Elastin-derived peptides favor type 2 innate lymphoid cells in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a condition characterized by chronic airway inflammation and obstruction, primarily caused by tobacco smoking. Although the involvement of immune cells in COPD pathogenesis is well established, the contribution of innate lymphoid cells (ILCs) remains poorly understood. ILCs are a type of innate immune cells that participate in tissue remodeling processes, but their specific role in COPD has not been fully elucidated. During COPD, the breakdown of pulmonary elastin generates elastin peptides that elicit biological activities on immune cells. This study aimed to investigate the presence of ILC in patients with COPD and examine the impact of elastin peptides on their functionality. Our findings revealed an elevated proportion of ILC2 in the peripheral blood of patients with COPD, and a general activation of ILC as indicated by an increase in their cytokine secretion capacity. Notably, our study demonstrated that serum from patients with COPD promotes ILC2 phenotype, likely due to the elevated concentration of IL-5, a cytokine known to favor ILC2 activation. Furthermore, we uncovered that this increase in IL-5 secretion is partially attributed to its secretion by macrophages upon stimulation by elastin peptides, suggesting an indirect role of elastin peptides on ILC in COPD. These findings shed light on the involvement of ILC in COPD and provide insights into the potential interplay between elastin breakdown, immune cells, and disease progression. Further understanding of the mechanisms underlying ILC activation and their interaction with elastin peptides could contribute to the development of novel therapeutic strategies for COPD management.NEW & NOTEWORTHY Elastin-derived peptides, generated following alveolar degradation during emphysema in patients with COPD, are able to influence the response of type 2 innate lymphoid cells. We show that the orientation of innate lymphoid cells in patients with COPD is shifted toward a type 2 profile and that elastin peptides are indirectly participating in that shift through their influence of macrophages, which in turn impact innate lymphoid cells.

Mild dyslipidemia accelerates tumorigenesis through expansion of Ly6Chi monocytes and differentiation to pro-angiogenic myeloid cells.

Cancer and cardiovascular disease (CVD) share common risk factors such as dyslipidemia, obesity and inflammation. However, the role of pro-atherogenic environment and its associated low-grade inflammation in tumor progression remains underexplored. Here we show that feeding C57BL/6J mice with a non-obesogenic high fat high cholesterol diet (HFHCD) for two weeks to induce mild dyslipidemia, increases the pool of circulating Ly6Chi monocytes available for initial melanoma development, in an IL-1ß-dependent manner. Descendants of circulating myeloid cells, which accumulate in the tumor microenvironment of mice under HFHCD, heighten pro-angiogenic and immunosuppressive activities locally. Limiting myeloid cell accumulation or targeting VEGF-A production by myeloid cells decrease HFHCD-induced tumor growth acceleration. Reverting the HFHCD to a chow diet at the time of tumor implantation protects against tumor growth. Together, these data shed light on cross-disease communication between cardiovascular pathologies and cancer.

The 13-Valent Pneumococcal Conjugate Vaccine Elicits Serological Response and Lasting Protection in Selected Patients With Primary Humoral Immunodeficiency.

Background: Patients with primary humoral immunodeficiency are more prone to invasive as well as recurrent pneumococcal infections. Therefore, anti-pneumococcal vaccination including the 13-valent conjugate vaccine is recommended. Nevertheless, to date, no data is available on immunogenicity of this vaccine in this population. Objective: To assess the immunogenicity and the persistence of protection up to one year after a 13-valent pneumococcal conjugate vaccine in patients with primary humoral immunodeficiency. Methods: Twenty-nine patients with common variable immunodeficiency or IgG subclass deficiency were vaccinated. Immune response and immune protection at baseline as well as at one, six and twelve months after vaccination were evaluated by measuring specific IgG serum concentrations (ELISA), and opsonophagocytic activities directed against selected pneumococcal (MOPA). Results: By ELISA, half of the patients had protective IgG concentrations before vaccination, 35.7% showed an immune response one month after vaccination, 71.4%, 66.7% and 56.0% of the patients were protected at one, six and twelve months respectively. Conversely, by MOPA, 3.4% of the patients were protected at baseline, 10.7% showed an immune response and 28.6%, 48.2% and 33.3% were protected at one, six and twelve months respectively. IgG subclass deficiency, Ig replacement therapy and higher IgG2 concentrations at diagnosis were associated with long-term protection. Conclusion: Pneumococcal conjugate vaccine improves immune protection and antibodies' functionality in a subset of patients with primary immunodeficiency. Prime-boost vaccine strategy needs to be better and individually adapted.

