific transgenic sequences or to rAd genome transcription. The rAd effect correlated with a rapid increase (1 h) in the NF-κB–DNA binding activity detected by electrophoretic mobility shift assays. rAd-induced DC maturation was blocked by the proteasome inhibitorNα-p-tosyl-l-lysine chloromethyl ketone (TLCK) or by infection with rAd-IκB, an rAd-encoding the dominant-negative form of IκB. In vivo studies showed that after intravenous administration, rAds were rapidly entrapped in the spleen by marginal zone DC that mobilized to T-cell areas, a phenomenon suggesting that rAd also induced DC differentiation in vivo. These findings may explain the immunogenicity of rAd and the difficulties in inducing long-term antigen-specific T-cell hyporesponsiveness with rAd-transduced DC.

Dermal-resident CD14+ cells differentiate into Langerhans cells

Epidermal Langerhans cells (LCs) show extraordinary immunostimulatory capacity and play a key role in the initiation and regulation of immune responses. Studies of LC biology are currently the focus of efforts to engineer immune responses and to better understand the immunopathology of cutaneous diseases. Here we identified and characterized a population of LC precursors that were resident in human skin. These immediate precursors expressed CD14, langerin and functional CCR6. When cultured with transforming growth factor-β1 alone, they had the potential to differentiate into epidermal LCs; when cultured in the presence of granulocyte macrophage–colony-stimulating factor and interleukin 4 they differentiated into functionally mature dendritic cells. Identification and characterization of these LC precursors provided insight into LC biology and the mechanism(s) through which LCs repopulate the epidermis.

Aspirin Inhibits In Vitro Maturation and In Vivo Immunostimulatory Function of Murine Myeloid Dendritic Cells

Aspirin is the most commonly used analgesic and antiinflammatory agent. In this study, at physiological concentrations, it profoundly inhibited CD40, CD80, CD86, and MHC class II expression on murine, GM-CSF + IL-4 stimulated, bone marrow-derived myeloid dendritic cells (DC). CD11c and MHC class I expression were unaffected. The inhibitory action was dose dependent and was evident at concentrations higher than those necessary to inhibit PG synthesis. Experiments with indomethacin revealed that the effects of aspirin on DC maturation were cyclooxygenase independent. Nuclear extracts of purified, aspirin-treated DC revealed a decreased NF-κB DNA-binding activity, whereas Ab supershift analysis indicated that aspirin targeted primarily NF-κB p50. Unexpectedly, aspirin promoted the generation of CD11c+ DC, due to apparent suppression of granulocyte development. The morphological and ultrastructural appearance of aspirin-treated cells was consistent with immaturity. Aspirin-treated DC were highly efficient at Ag capture, via both mannose receptor-mediated endocytosis and macropinocytosis. By contrast, they were poor stimulators of naive allogeneic T cell proliferation and induced lower levels of IL-2 in responding T cells. They also exhibited impaired IL-12 expression and did not produce IL-10 after LPS stimulation. Assessment of the in vivo function of aspirin-treated DC, pulsed with the hapten trinitrobenzenesulfonic acid, revealed an inability to induce normal cell-mediated contact hypersensitivity, despite the ability of the cells to migrate to T cell areas of draining lymphoid tissue. These data provide new insight into the immunopharmacology of aspirin and suggest a novel approach to the manipulation of DC for therapeutic application.

The in vitro effects of bupivacaine on articular chondrocytes

We have studied the effects of bupivacaine on human and bovine articular chondrocytes in vitro. Time-lapse confocal microscopy of human articular chondrocytes showed > 95% cellular death after exposure to 0.5% bupivacaine for 30 minutes. Human and bovine chondrocytes exposed to 0.25% bupivacaine had a time-dependent reduction in viability, with longer exposure times resulting in higher cytotoxicity. Cellular death continued even after removal of 0.25% bupivacaine. After exposure to 0.25% bupivacaine for 15 minutes, flow cytometry showed bovine chondrocyte viability to be 41% of saline control after seven days. After exposure to 0.125% bupivacaine for up to 60 minutes, the viability of both bovine and human chondrocytes was similar to that of control groups. These data show that prolonged exposure 0.5% and 0.25% bupivacaine solutions are potentially chondrotoxic.

