Josalyn L. Cho

Dedifferentiation of committed epithelial cells into stem cells in vivo

Cellular plasticity contributes to the regenerative capacity of plants, invertebrates, teleost fishes and amphibians. In vertebrates, differentiated cells are known to revert into replicating progenitors, but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells, we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming, the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states, notably cancer.

18F-FDG Uptake Rate Is a Biomarker of Eosinophilic Inflammation and Airway Response in Asthma

In asthma, the relationship among airway inflammation, airway hyperresponsiveness, and lung function is poorly understood. Methods to noninvasively assess these relationships in human subjects are needed. We sought to determine whether 18F-FDG uptake rate (Ki, min−1) could serve as a biomarker of eosinophilic inflammation and local lung function. Methods: We used PET/CT to assess regional pulmonary perfusion (Q˙), specific ventilation per unit volume (sV˙A), fractional gas content (Fgas), airway wall thickness, and regional Ki 10 h after segmental allergen challenge to the right middle lobe in 6 asthmatic subjects with demonstrated atopy. Q˙, sV˙A, and Fgas in the allergen-challenged lobe were compared with the right upper lobe, where diluent was applied as a control. The airway wall thickness aspect ratio (ω) of the allergen-challenged airway was compared with those of similarly sized airways from unaffected areas of the lung. Differences in Ki between allergen and diluent segments were compared with those in cell counts obtained 24 h after the allergen challenge by a bronchoalveolar lavage of the respective segments. Results: We found systematic reductions in regional Q˙, sV˙A, and Fgas and increased ω in all subjects. The ratio of eosinophil count (allergen to diluent) was linearly related (R2 = 0.9917, P < 0.001) to the ratio of Ki. Conclusion: Regional Ki measured with PET is a noninvasive and highly predictive biomarker of eosinophilic airway inflammation and its functional effects. This method may serve to help in the understanding of allergic inflammation and test the therapeutic effectiveness of novel drugs or treatments.

Compartmentalized chemokine-dependent regulatory T-cell inhibition of allergic pulmonary inflammation

BACKGROUND Induction of endogenous regulatory T (Treg) cells represents an exciting new potential modality for treating allergic diseases, such as asthma. Treg cells have been implicated in the regulation of asthma, but the anatomic location in which they exert their regulatory function and the mechanisms controlling the migration necessary for their suppressive function in asthma are not known. Understanding these aspects of Treg cell biology will be important for harnessing their power in the clinic. OBJECTIVE We sought to determine the anatomic location at which Treg cells exert their regulatory function in the sensitization and effector phases of allergic asthma and to determine the chemokine receptors that control the migration of Treg cells to these sites in vivo in both mice and human subjects. METHODS The clinical efficacy and anatomic location of adoptively transferred chemokine receptor-deficient CD4(+)CD25(+) forkhead box protein 3-positive Treg cells was determined in the sensitization and effector phases of allergic airway inflammation in mice. The chemokine receptor expression profile was determined on Treg cells recruited into the human airway after bronchoscopic segmental allergen challenge of asthmatic patients. RESULTS We show that CCR7, but not CCR4, is required on Treg cells to suppress allergic airway inflammation during the sensitization phase. In contrast, CCR4, but not CCR7, is required on Treg cells to suppress allergic airway inflammation during the effector phase. Consistent with our murine studies, human subjects with allergic asthma had an increase in CCR4-expressing functional Treg cells in the lungs after segmental allergen challenge. CONCLUSION The location of Treg cell function differs during allergic sensitization and allergen-induced recall responses in the lung, and this differential localization is critically dependent on differential chemokine function.

Birefringence microscopy platform for assessing airway smooth muscle structure and function in vivo

A birefringence microscopy technique provides structural and functional information about airway smooth muscle in healthy and mild asthmatic subjects. Advanced analysis of asthma Not all individuals who have respiratory reactions to allergens progress to asthma. In this issue, Cho et al. found that although allergic asthmatics and allergic nonasthmatics both experienced inflammation after allergen challenge, asthmatics had more mucin and type 2 cytokines, and allergen-specific T cells sampled from the airspace had increased innate type 2 receptors. Using orientation-resolved optical coherence tomography, described by Adams et al., they demonstrated that allergic asthmatics also had increased airway smooth muscle mass. This technique allows for in vivo imaging of airway smooth muscle structure and function, which could shed light on the pathogenesis of many respiratory diseases. The inability to visualize airway smooth muscle (ASM) cells in vivo is a major obstacle in understanding their role in normal physiology and diseases. At present, there is no imaging modality available to assess ASM in vivo. Confocal endomicroscopy lacks the penetration depth and field of view, and conventional optical coherence tomography (OCT) does not have sufficient contrast to differentiate ASM from surrounding tissues. We have developed a birefringence microscopy platform that leverages the micro-organization of tissue to add further dimension to traditional OCT. We have used this technology to validate ASM measurements in ex vivo swine and canine studies, visualize and characterize volumetric representations of ASM in vivo, and quantify and predict ASM contractile force as a function of optical retardation. We provide in vivo images and volumetric assessments of ASM in living humans and document structural disease variations in subjects with mild asthma. The opportunity to link inflammatory responses to ASM responses and to link ASM responses to clinical responses and outcomes could lead to an increased understanding of diseases of the airway and, ultimately, to improved patient outcomes.