The elastin peptide VGVAPG increases CD4+ T-cell IL-4 production in patients with chronic obstructive pulmonary disease.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), lung-infiltrating inflammatory cells secrete proteases and participate in elastin breakdown and genesis of elastin-derived peptides (EP). In the present study, we hypothesized that the pattern of T lymphocytes cytokine expression may be modulated by EP in COPD patients. METHODS: CD4+ and CD8+ T-cells, sorted from peripheral blood mononuclear cells (PBMC) collected from COPD patients (n = 29) and controls (n = 13) were cultured with or without EP. Cytokine expression in T-cell phenotypes was analyzed by multicolor flow cytometry, whereas desmosine concentration, a specific marker of elastin degradation, was measured in sera. RESULTS: Compared with control, the percentage of IL-4 (Th2) producing CD4+ T-cells was decreased in COPD patients (35.3 ± 3.4% and 26.3 ± 2.4%, respectively, p < 0.05), whereas no significant differences were found with IFN-γ (Th1) and IL-17A (Th17). Among COPD patients, two subpopulations were observed based on the percentage of IL-4 (Th2) producing CD4+ T-cells, of which only one expressed high IL-4 levels in association with high levels of desmosine and strong smoking exposure (n = 7). Upon stimulation with VGVAPG, a bioactive EP motif, the percentage of CD4+ T cells expressing IL-4 significantly increased in COPD patients (p < 0.05), but not in controls. The VGVAPG-induced increase in IL-4 was inhibited in the presence of analogous peptide antagonizing VGVAPG/elastin receptor (S-gal) interactions. CONCLUSIONS: The present study demonstrates that the VGVAPG elastin peptide modulates CD4+ T-cells IL-4 production in COPD. Monitoring IL-4 in circulating CD4+ T-cells may help to better characterize COPD phenotypes and could open a new pharmacologic opportunity through CD4+ T-cells stimulation via the VGVAPG/S-gal receptor in order to favor an anti-inflammatory response in those COPD patients.

Neutrophil Extracellular Traps Generation Relates with Early Stage and Vascular Complications in Systemic Sclerosis.

Systemic sclerosis (SSc) is a systemic disease characterized by a great clinical and immunological heterogeneity whose pathophysiology is still being unraveled. Recently, innate immunity has been proposed to participate to the pathogenesis of SSc. In this study, we investigated the release of neutrophil extracellular traps (NETs) according to patient phenotype. Polymorphonuclear neutrophils (PMN) from 34 SSc patients and 26 healthy controls were stimulated by serum from SSc or healthy subject. NETs were visualized using epifluorescence microscope after DNA, myeloperoxidase, and Histone H3 tagging. Area of NETs were quantified using an original macro running in ImageJ® software. PMN from SSc patients were significantly more prone to releasing NETs than control PMN after autologous stimulation. PMN from patients with severe vascular complications (pulmonary arterial hypertension, digital ulcers) produced more NETs than PMN from other SSc patients and their aberrant NET production appeared to be sustained over time. In patients with pulmonary interstitial disease or extensive cutaneous fibrosis, NET production was high at an early stage of the disease before progressively decreasing. Both serum factors and PMN activation status were involved in the enhanced production of NETs in SSc. Consequently, neutrophils and especially NETosis represent new physiopathological and therapeutic fields in SSc.

Characteristic Pattern of IL-17RA, IL-17RB, and IL-17RC in Monocytes/Macrophages and Mast Cells From Patients With Bullous Pemphigoid.

