Lionel Loubaki

Crosstalk between T cells and bronchial fibroblasts obtained from asthmatic subjects involves CD40L/α5β1 interaction

BACKGROUND Allergic asthma is characterized by infiltration of inflammatory cells into the airways. T cell-derived cytokines regulate both airway inflammation and remodelling. In the human airways, T cell-fibroblast interactions may have a role in regulating inflammation and remodelling. OBJECTIVES To evaluate the effect of bronchial fibroblast-T cell interaction on profibrogenic cytokine release and determine the nature of the molecules involved in this interaction. METHODS Human bronchial fibroblasts obtained from healthy and asthmatic donors were co-cultured with purified T cells derived from peripheral blood of the same subjects. IL-6 mRNA and protein levels were measured by real time PCR and ELISA. CD40, CD40L and alpha 5 beta 1 were evaluated by flow cytometry. Bronchial fibroblasts were stimulated with rsCD40L. Neutralisation was performed using neutralizing antibodies anti-CD40L and anti-alpha 5. RESULTS Contact of T cells with bronchial fibroblasts up-regulated IL-6 at both gene and protein levels. This effect was significantly higher in fibroblasts from asthmatics than those from controls. Blocking CD40L and alpha 5 beta 1 integrin showed a significant inhibition of IL-6 expression in asthmatics but not in healthy controls. Stimulation of fibroblasts with recombinant soluble CD40L up-regulated IL-6 production in asthmatics but not in controls. Adhesion to fibronectin, a alpha 5 beta 1 integrin ligand, is increased in fibroblasts from asthmatics compared to fibroblasts from controls. CONCLUSION These results showed that interaction of bronchial fibroblasts with T cells increases the production of profibrogenic cytokine IL-6. In asthmatic condition this interaction involves CD40L/alpha 5 beta 1. These results suggest that T cells and structural cells crosstalk in asthma may maintain local mucosal inflammation.

Tumor Necrosis Factor-α Expression in Uvular Tissues Differs Between Snorers and Apneic Patients

BACKGROUND Inflammatory changes such as subepithelial edema and excessive inflammatory cell infiltration have been observed in uvular tissues of obstructive sleep apnea (OSA) subjects. The levels of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin-6 are elevated in the serum of apneic patients and have been proposed as mediators of muscle weakness. TNF-alpha has been shown to affect diaphragm contractility in mice and rabbit in vivo. OBJECTIVES To assess total and compartmental TNF-alpha expression in uvular tissues of apneic and nonapneic patients. METHODS Uvular tissues were collected from 14 snorers without sleep disorders breathing, 14 subjects with OSA (OSA 1 group) whose body mass index (BMI) was similar to that of snorers, and 12 additional obese OSA subjects (OSA 2 group) who underwent an uvulopalatopharyngoplasty. Sections were examined using immunohistochemistry and Western blot analysis. TNF-alpha expression was evaluated in the musculus uvulae (MU), epithelial layer, and perimuscular tissues from proximal uvular sections. RESULTS TNF-alpha was more highly expressed in whole uvular protein extracts of apneic groups than in snorers ([mean +/- SEM] snorers, 100.5 +/- 3.0%; OSA 1 group, 127.1 +/- 6.9%; OSA 2 group, 140.7 +/- 11.0%; p = 0.01). In the muscular area, TNF-alpha levels were higher in the more obese OSA subjects than in the other two groups (snorers, 100.3 +/- 3%; OSA 1 group, 107.4 +/- 0.7%; OSA 2 group, 124.1 +/- 4.2%; p = 0.007). In the muscular area, TNF-alpha was correlated with BMI, but no relationship was found with the apnea-hypopnea index. CONCLUSIONS We conclude that MU is the major TNF-alpha source in uvular tissue and that TNF-alpha is more highly expressed in the heaviest OSA patients compared to less obese OSA patients and nonapneic snorers.

α2β1 Integrin Regulates Th17 Cell Activity and Its Neutralization Decreases the Severity of Collagen-Induced Arthritis

Th17 cells play a critical role in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms by which these cells regulate the development of RA are not fully understood. We have recently shown that α2β1 integrin, the receptor of type I collagen, is the major collagen-binding integrin expressed by human Th17 cells. In this study, we examined the role of α2β1 integrin in Th17-mediated destructive arthritis in the murine model of collagen-induced arthritis (CIA). We found that α2β1 integrin is expressed on synovial Th17 cells from CIA mice and its neutralization with a specific mAb significantly reduced inflammation and cartilage degradation, and protected the mice from bone erosion. Blockade of α2β1 integrin led to a decrease in the number of Th17 cells in the joints and to a reduction of IL-17 levels in CIA mice. This was associated with an inhibition of receptor activator of NF-κB ligand levels and osteoclast numbers, and reduction of bone loss. We further show that α2β1 integrin is expressed on synovial Th17 cells from RA patients, and that its ligation with collagen costimulated the production of IL-17 by polarized human Th17 cells by enhancing the expression of retinoic acid receptor–related orphan receptor C through ERK and PI3K/AKT. Our findings provide the first evidence, to our knowledge, that α2β1 integrin is an important pathway in Th17 cell activation in the pathogenesis of CIA, suggesting that its blockade can be beneficial for the treatment of RA and other Th17-associated autoimmune diseases.

