CCL18 as an indicator of pulmonary fibrotic activity in idiopathic interstitial pneumonias and systemic sclerosis

Objective In diffuse parenchymal lung diseases, the evolution of pulmonary fibrosis is often devastating and may result in death. In this study the role of CCL18 as a biomarker of disease activity in idiopathic interstitial pneumonias (IIPs) and systemic sclerosis (SSc) with lung involvement was evaluated. Methods CCL18 was assessed in supernatants of cultured bronchoalveolar lavage (BAL) cells as well as BAL fluid and serum samples from 43 patients with IIPs, 12 patients with SSc, and 23 healthy control subjects. Concentrations of CCL18 were measured by enzyme-linked immunosorbent assay, and expression of CCL18 was assessed by flow cytometry. Results CCL18 concentrations were statistically significantly increased in all patients with fibrotic lung diseases. Spontaneous CCL18 production by BAL cells was negatively correlated with total lung capacity and the diffusion capacity for carbon monoxide, whereas there was a positive correlation of CCL18 concentrations with BAL neutrophil and eosinophil cell counts. Flow cytometry revealed an increase in the percentage of CCL18-positive alveolar macrophages and an increase in the CCL18 fluorescence intensity per cell in patients with fibrotic lung diseases. In a cohort of patients who were followed up for at least 6 months (n = 40), a close negative correlation was observed between changes in the predicted total lung capacity and changes in CCL18 serum concentrations. Conclusion These findings suggest that CCL18 production by BAL cells and serum CCL18 concentrations reflect pulmonary fibrotic activity in patients with IIPs and those with SSc. Monitoring changes in CCL18 production might be an extraordinarily useful tool in clinical practice and in studies aimed at evaluating new approaches for treatment of fibrotic lung diseases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56037/1/22559_ftp.pd

Decreased Frequencies of Circulating CD4+ T Follicular Helper Cells Associated with Diminished Plasma IL-21 in Active Pulmonary Tuberculosis

Circulating T follicular helper (Tfh) cells represent a distinct subset of CD4+ T cells and are important in immunity to infections. Although they have been shown to play a role in experimental models of tuberculosis infection, their role in human tuberculosis remains unexplored.To determine the distribution of circulating Tfh cells in human TB, we measured the frequencies of Tfh cells ex vivo and following TB - antigen or polyclonal stimulation in pulmonary TB (PTB; n = 30) and latent TB (LTB; n = 20) individuals, using the markers CXCR5, PD-1 and ICOS.We found that both ex vivo and TB - antigen induced frequencies of Tfh cell subsets was significantly lower in PTB compared to LTB individuals. Similarly, antigen induced frequencies of Tfh cells expressing IL-21 was also significantly lower in PTB individuals and this was reflected in diminished circulating levels of IL-21 and IFNγ. This was not accompanied by diminished frequencies of activated or memory B cell subsets. Finally, the diminution in frequency of Tfh cells in PTB individuals was dependent on IL-10, CTLA-4 and PD-L1 in vitro.Thus, PTB is characterized by adiminution in the frequency of Tfh cell subsets

P2Y6 Receptor Activation Promotes Inflammation and Tissue Remodeling in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and very few available treatment options. The involvement of the purinergic receptor subtypes P2Y2 and P2X7 in fibrotic lung disease has been demonstrated recently. In this study, we investigated the role of P2Y6 receptors in the pathogenesis of IPF in humans and in the animal model of bleomycin-induced lung injury. P2Y6R expression was upregulated in lung structural cells but not in bronchoalveolar lavage (BAL) cells derived from IPF patients as well as in animals following bleomycin administration. Furthermore, BAL fluid levels of the P2Y6R agonist uridine-5′-diphosphate were elevated in animals with bleomycin-induced pulmonary fibrosis. Inflammation and fibrosis following bleomycin administration were reduced in P2Y6R-deficient compared to wild-type animals confirming the pathophysiological relevance of P2Y6R subtypes for fibrotic lung diseases. Experiments with bone marrow chimeras revealed the importance of P2Y6R expression on lung structural cells for pulmonary inflammation and fibrosis. Similar effects were obtained when animals were treated with the P2Y6R antagonist MRS2578. In vitro studies demonstrated that proliferation and secretion of the pro-inflammatory/pro-fibrotic cytokine IL-6 by lung fibroblasts are P2Y6R-mediated processes. In summary, our results clearly demonstrate the involvement of P2Y6R subtypes in the pathogenesis of pulmonary fibrosis. Thus, blocking pulmonary P2Y6 receptors might be a new target for the treatment of IPF

The purinergic receptor subtype P2Y2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with few available treatment options. Recently, the involvement of purinergic receptor subtypes in the pathogenesis of different lung diseases has been demonstrated. Here we investigated the role of the purinergic receptor subtype P2Y2 in the context of fibrotic lung diseases.The concentration of different nucleotides was measured in the broncho-alveolar lavage (BAL) fluid derived from IPF patients and animals with bleomycin-induced pulmonary fibrosis. In addition expression of P2Y2 receptors by different cell types was determined. To investigate the functional relevance of P2Y2 receptors for the pathogenesis of the disease the bleomycin model of pulmonary fibrosis was used. Finally, experiments were performed in pursuit of the involved mechanisms.Compared to healthy individuals or vehicle treated animals, extracellular nucleotide levels in the BAL fluid were increased in patients with IPF and in mice after bleomycin administration, paralleled by a functional up-regulation of P2Y2R expression. Both bleomycin-induced inflammation and fibrosis were reduced in P2Y2R-deficient compared to wild type animals. Mechanistic studies demonstrated that recruitment of neutrophils into the lungs, proliferation and migration of lung fibroblasts as well as IL6 production are key P2Y2R mediated processes.Our results clearly demonstrate the involvement of P2Y2R subtypes in the pathogenesis of fibrotic lung diseases in humans and mice and hence support the development of selective P2Y2R antagonists for the treatment of IPF