Inflammation is largely implicated in bullous pemphigoid (BP), the most frequent skin auto-immune blistering disease. IL-17, essentially IL-17A/F, has been involved in blister formation through regulation of protease production, and its specific serum profile within BP was related to disease outcome. However, relationships between IL-17 family ligands and receptors are quite complex with six different IL-17 isoforms, and five different receptors. We here aimed at clarifying the contribution of the IL-17 axis in BP by characterizing not only the expression of IL-17 receptor (IL-17R) members within immune cells isolated from BP patients (PMNs, n = 9; T-lymphocytes, n = 10; and monocytes, n = 10) but also the expression of IL-17 isoforms in sera (n = 83), and blister fluid (n = 31) of BP patients. We showed that at diagnosis, IL-17RA and IL-17RC expression were significantly increased in monocytes isolated from BP patients as compared to those from control subjects (p = 0.006 and p = 0.016, respectively). Notably, both IL-17RA and IL-17RC mRNA expression remained elevated in BP monocytes at time of relapse. We further demonstrated a significant increase of all IL-17 isoforms tested in BP blister fluid compared with BP serum (IL-17A, p < 0.0001; IL-17A/F, p < 0.0001; IL-17B, p = 0.0023; IL-17C, p = 0.0022; IL-17E, p < 0.0001). Among all, IL-17B was the only cytokine for which a significant decreased concentration within blister fluid was observed in BP patients with severe disease compared to patients with moderate disease (p = 0.012). We further evidenced a significant negative correlation between IL-17B levels and blister/erosion BPDAI subscore (r = -0.52, p = 0.003). We finally identified mast cells as a potential target of IL-17B in lesional skin of BP patients. In conclusion, we showed here that IL-17RA and IL-17RC expression in monocyte was associated with disease activity and evidenced in situ a negative correlation between BP disease activity and IL-17B, whose effects could be mediated by IL-17RB expressed by mast cell in BP lesional skin.

Impact of aging on inflammatory and immune responses during elastin peptide-induced murine emphysema.

Deterioration of lung functions and degradation of elastin fibers with age are accelerated during chronic obstructive pulmonary disease (COPD). Excessive genesis of soluble elastin peptides (EP) is a key factor in the pathophysiology of COPD. We have previously demonstrated that 6-wk-old mice exhibited emphysematous structural changes associated with proinflammatory immune response after EP instillation. In this study, we investigated the consequences of aging on inflammatory, immune, and histological criteria associated with murine emphysema progression after EP exposure. Young (6 wk old) and elderly (15 mo old) C57BL/6J mice were endotracheally instilled with EP, and, at various time points after treatment, the inflammatory cell profiles from bronchoalveolar lavage fluids (BALF) and the T-lymphocyte phenotypes, at local and systemic levels, were analyzed by flow cytometry. Lungs were also prepared to allow morphological and histological analysis by confocal microscopy. Elderly mice exhibited an earlier development of pulmonary emphysema, characterized by an increase of the inflammatory and lymphocytic infiltrates, extracellular matrix breakdown, and airspace enlargement compared with young mice. This age-dependent parenchymal tissue remodeling was associated with an increase of the matrix metalloproteinase expressions and desmosine levels in BALF and/or sera of EP-treated mice. In addition, both the proportion of CD4+CD28- and CD8+CD28- T cells in the tissues of EP-treated mice and the interferon-γ levels in the EP-specific memory T-cell clones were significantly higher in elderly versus younger mice. This study demonstrates that aging accelerates emphysema development and that this effect is linked to increased EP production and their effects on inflammatory and immune response.

Elastin receptor (S-gal) occupancy by elastin peptides modulates T-cell response during murine emphysema.