Co-culture of human bronchial fibroblasts and CD4+ T cells increases Th17 cytokine signature.

Background Airway inflammation is an important characteristic of asthma and has been associated with airway remodelling and bronchial hyperreactivity. The mucosal microenvironment composed of structural cells and highly specialised extracellular matrix is able to amplify and promote inflammation. This microenvironment leads to the development and maintenance of a specific adaptive response characterized by Th2 and Th17. Bronchial fibroblasts produce multiple mediators that may play a role in maintaining and amplifying this response in asthma. Objective To investigate the role of bronchial fibroblasts obtained from asthmatic subjects and healthy controls in regulating Th17 response by creating a local micro-environment that promotes this response in the airways. Methods Human bronchial fibroblasts and CD4+T cells were isolated from atopic asthmatics and non-atopic healthy controls. CD4+T were co-cultured with bronchial fibroblasts of asthmatic subjects and healthy controls. RORc gene expression was detected by qPCR. Phosphorylated STAT-3 and RORγt were evaluated by western blots. Th17 phenotype was measured by flow cytometry. IL-22, IL17, IL-6 TGF-β and IL1-β were assessed by qPCR and ELISA. Results Co-culture of CD4+T cells with bronchial fibroblasts significantly stimulated RORc expression and induced a significant increase in Th17 cells as characterized by the percentage of IL-17+/CCR6+ staining in asthmatic conditions. IL-17 and IL-22 were increased in both normal and asthmatic conditions with a significantly higher amount in asthmatics compared to controls. IL-6, IL-1β, TGF-β and IL-23 were significantly elevated in fibroblasts from asthmatic subjects upon co-culture with CD4+T cells. IL-23 stimulates IL-6 and IL-1β expression by bronchial fibroblasts. Conclusion Interaction between bronchial fibroblasts and T cells seems to promote specifically Th17 cells profile in asthma. These results suggest that cellular interaction particularly between T cells and fibroblasts may play a pivotal role in the regulation of the inflammatory response in asthma.

MiR-146a potentially promotes IVIg-mediated inhibition of TLR4 signaling in LPS-activated human monocytes

IVIg is used as an immunomodulatory agent in inflammatory disorders such as sepsis. IVIg also affects monocyte differentiation and functions, two processes in which microRNAs play a crucial role. Monocytes detect microorganisms through pathogen recognition receptors (PRRs) such as TLR4. MiR-146a has been shown to supress NF-κB and IRF3 activity, two key components of TLR4 signaling. To evaluate whether miR-146a is involved in the anti-inflammatory effects of IVIg, monocytes were treated with LPS or IVIg alone or, alternately, first activated with LPS followed by washing and addition of IVIg. MiR-146a, IRF3, TNF-α, IL-1β, IL-6, IL-10, IFN-β, TGF-β1 and IL-1Ra expression was analyzed by qPCR, while IRAK1, TRAF6 and IκBα expression was measured by Western blotting. We found that addition of IVIg to LPS-activated monocytes significantly upregulated the expression of miR-146a, which was associated with a significant reduction in the expression of its targets IRF3 and its regulated gene IFN-β. Furthermore, expression of IRAK1, TRAF6, and consequently NF-κB activation, was also reduced in LPS-activated monocytes following addition of IVIg, whereas TGF-β1, IL-10 and IL-1Ra were increased. Our results thus suggest that miR-146a is a mediator of IVIg effects in inflammatory disorders, point to an important role for miR-146a in the control of inflammation during sepsis and highlight a new mechanism by which IVIg exerts its anti-inflammatory effects in sepsis.

Transient warming events occurring after freezing impairs umbilical cord–derived mesenchymal stromal cells functionality

BACKGROUND Mesenchymal stromal cells (MSCs) have shown promising results for the treatment of refractory acute graft-versus-host disease. While safety of MSC infusion has been demonstrated, the use of cryopreserved MSCs in clinical trials has raised concerns regarding the retention of their functional activity. This has led to the recommendation by experts in the field to use freshly harvested MSCs, even though this approach is much less practical from a logistic point of view. In the present study, we revisited the impact of cryopreservation on MSC functionality and addressed the possibility that warming events on frozen cells rather than cryopreservation per se could impact MSC functionality. METHODS Following controlled-rate freezing to -130°C, umbilical cord-derived MSCs were left at room temperature (RT) for 2-10 min or on dry ice for 10 min, before being transferred into liquid nitrogen (LqN2). MSCs of each group were subsequently tested (viability, functionality and cellular damage) and compared with their freshly harvested counterparts. RESULTS We demonstrated that freshly harvested MSCs as well as cryopreserved MSCs that were left on dry ice following step-down freezing have comparable viability, functionality and integrity. In contrast, cryopreserved MSCs that were left at RT before being transferred into LqN2 were functionally impaired and showed cellular damage upon thawing even though they exhibited high viability. DISCUSSION Warming events after freezing and not cryopreservation per se significantly impair MSC functionality, indicating that cryopreserved MSCs can be an advantageous alternative to freshly harvested cells for therapeutic purposes.