Mycoplasma hyorhinis-contaminated cell lines activate primary innate immune cells via a protease-sensitive factor

Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments

Association between Variations in Cell Cycle Genes and Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating and progressive lung disease. Its aetiology is thought to involve damage to the epithelium and abnormal repair. Alveolar epithelial cells near areas of remodelling show an increased expression of proapoptotic molecules. Therefore, we investigated the role of genes involved in cell cycle control in IPF. Genotypes for five single nucleotide polymorphisms (SNPs) in the tumour protein 53 (TP53) gene and four SNPs in cyclin-dependent kinase inhibitor 1A (CDKN1A), the gene encoding p21, were determined in 77 IPF patients and 353 controls. In peripheral blood mononuclear cells (PBMC) from 16 healthy controls mRNA expression of TP53 and CDKN1A was determined

Bayesian Approach to Model CD137 Signaling in Human M.tuberculosis in vitro Responses

Abstract Immune responses are qualitatively and quantitatively influenced by a complex network of receptor-ligand interactions. Among them, the CD137:CD137L pathway is known to modulate innate and adaptive human responses against Mycobacterium tuberculosis. However, the underlying mechanisms of this regulation remain unclear. In this work, we developed a Bayesian Computational Model (BCM) of in vitro CD137 signaling, devised to fit previously gathered experimental data. The BCM is fed with the data and the prior distribution of the model parameters and it returns theirposterior distribution and the model evidence, which allows comparing alternative signaling mechanisms. The BCM uses a coupled system of non-linear differential equations to describe the dynamics of Antigen Presenting Cells, Natural Killer and T Cells together with the interpheron (IFN)-c and tumor necrosis factor (TNF)-a levels in the media culture. Fast and complete mixing of the media is assumed. The prior distribution of the parameters that describe the dynamics of the immunological response was obtained from the literature and theoretical considerations Our BCM applies successively the Levenberg-Marquardt algorithm to find the maximum a posteriori likelihood (MAP); the Metropolis Markov Chain Monte Carlo method to approximate the posterior distribution of the parameters and Thermodynamic Integration to calculate the evidence of alternative hypothesis. Bayes factors provided decisive evidence favoring direct CD137 signaling on T cells. Moreover, the posterior distribution of the parameters that describe the CD137 signaling showed that the regulation of IFNc levels is based more on T cells survival than on direct induction. Furthermore, the mechanisms that account for the effect of CD137 signaling on TNF-a production were based on a decrease of TNF-a production by APC and, perhaps, on the increase in APC apoptosis. BCM proved to be a useful tool to gain insight on the mechanisms of CD137 signaling during human response against Mycobacterium tuberculosis.Fil: Darío A Fernández Do Porto. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG.Fil: Jerónimo Auzmendi. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG.Fil: Delfina Peña. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. CONSEJO NAC.DE INVEST.CIENTIF.Y TECNICAS. OFICINA DE COORDINACION ADMINISTRATIVA CIUDAD UNIVERSITARIA. INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CS. EXACTAS Y NATURALES. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. DTO.DE QUIMICA BIOLOGICA.Fil: Veronica E Garcia. CONSEJO NAC.DE INVEST.CIENTIF.Y TECNICAS. OFICINA DE COORDINACION ADMINISTRATIVA CIUDAD UNIVERSITARIA. INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CS. EXACTAS Y NATURALES.Fil: Luciano Moffatt. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG

Common Gamma Chain Cytokines Promote Rapid In Vitro Expansion of Allo-Specific Human CD8+ Suppressor T Cells

Human CD8+ regulatory T cells, particularly the CD8+CD28− T suppressor cells, have emerged as an important modulator of alloimmunity. Understanding the conditions under which these cells are induced and/or expanded would greatly facilitate their application in future clinical trials. In the current study, we develop a novel strategy that combines common gamma chain (γc) cytokines IL-2, IL-7 and IL-15 and donor antigen presenting cells (APCs) to stimulate full HLA-mismatched allogeneic human CD8+ T cells which results in significant expansions of donor-specific CD8+CD28− T suppressor cells in vitro. The expanded CD8+CD28− T cells exhibit increased expressions of CTLA-4, FoxP3, and CD25, while down-regulate expressions of CD56, CD57, CD127, and perforin. Furthermore, these cells suppress proliferation of CD4+ T cells in a contact-dependent and cytokine-independent manner. Interestingly, the specificity of suppression is restricted by the donor HLA class I antigens but promiscuous to HLA class II antigens, providing a potential mechanism for linked suppression. Taken together, our results demonstrate a novel role for common γc cytokines in combination with donor APCs in the expansion of donor-specific CD8+CD28− T suppressor cells, and represent a robust strategy for in vitro generation of such cells for adoptive cellular immunotherapy in transplantation