Chronic obstructive pulmonary disease and emphysema are associated with increased elastin peptides (EP) production because of excessive breakdown of lung connective tissue. We recently reported that exposure of mice to EP elicited hallmark features of emphysema. EP effects are largely mediated through a receptor complex that includes the elastin-binding protein spliced-galactosidase (S-gal). In previous studies, we established a correlation between cytokine production and S-gal protein expression in EP-treated immune cells. In this study, we investigated the S-gal-dependent EP effects on T-helper (Th) and T-cytotoxic (Tc) responses during murine EP-triggered pulmonary inflammation. C57BL/6J mice were endotracheally instilled with the valine-glycine-valine-alanine-proline-glycine (VGVAPG) elastin peptide, and, 21 days after treatment, local and systemic T-lymphocyte phenotypes were analyzed at cytokine and transcription factor expression levels by multicolor flow cytometry. Exposure of mice to the VGVAPG peptide resulted in a significant increase in the proportion of the CD4+ and CD8+ T cells expressing the cytokines IFN-γ or IL-17a and the transcription factors T-box expressed in T cells or retinoic acid-related orphan receptor-γt (RORγt) without effects on IL-4 and Gata-binding protein 3 to DNA sequence [A/T]GATA[A/G] expression. These effects were maximized when each T-cell subpopulation was challenged ex vivo with EP, and they were inhibited in vivo when an analogous peptide antagonizing the EP/S-gal interactions was instilled together with the VGVAPG peptide. This study demonstrates that, during murine emphysema, EP-S-gal interactions contribute to a Th-1 and Th-17 proinflammatory T-cell response combined with a Tc-1 response. Our study also highlights the S-gal receptor as a putative pharmacological target to modulate such an immune response.

Bullous pemphigoid outcome is associated with CXCL10-induced matrix metalloproteinase 9 secretion from monocytes and neutrophils but not lymphocytes.

BACKGROUND: The outcome of bullous pemphigoid (BP), the most frequent autoimmune skin-blistering disease, involves matrix metalloproteinase 9 (MMP-9), IL-17, and IL-23 release from infiltrated inflammatory cells. The chemokine CXCL10 has been associated with several autoimmune diseases, but its participation in BP pathophysiology still needs to be clarified. OBJECTIVE: We sought to assess whether BP outcome was associated with different CXCL10 levels and to evaluate the contribution of CXCL10 to the described cytokine/protease inflammatory loop associated with disease outcome. METHODS: Skin biopsy specimens (n = 16), serum (n = 114), blister fluid (n = 23), and primary inflammatory cells from patients with BP were used to investigate CXCL10 expression and function. RESULTS: At baseline, both resident cells, such as keratinocytes and fibroblasts, and infiltrating immune cells expressed CXCL10 at lesional sites in skin of patients with BP. CXCL10 levels were higher in blister fluid (P < .0001) and serum (P < .005) from patients with BP than in serum from age- and sex-matched control subjects (n = 34). Furthermore, CXCL10 serum levels increased at day 60 only in patients who relapsed within the first year of treatment (n = 33, P < .005). Interestingly, CXCL10 expression could be upregulated by itself and IL-17 in inflammatory cells. Notably, neutrophils and monocytes from patients with BP, but not lymphocytes, responded to CXCL10 by increasing MMP-9 secretion through the activation of extracellular signal-regulated kinase 1/2, p38, phosphoinositide-3 kinase signaling pathways. Finally, CXCL10-increased MMP-9 secretion was inhibited by methylprednisolone and also by compound A, a novel nonsteroidal glucocorticoid receptor ligand. CONCLUSION: We showed that increased levels of inflammatory biomarkers in patients with BP, such as CXCL10, favor neutrophil- and monocyte-associated MMP-9 release and disease relapse and opened new therapeutic horizons in patients with this autoimmune disease.

Induction and regulation of murine emphysema by elastin peptides.

