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Cystic fibrosis (CF) is characterized by an excessive neutrophilic inflammatory response within the airway as a result of defective cystic fibrosis transmembrane receptor (CFTR) expression and function. Interleukin-17A induces airway neutrophilia and mucin production associated with Pseudomonas aeruginosa colonization, which is associated with the pathophysiology of cystic fibrosis. The objectives of this study were to use the preclinical murine model of cystic fibrosis lung infection and inflammation to investigate the role of IL-17 in CF lung pathophysiology and explore therapeutic intervention with a focus on IL-17. Cftr-deficient mice (CF mice) and wild-type mice (WT mice) infected with P. aeruginosa had robust IL-17 production early in the infection associated with a persistent elevated inflammatory response. Intratracheal administration of IL-17 provoked a neutrophilic response in the airways of WT and CF animals which was similar to that observed with P. aeruginosa infection. The neutralization of IL-17 prior to infection significantly improved the outcomes in the CF mice, suggesting that IL-17 may be a therapeutic target. We demonstrate in this report that the pathophysiological contribution of IL-17 may be due to the induction of chemokines from the epithelium which is augmented by a deficiency of Cftr and ongoing inflammation. These studies demonstrate the in vivo contribution of IL-17 in cystic fibrosis lung disease and the therapeutic validity of attenuating IL-17 activity in cystic fibrosis.

• Human mesenchymal stem cells have anti-inflammatory and immunomodulatory properties. • Preclinical models of CF show mesenchymal stem cells attenuate pulmonary inflammation. • Mesenchymal stem cells were safe and well-tolerated in this phase 1 clinical trial. Mesenchymal stem cells are of particular interest in cystic fibrosis (CF) as a potential therapeutic. Data from pre-clinical studies suggest that allogeneic bone marrow-derived human mesenchymal stem cells (hMSCs) may provide a new therapeutic treatment for CF lung disease by attenuating pulmonary inflammation while decreasing bacterial growth and enhancing antibiotic efficacy. Fifteen adults with CF were enrolled in a phase 1 dose-escalation trial of a single intravenous infusion of hMSCs derived from bone marrow aspirates obtained from a single pre-clinically validated healthy volunteer donor. The study employed a 3+3 dose escalation design with subjects receiving a single, intravenous dose of either 1×106, 3×106, or 5×106 hMSCs/kg. Subjects were monitored inpatient for 24 hours and by outpatient visits and telephone calls for 12 months after the infusion. Safety and tolerability were evaluated by monitoring symptoms, patient reported outcome questionnaires, adverse events (AEs), physical exam findings, spirometry, and analyses of safety laboratories. Preliminary evidence for potential efficacy using inflammatory markers in the blood and sputum were also evaluated. No dose-limiting toxicities, deaths or life-threatening adverse events were observed. Most AEs and serious adverse events (SAEs) were consistent with underlying CF. Vital signs, physical exam findings, spirometry and safety laboratory results showed no significant change from baseline. No trends over time were seen in serum or sputum inflammatory markers nor with clinical spirometry. Allogeneic hMSC intravenous infusions were safe and well-tolerated in this phase 1 study and warrant additional clinical testing as a potential therapeutic for CF lung disease. [ABSTRACT FROM AUTHOR]