Inhibiting CXCR3-Dependent CD8+ T Cell Trafficking Enhances Tolerance Induction in a Mouse Model of Lung Rejection

Lung transplantation remains the only effective therapy for patients with end-stage pulmonary diseases. Unfortunately, acute rejection of the lung remains a frequent complication and is an important cause of morbidity and mortality. The induction of transplant tolerance is thought to be dependent, in part, on the balance between allograft effector mechanisms mediated by effector T lymphocytes (Teff), and regulatory mechanisms mediated by FOXP3+ regulatory T cells (Treg). In this study, we explored an approach to tip the balance in favor of regulatory mechanisms by modulating chemokine activity. We demonstrate in an adoptive transfer model of lung rejection that CXCR3-deficient CD8+ Teff have impaired migration into the lungs compared with wild-type Teff, which results in a dramatic reduction in fatal pulmonary inflammation. The lungs of surviving mice contained tolerized CXCR3-deficient Teff, as well as a large increase in Treg. We confirmed that Treg were needed for tolerance and that their ability to induce tolerance was dependent on their numbers in the lung relative to the numbers of Teff. These data suggest that transplantation tolerance can be achieved by reducing the recruitment of some, but not necessarily all, CD8+ Teff into the target organ and suggest a novel approach to achieve transplant tolerance.

Standardization and quality control for high‐dimensional mass cytometry studies of human samples

Mass cytometry (CyTOF), a mass spectrometry‐based single cell phenotyping technology, allows utilization of over 35 antibodies in a single sample and is a promising tool for translational human immunology studies. Although several analysis tools are available to interpret the complex data sets generated, a robust method for standardization and quality control within and across studies is needed. Here we report an efficient and easily adaptable method to monitor quality of individual samples in human immunology studies and to facilitate reproducible data analysis. Samples to be assessed are spiked with a defined amount of reference peripheral blood mononuclear cells from a healthy donor, derived from a single large blood draw. The presence of known standardized numbers and phenotypic profiles of these reference cells greatly facilitates sample analysis by allowing for: 1) quality control for consistent staining of each antibody in the panel, 2) identification of potential batch effects, and 3) implementation of a robust gating strategy. We demonstrate the utility of this method using peripheral blood and bronchoalveolar lavage samples from HIV+ patients by characterizing their CD8+ T‐cell phenotypes and cytokine expression, respectively. Our results indicate that this method allows quality control of experimental conditions and results in highly reproducible population frequencies through a robust gating strategy.

Enhanced Tim3 Activity Improves Survival after Influenza Infection

Influenza is a major cause of morbidity and mortality in the United States. Studies have shown that excessive T cell activity can mediate pneumonitis in the setting of influenza infection, and data from the 2009 H1N1 pandemic indicate that critical illness and respiratory failure postinfection were associated with greater infiltration of the lungs with CD8+ T cells. T cell Ig and mucin domain 3 (Tim3) is a negative regulator of Th1/Tc1-type immune responses. Activation of Tim3 on effector T cells has been shown to downregulate proliferation, cell-mediated cytotoxicity, and IFN-γ production, as well as induce apoptosis. In this article, we demonstrate that deletion of the terminal cytoplasmic domain of the Tim3 gene potentiates its ability to downregulate Tc1 inflammation, and that this enhanced Tim3 activity is associated with decreased phosphorylation of the TCR–CD3ζ-chain. We then show that mice with this Tim3 mutation infected with influenza are protected from morbidity and mortality without impairment in viral clearance or functional heterotypic immunity. This protection is associated with decreased CD8+ T cell proliferation and decreased production of inflammatory cytokines, including IFN-γ. Furthermore, the Tim3 mutation was protective against mortality in a CD8+ T cell-specific model of pneumonitis. These data suggest that Tim3 could be targeted to prevent immunopathology during influenza infection and demonstrate a potentially novel signaling mechanism used by Tim3 to downregulate the Tc1 response.