Emphysema is the major component of chronic obstructive pulmonary disease (COPD). During emphysema, elastin breakdown in the lung tissue originates from the release of large amounts of elastase by inflammatory cells. Elevated levels of elastin-derived peptides (EP) reflect massive pulmonary elastin breakdown in COPD patients. Only the EP containing the GXXPG conformational motif with a type VIII ß-turn are elastin receptor ligands inducing biological activities. In addition, the COOH-terminal glycine residue of the GXXPG motif seems a prerequisite to the biological activity. In this study, we endotracheally instilled C57BL/6J mice with GXXPG EP and/or COOH-terminal glycine deleted-EP whose sequences were designed by molecular dynamics and docking simulations. We investigated their effect on all criteria associated with the progression of murine emphysema. Bronchoalveolar lavages were recovered to analyze cell profiles by flow cytometry and lungs were prepared to allow morphological and histological analysis by immunostaining and confocal microscopy. We observed that exposure of mice to EP elicited hallmark features of emphysema with inflammatory cell accumulation associated with increased matrix metalloproteinases and desmosine expression and of remodeling of parenchymal tissue. We also identified an inactive COOH-terminal glycine deleted-EP that retains its binding-activity to EBP and that is able to inhibit the in vitro and in vivo activities of emphysema-inducing EP. This study demonstrates that EP are key actors in the development of emphysema and that they represent pharmacological targets for an alternative treatment of emphysema based on the identification of EP analogous antagonists by molecular modeling studies.

Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10(6) tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me2SO without incubation. A cooling rate of ∼1 °C/min to -80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.

Deletion of receptor for advanced glycation end products exacerbates lymphoproliferative syndrome and lupus nephritis in B6-MRL Fas lpr/j mice.

The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor that interacts with advanced glycation end products, but also with C3a, CpG DNA oligonucleotides, and alarmin molecules such as HMGB1 to initiate a proinflammatory reaction. Systemic lupus erythematosus is an autoimmune disorder associated with the accumulation of RAGE ligands. We generated mice invalidated for RAGE in the lupus-prone B6-MRL Fas lpr/j background to determine the role of RAGE in the pathogenesis of systemic lupus erythematosus. We compared the phenotype of these mice with that of their wild-type and B6-MRL Fas lpr/j littermates. Lymphoproliferative syndrome, production of anti-dsDNA Abs, lupus nephritis, and accumulation of CD3(+)B220(+)CD4(-)CD8(-) autoreactive T cells (in the peripheral blood and the spleen) were significantly increased in B6-MRL Fas lpr/j RAGE(-/-) mice compared with B6-MRL Fas lpr/j mice (respectively p < 0.005, p < 0.05, p < 0.001, and p < 0.001). A large proportion of autoreactive T cells from B6-MRL Fas lpr/j mice expressed RAGE at their surface. Time course studies of annexin V expression revealed that autoreactive T cells in the spleen of B6-MRL Fas lpr/j-RAGE(-/-) mice exhibited a delay in apoptosis and expressed significantly less activated caspase 3 (39.5 ± 4.3%) than T cells in B6-MRL Fas lpr/j mice (65.5 ± 5.2%) or wild-type mice (75.3 ± 2.64%) (p = 0.02). We conclude that the deletion of RAGE in B6-MRL Fas lpr/j mice promotes the accumulation of autoreactive CD3(+)B220(+)CD4(-)CD8(-) T cells, therefore exacerbating lymphoproliferative syndrome, autoimmunity, and organ injury. This suggests that RAGE rescues the apoptosis of T lymphocytes when the death receptor Fas/CD95 is dysfunctional.

Increased iron sequestration in alveolar macrophages in chronic obstructive pulmonary disease.