Allogeneic human mesenchymal stem cells (hMSCs) introduced intravenously can have profound anti-inflammatory activity resulting in suppression of graft vs. host disease as well as regenerative events in the case of stroke, infarct, spinal cord injury, meniscus regeneration, tendinitis, acute renal failure, and heart disease in human and animal models of these diseases, hMSCs produce bioactive factors that provide molecular cuing for: 1) immunosuppression of T cells; 2) antiscarring; 3) angiogenesis; 4) antiapoptosis; and 5) regeneration (i.e., mitotic for host-derived progenitor cells). Studies have shown that hMSCs have profound effects on the immune system and are well-tolerated and therapeutically active in immunocompetent rodent models of multiple sclerosis and stroke. Furthermore, intravenous administration of MSCs results in pulmonary localization. Asthma is a major debilitating pulmonary disease that impacts in excess of 150 million people in the world with uncontrolled asthma potentially leading to death. In addition, the socioeconomic impact of asthma-associated illnesses at the pediatric and adult level are in the millions of dollars in healthcare costs and lost days of work. hMSCs may provide a viable multiaction therapeutic for this inflammatory lung disease by secreting bioactive factors or directing cellular activity. Our studies show the effectiveness and specificity of the hMSCs on decreasing chronic airway inflammation associated with the murine ovalbumin model of asthma. In addition, the results from these studies verify the in vivo immunoeffectiveness of hMSCs in rodents and support the potential therapeutic use of hMSCs for the treatment of airway inflammation associated with chronic asthma. models of asthma; stem cell therapy doi: 10.1152/ajplung.00182.2009.

Bonfield, Tracey L., Baisakhi Raychaudhuri, Anagha Malur, Susamma Abraham, Bruce C. Trapnell, Mani S. Kavuru, and Mary Jane Thomassen. PU.1 regulation of human alveolar macrophage differentiation requires granulocyte-macrophage colony-stimulating factor. Am J Physiol Lung Cell Mol Physiol 285: L1132-L1136, 2003. First published August 1, 2003; 10.1152/ajplung.00216.2003.--Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically implicated in lung homeostasis in the GM-CSF knockout mouse model. These animals develop an isolated lung lesion reminiscent of pulmonary alveolar proteinosis (PAP) seen in humans. The development of the adult form of human alveolar proteinosis is not due to the absence of a GM-CSF gene or receptor defect but to the development of an anti-GM-CSF autoimmunity. The role of GM-CSF in the development of PAP is unknown. Studies in the GM-CSF knockout mouse have shown that lack of PU.1 protein expression in alveolar macrophages is correlated with decreased maturation, differentiation, and surfactant catabolism. This study investigates PU. 1 expression in vitro and in vivo in human PAP alveolar macrophages as well as the regulation of PU.1 by GM-CSF. We show for the first time that PU.1 mRNA expression in PAP bronchoalveolar lavage cells is deficient compared with healthy controls. PU.1-dependent terminal differentiation markers CD32 (FC[gamma]II), mannose receptor, and macrophage colony-stimulating factor receptor (M-CSFR) are decreased in PAP alveolar macrophages. In vitro studies demonstrate that exogenous GM-CSF treatment upregulated PU.1 and M-CSFR gene expression in PAP alveolar macrophages. Finally, in vivo studies showed that PAP patients treated with GM-CSF therapy have higher levels of PU.1 and M-CSFR expression in alveolar macrophages compared with healthy control and PAP patients before GM-CSF therapy. These observations suggest that PU.1 is critical in the terminal differentiation of human alveolar macrophages. PU-1; c-fms; alveolar proteinosis

Objective: Seropositive rheumatoid arthritis (RA) is a chronic autoimmune disease that is rarely "cured." Human mesenchymal stem cells (hMSCs) are known to reduce inflammation and restore immune homeostasis. However, methods for predicting therapeutic hMSC potency have not been established. The goal of these studies was to use and refine an ex vivo functional assay that determines potency of hMSCs and can then be validated in clinical trials as a potency measure of hMSCs used therapeutically to treat RA. Methods: Allogeneic hMSCs were cytokine‐stimulated, and a conditioned medium (CM) was harvested. The CM was tested for the potential to attenuate RA CD4+ T cell proliferation using suppression assays. Indoleamine 2, 3‐dioxygenase (IDO) mRNA, and protein were quantified in hMSCs as a measure to compare hMSCs across (prior) studies. Results: To mimic a proinflammatory environment that resembles that in RA, interleukin‐1(IL1β), tumor necrosis factor α (TNFα), and interferon γ (IFNγ) (alone or in combination) were used to precondition hMSCs. Treating hMSCs with a combination of these cytokines generated a CM "secretome" that suppressed T cell proliferation between 70 and 83%. Forty‐eight hours of cytokine preconditioning hMSCs was required to maximize this effect. T cell suppression positively correlated with increases in hMSC cellular IDO mRNA and protein. Conclusion: By standardizing assays to measure hMSC effects, their potency on T cell suppression can be quantified. These studies demonstrate that hMSCs can be compared functionally to identify optimal preparation(s) for therapeutic use in RA and that the potency of hMSC‐dependent T cell suppression may differ between hMSC donors. Clinical studies are warranted to validate the hypothesis that ex vivo potency in suppressing T cells will positively correlate with a reduction in RA disease activity and increase in immunological quiescence. [ABSTRACT FROM AUTHOR]