Allergic asthma is distinguished by sensitivity of allergen-specific CD4+ T cells and airway structural cells to type 2 inflammation

The development of allergic asthma requires type 2 airway inflammation as well as increased sensitivity of airway epithelial cells and smooth muscle to inflammation. Advanced analysis of asthma Not all individuals who have respiratory reactions to allergens progress to asthma. In this issue, Cho et al. found that although allergic asthmatics and allergic nonasthmatics both experienced inflammation after allergen challenge, asthmatics had more mucin and type 2 cytokines, and allergen-specific T cells sampled from the airspace had increased innate type 2 receptors. Using orientation-resolved optical coherence tomography, described by Adams et al., they demonstrated that allergic asthmatics also had increased airway smooth muscle mass. This technique allows for in vivo imaging of airway smooth muscle structure and function, which could shed light on the pathogenesis of many respiratory diseases. Despite systemic sensitization, not all allergic individuals develop asthma symptoms upon airborne allergen exposure. Determination of the factors that lead to the asthma phenotype in allergic individuals could guide treatment and identify novel therapeutic targets. We used segmental allergen challenge of allergic asthmatics (AA) and allergic nonasthmatic controls (AC) to determine whether there are differences in the airway immune response or airway structural cells that could drive the development of asthma. Both groups developed prominent allergic airway inflammation in response to allergen. However, asthmatic subjects had markedly higher levels of innate type 2 receptors on allergen-specific CD4+ T cells recruited into the airway. There were also increased levels of type 2 cytokines, increased total mucin, and increased mucin MUC5AC in response to allergen in the airways of AA subjects. Furthermore, type 2 cytokine levels correlated with the mucin response in AA but not AC subjects, suggesting differences in the airway epithelial response to inflammation. Finally, AA subjects had increased airway smooth muscle mass at baseline measured in vivo using novel orientation-resolved optical coherence tomography. Our data demonstrate that the development of allergic asthma is dependent on the responsiveness of allergen-specific CD4+ T cells to innate type 2 mediators as well as increased sensitivity of airway epithelial cells and smooth muscle to type 2 inflammation.

CARMA3 Is Critical for the Initiation of Allergic Airway Inflammation

Innate immune responses to allergens by airway epithelial cells (AECs) help initiate and propagate the adaptive immune response associated with allergic airway inflammation in asthma. Activation of the transcription factor NF-κB in AECs by allergens or secondary mediators via G protein–coupled receptors (GPCRs) is an important component of this multifaceted inflammatory cascade. Members of the caspase recruitment domain family of proteins display tissue-specific expression and help mediate NF-κB activity in response to numerous stimuli. We have previously shown that caspase recruitment domain–containing membrane-associated guanylate kinase protein (CARMA)3 is specifically expressed in AECs and mediates NF-κB activation in these cells in response to stimulation with the GPCR agonist lysophosphatidic acid. In this study, we demonstrate that reduced levels of CARMA3 in normal human bronchial epithelial cells decreases the production of proasthmatic mediators in response to a panel of asthma-relevant GPCR ligands such as lysophosphatidic acid, adenosine triphosphate, and allergens that activate GPCRs such as Alternaria alternata and house dust mite. We then show that genetically modified mice with CARMA3-deficient AECs have reduced airway eosinophilia and proinflammatory cytokine production in a murine model of allergic airway inflammation. Additionally, we demonstrate that these mice have impaired dendritic cell maturation in the lung and that dendritic cells from mice with CARMA3-deficient AECs have impaired Ag processing. In conclusion, we show that AEC CARMA3 helps mediate allergic airway inflammation, and that CARMA3 is a critical signaling molecule bridging the innate and adaptive immune responses in the lung.

CARMA3 Mediates Allergic Lung Inflammation in Response to Alternaria alternata

&NA; The airway epithelial cell (AEC) response to allergens helps initiate and propagate allergic inflammation in asthma. CARMA3 is a scaffold protein that mediates G protein‐coupled receptor‐induced NF‐&kgr;B activation in airway epithelium. In this study, we demonstrate that mice with CARMA3‐deficient AECs have reduced airway inflammation, as well as reduced type 2 cytokine levels in response to Alternaria alternata. These mice also have reduced production of IL‐33 and IL‐25, and reduced numbers of innate lymphoid cells in the lung. We also show that CARMA3‐deficient human AECs have decreased production of proasthmatic mediators in response to A. alternata. Finally, we show that CARMA3 interacts with inositol 1,4,5‐trisphosphate receptors in AECs, and that inhibition of CARMA3 signaling reduces A. alternata‐induced intracellular calcium release. In conclusion, we show that CARMA3 signaling in AECs helps mediate A. alternata‐induced allergic airway inflammation, and that CARMA3 is an important signaling molecule for type 2 immune responses in the lung.