Free iron in lung can cause the generation of reactive oxygen species, an important factor in chronic obstructive pulmonary disease (COPD) pathogenesis. Iron accumulation has been implicated in oxidative stress in other diseases, such as Alzheimer's and Parkinson's diseases, but little is known about iron accumulation in COPD. We sought to determine if iron content and the expression of iron transport and/or storage genes in lung differ between controls and COPD subjects, and whether changes in these correlate with airway obstruction. Explanted lung tissue was obtained from transplant donors, GOLD 2-3 COPD subjects, and GOLD 4 lung transplant recipients, and bronchoalveolar lavage (BAL) cells were obtained from non-smokers, healthy smokers, and GOLD 1-3 COPD subjects. Iron-positive cells were quantified histologically, and the expression of iron uptake (transferrin and transferrin receptor), storage (ferritin) and export (ferroportin) genes was examined by real-time RT-PCR assay. Percentage of iron-positive cells and expression levels of iron metabolism genes were examined for correlations with airflow limitation indices (forced expiratory volume in the first second (FEV1) and the ratio between FEV1 and forced vital capacity (FEV1/FVC)). The alveolar macrophage was identified as the predominant iron-positive cell type in lung tissues. Furthermore, the quantity of iron deposit and the percentage of iron positive macrophages were increased with COPD and emphysema severity. The mRNA expression of iron uptake and storage genes transferrin and ferritin were significantly increased in GOLD 4 COPD lungs compared to donors (6.9 and 3.22 fold increase, respectively). In BAL cells, the mRNA expression of transferrin, transferrin receptor and ferritin correlated with airway obstruction. These results support activation of an iron sequestration mechanism by alveolar macrophages in COPD, which we postulate is a protective mechanism against iron induced oxidative stress.

Impairment of neutrophil reactivity to elastin peptides in COPD.

RATIONALE: Neutrophils play an important role in the inflammatory process associated with chronic obstructive pulmonary disease (COPD). Lung-infiltrating neutrophils secrete elastinolytic proteases that participate in elastin breakdown and the formation of elastin peptides (EPs). OBJECTIVES: We hypothesized that circulating neutrophil-associated immune response may be modulated by EPs during COPD. METHODS: Neutrophils obtained from patients with either stable or exacerbated COPD and controls were cultured with or without EPs. Cell chemotaxis was analysed by the Boyden method and cytokine expression was analysed by ELISA and real-time reverse transcriptase PCR. Bacterial phagocytosis and killing of ingested bacteria were evaluated after incubation with Pseudomonas aeruginosa. Reactive oxygen species (ROS) measurement and elastin receptor expression were determined by flow cytometry. RESULTS: Chemotactic activity of neutrophils from patients with COPD towards the VGVAPG EP was reduced compared with controls. VGVAPG increased proinflammatory cytokine synthesis and bacterial load, but reduced ROS production in neutrophils from controls and from patients with stable COPD. Patients with exacerbated COPD were unresponsive to VGVAPG treatment. These findings were associated with a decreased or almost complete loss of S-Gal elastin receptor expression in neutrophils from patients with stable or exacerbated COPD, respectively. CONCLUSIONS: The study demonstrates that the response of neutrophils from patients with COPD to VGVAPG varied according to COPD phase and critical level of S-Gal expression. S-Gal downregulation could result from a feedback mechanism induced by high levels of EPs.

Rapid method of quantification of tight-junction organization using image analysis.

The spatial organization of proteins in a cell population or in tissues is an important parameter to study the functionality of biological specimens. In this article, we have focused on tight junctions which form network-like features in immunofluorescence microscopy images. Usually, the organization or disorganization of tight junctions is noticed qualitatively. The aim of this article is to present a simple method to quantify the organization level of tight junction network using image analysis with a dedicated macro developed with Image J software. The method has been validated with simulated images displaying regular decrease of network organization. Then, the macro has been applied to immunofluorescence microscopy images of cells in culture and of tissue sections.

Development of a recombinant CHO cell model for the investigation of CAR and DAF role during early steps of echovirus 6 infection.