Abstract Background Excessive secretion of IL-8 characterizes cystic fibrosis (CF). This has been attributed to excessive activation of epithelial cell I-κB Kinase and/or NFκB. Maximum IL-8 production requires 3 cooperative mechanisms: 1) release of the promoter from repression; 2) activation of transcription by NFκB and AP-1; 3) stabilization of mRNA by p38-MAPK. Little is known about regulation of IL-8 by MAPKs or AP-1 in CF. Methods We studied our hypothesis in vitro using 3-cellular models. Two of these models are transformed cell lines with defective versus normal cystic fibrosis transmembrane conductance regulator (CFTR) expression: an antisense/sense transfected cell line and the patient derived IB3-1/S9. In the third series of studies, we studied primary necropsy human tracheal epithelial cells treated with an inhibitor of CFTR function. All cell lines were pretreated with parthenolide and then stimulated with TNFα and/or IL-1β. Results In response to stimulation with TNFα and/or IL-1β, IL-8 production and mRNA expression was greater in CF-type cells than in non-CF controls. This was associated with enhanced phosphorylation of p38, ERK1/2 and JNK and increased activation of AP-1. Since we previously showed that parthenolide inhibits excessive IL-8 production by CF cells, we evaluated its effects on MAPK and AP-1 activation and showed that parthenolide inhibited ERK and AP-1 activation. Using a luciferase promoter assay, our studies showed that parthenolide decreased activation of the IL-8 promoter in CF cells stimulated with TNFα/IL-1β. Conclusions In addition to NFκB MAPKs ERK, JNK and p38 and the transcription factor AP-1 are also dysregulated in CF epithelial cells. Parthenolide inhibited both NFκB and MAPK/AP-1 pathways contributing to the inhibition of IL-8 production.

Abstract Allogeneic human mesenchymal stem cells (hMSCs) can suppress graft versus host disease (GvHD) and have profound anti-inflammatory and regenerative capacity in stroke, infarct, spinal cord injury, meniscus regeneration, tendinitis, acute renal failure, and heart disease in human and animal models of disease. There is significant clinical hMSC variability in efficacy and the ultimate response in vivo. The challenge in hMSC based therapy is defining the efficacy of hMSC in vivo. Models which may provide insight into hMSC bioactivity in vivo would provide a means to distinguish hMSCs for clinical utility. hMSC function has been described as both regenerative and trophic through the production of bioactive factors. The regenerative component involves the multi-potentiality of hMSC progenitor differentiation. The secreted factors generated by the hMSCs are milieu and injury specific providing unique niches for responses in vivo. These bioactive factors are anti-scarring, angiogenic, anti-apoptotic as well as regenerative. Further, from an immunological standpoint, hMSC's can avoid host immune response, providing xenographic applications. To study the in vivo immuno-regulatory effectiveness of hMSCs, we used the ovalbumin challenge model of acute asthma. This is a quick 3 week in vivo pulmonary inflammation model with readily accessible ways of measuring effectiveness of hMSCs. Our data show that there is a direct correlation between the traditional ceramic cube score to hMSCs attenuation of cellular recruitment due to ovalbumin challenge. The results from these studies verify the in vivo immuno-modulator effectiveness of hMSCs and support the potential use of the ovalbumin model as an in vivo model of hMSC potency and efficacy. Our data also support future directions toward exploring hMSCs as an alternative therapeutic for the treatment of airway inflammation associated with asthma.