A Reservoir of Brown Adipocyte Progenitors in Human Skeletal Muscle

Brown adipose tissue uncoupling protein‐1 (UCP1) plays a major role in the control of energy balance in rodents. It has long been thought, however, that there is no physiologically relevant UCP1 expression in adult humans. In this study we show, using an original approach consisting of sorting cells from various tissues and differentiating them in an adipogenic medium, that a stationary population of skeletal muscle cells expressing the CD34 surface protein can differentiate in vitro into genuine brown adipocytes with a high level of UCP1 expression and uncoupled respiration. These cells can be expanded in culture, and their UCP1 mRNA expression is strongly increased by cell‐permeating cAMP derivatives and a peroxisome‐proliferator‐activated receptor‐γ (PPARγ) agonist. Furthermore, UCP1 mRNA was detected in the skeletal muscle of adult humans, and its expression was increased in vivo by PPARγ agonist treatment. All the studies concerning UCP1 expression in adult humans have until now been focused on the white adipose tissue. Here we show for the first time the existence in human skeletal muscle and the prospective isolation of progenitor cells with a high potential for UCP1 expression. The discovery of this reservoir generates a new hope of treating obesity by acting on energy dissipation.

Immature and mature CD8α+ dendritic cells prolong the survival of vascularized heart allografts

CD8α+ and CD8α− dendritic cells (DCs) arise from committed bone marrow progenitors and can induce or regulate immune reactivity. Previously, the maturational status of CD8α− (myeloid) DCs has been shown to influence allogeneic T cell responses and allograft survival. Although CD8α+ DCs have been implicated in central tolerance and found to modulate peripheral T cell function, their influence on the outcome of organ transplantation has not been examined. Consistent with their equivalent high surface expression of MHC and costimulatory molecules, sorted mature C57BL/10J (B10; H2b) DCs of either subset primed naive, allogeneic C3H/HeJ (C3H; H2k) recipients for Th1 responses. Paradoxically and in contrast to their CD8α− counterparts, mature CD8α+ B10 DCs given systemically 7 days before transplant markedly prolonged B10 heart graft survival in C3H recipients. This effect was associated with specific impairment of ex vivo antidonor T cell proliferative responses, which was not reversed by exogenous IL-2. Further analyses of possible underlying mechanisms indicated that neither immune deviation nor induction of regulatory cells was a significant contributory factor. In contrast to the differential capacity of the mature DC subsets to affect graft outcome, immature CD8α+ and CD8α− DCs administered under the same experimental conditions significantly prolonged transplant survival. These observations demonstrate for the first time the innate capacity of CD8α+ DCs to regulate alloimmune reactivity and transplant survival, independent of their maturation status. Mobilization of such a donor DC subset with capacity to modulate antidonor immunity may have significant implications for the therapy of allograft rejection.

Vitamin D3 attenuates Th2 responses to Aspergillus fumigatus mounted by CD4+ T cells from cystic fibrosis patients with allergic bronchopulmonary aspergillosis

Allergic bronchopulmonary aspergillosis (ABPA) is caused by a dominant Th2 immune response to antigens derived from the opportunistic mold Aspergillus, most commonly Aspergillus fumigatus. It occurs in 4%-15% of patients with cystic fibrosis (CF); however, not all patients with CF infected with A. fumigatus develop ABPA. Therefore, we compared cohorts of A. fumigatus-colonized CF patients with and without ABPA to identify factors mediating tolerance versus sensitization. We found that the costimulatory molecule OX40 ligand (OX40L) was critical in driving Th2 responses to A. fumigatus in peripheral CD4+ T cells isolated from patients with ABPA. In contrast, CD4+ T cells from the non-ABPA cohort did not mount enhanced Th2 responses in vitro and contained a higher frequency of TGF-beta-expressing regulatory T cells. Heightened Th2 reactivity in the ABPA cohort correlated with lower mean serum vitamin D levels. Further, in vitro addition of 1,25 OH-vitamin D3 substantially reduced DC expression of OX40L and increased DC expression of TGF-beta. This in vitro treatment also resulted in increased Treg TGF-beta expression and reduced Th2 responses by CD4+ T cells from patients with ABPA. These data provide rationale for a therapeutic trial of vitamin D to prevent or treat ABPA in patients with CF.