Functional impairment of MSC induced by transient warming events: Correlation with loss of adhesion and altered cell size

BACKGROUND We recently showed that transient warming effects decreased the functional and adhesion properties of mesenchymal stromal cells (MSC) while post-thaw viability remained high. In an attempt to better predict functional impairment of cryopreserved MSC, we further analysed the correlation between viability, immunosuppressive activity and adhesion of cells exposed or not to warming events. METHODS MSC prepared from six umbilical cords were frozen to -130°C and immediately transferred in a dry ice container or exposed to room temperature for 2 to 10 min (warming events) prior to storage in liquid nitrogen. Viability, functionality (inhibition of T-cell proliferation), adhesion and expression of various integrins were evaluated. RESULTS The monotonic loss of functional activity with time was proportional to the length of warming events to which MSC were subjected and correlated with the monotonic loss of adhesion capacity. In contrast, post-thaw viability assessment did not predict functional impairment. Interestingly, flow cytometry analyses revealed the emergence of a FSClow population present in the viable cell fraction of freshly thawed MSC, which displayed poor adhesion capacity and expressed low levels of integrin β5. The prevalence of this FSClow population increased with the length of warming events and correlated with impaired functional and adhesion properties. DISCUSSION Our results reveal that loss of functional activity (4-day test) induced by transient warming events could be predicted by evaluating adhesion (2-hr test) or FSC profile (10-min test) of MSC immediately post-thaw. These observations could lead to the development of surrogate tests for rapidly assessing the functional quality of cryopreserved MSC.

NLRP10 A Potential Contributor to the Anti-Inflammatory Effects of IVIg in Sepsis

Intravenous immunoglobulin (IVIg) is used to treat a number of inflammatory diseases including sepsis. The objective of this study was to evaluate whether NLRP10, postulated as a negative regulator of inflammasome activation, can play a role in the anti-inflammatory effects observed in the treatment of severe sepsis with IVIg. Human peripheral blood monocytes were isolated and treated either with LPS or IVIg, or alternatively treated first with LPS followed by addition of IVIg. The induction of IL-1β and IL-18 was used as an index of inflammasome activation. Treatment of resting monocytes with IVIg resulted in a significant increase in the expression of NLRP10. LPS but not IVIg resulted in a significant increase in both IL-1β and IL-18 protein release, consistent with inflammasome activation. Furthermore, addition of IVIg to LPS-treated monocytes resulted in a significant increase in the expression of NLRP10 which was associated with a complete inhibition of both IL-1β and IL-18 increased expression. These data suggest that the IVIg-mediated induction of NLRP10 expression in monocytes contributes to the control of inflammation during sepsis and thus highlight a complementary mechanism by which IVIg exerts their anti-inflammatory effects in sepsis.

Role of the CD62L Expression Pathway in the IVIg Inhibition of Cytotoxic T Cell Responses

Intravenous immunoglobulin (IVIg) is successfully used in the treatment of a number of immune-mediated disorders, including diseases in which self-reactive cytotoxic CD8+ T lymphocytes (CTLs) play an important pathological role. However, the exact molecular and cellular mechanisms underlying its effects on the cytotoxic response remain undefined. Using a mouse model of ovalbumin (OVA) immunization, we recently showed that IVIg treatment decreases the in vivo generation of OVA-specific CD8+ T cells, as well as the proportion of CTLs expressing the extracellular cytotoxic marker CD107a. In the present work, we demonstrate that during the course of an active immune response in mice, IVIg treatment is associated with the presence of splenic CD8+ T cells expressing high levels of CD62L and by an increased plasma concentration of soluble CD62L. Because cell surface expression of CD62L negatively correlates with the cytotoxic activity of CD8+ T cells and that soluble CD62L exhibits anti-inflammatory effects, we herein propose that the CD62L expression pathway plays a key role in the therapeutic effects of IVIg in CD8-mediated autoimmune and inflammatory disorders.

Bronchial fibroblasts modulate CD4+Tcells phenotype towards Th17 in asthma

Objective/purpose In asthma, CD4+T cells are selectively recruited into the bronchial mucosa. CD4+ T cells consist of different subsets that express lineage specific transcription factors and play different roles either in initiating and supporting the development of immune response, but also in orchestrating and regulating them. The aim of our study was to evaluate the effect of T cells-bronchial fibroblasts interaction on CD4+T cell phenotype.