The early steps of echovirus 6 (E6) infection remain poorly understood and the only described receptor for haemagglutinating E6 strains is the decay accelerating factor (DAF). There is, however, accumulating evidence suggesting that E6 interaction with DAF is necessary but not sufficient for infection. In this report, we investigated the role of the coxsackie-adenovirus-receptor (CAR) as a potential DAF co-receptor during E6 infection. Using stably transfected Chinese Hamster Ovary (CHO) cells expressing CAR and DAF receptors, we found that DAF expression allowed attachment of both haemagglutinating and non-haemagglutinating E6 strains but was not sufficient for promoting E6 cell entry. Interestingly, the co-expression of DAF and CAR rendered 0.1-0.2% of cells permissive to some E6 strains' infection. Although our results did not show a major role of the CAR/DAF cooperation for E6 infection, it nevertheless indicated the use of CAR in the cell entry step of some minor E6 quasispecies. Moreover, the present report validates the use of recombinant CHO cells as valuable cellular model for the further characterisation of E6 receptors.

Elastin peptides signaling relies on neuraminidase-1-dependent lactosylceramide generation.

The sialidase activity of neuraminidase-1 (Neu-1) is responsible for ERK 1/2 pathway activation following binding of elastin peptide on the elastin receptor complex. In this work, we demonstrate that the receptor and lipid rafts colocalize at the plasma membrane. We also show that the disruption of these microdomains as well as their depletion in glycolipids blocks the receptor signaling. Following elastin peptide treatment, the cellular GM(3) level decreases while lactosylceramide (LacCer) content increases consistently with a GM(3)/LacCer conversion. The use of lactose or Neu-1 siRNA blocks this process suggesting that the elastin receptor complex is responsible for this lipid conversion. Flow cytometry analysis confirms this elastin peptide-driven LacCer generation. Further, the use of a monoclonal anti-GM(3) blocking antibody shows that GM(3) is required for signaling. In conclusion, our data strongly suggest that Neu-1-dependent GM(3)/LacCer conversion is the key event leading to signaling by the elastin receptor complex. As a consequence, we propose that LacCer is an early messenger for this receptor.

Elastin receptor (spliced galactosidase) occupancy by elastin peptides counteracts proinflammatory cytokine expression in lipopolysaccharide-stimulated human monocytes through NF-kappaB down-regulation.

In inflammatory diseases, strong release of elastinolytic proteases results in elastin fiber degradation generating elastin peptides (EPs). Chemotactic activity for inflammatory cells was, among wide range of properties, the former identified biological activity exerted by EPs. Recently, we demonstrated the ability of EPs to favor a Th1 cytokine (IL-2, IFN-gamma) cell response in lymphocytes and to regulate IL-1beta expression in melanoma cells. We hypothesized that EPs might also influence inflammatory cell properties by regulating cytokine expression by these cells. Therefore, we investigated the influence of EPs on inflammatory cytokine synthesis by human monocytes. We evidenced that EPs down-regulated both at the mRNA and protein levels the proinflammatory TNF-alpha, IL-1beta, and IL-6 expression in LPS-activated monocytes. Such negative feedback loop could be accounted solely for EP-mediated effects on proinflammatory cytokine production because EPs did not affect anti-inflammatory IL-10 or TGF-beta secretion by LPS-activated monocytes. Furthermore, we demonstrated that EP effect on proinflammatory cytokine expression by LPS-stimulated monocytes could not be due either to a decrease of LPS receptor expression or to an alteration of LPS binding to its receptor. The inhibitory effects of EPs on cytokine expression were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose, and to be associated with the decrease of NF-kappaB-DNA complex formation. As a whole, these results demonstrated that EP/spliced galactosidase interaction on human monocytes down-regulated NF-kappaB-dependent proinflammatory cytokine expression and pointed out the critical role of EPs in the regulation of inflammatory response.

UVB-induced IL-18 production in human keratinocyte cell line NCTC 2544 through NF-kappaB activation.

In the present study, we investigated the implication of NF-kappaB in the production of pro-inflammatory cytokine IL-18 by human keratinocytes stimulated by UVB. We demonstrated that NCTC 2544 keratinocyte cell line irradiated by UVB enhanced the IL-18 mRNA and protein secretion under its bioactive form. Overexpression of IL-18 by UVB irradiation was accompanied by NF-kappaB transcription factor activation using specific IL-18 gene sequence corresponding to NF-kappaB DNA binding site. The relationship between these transcription factors and IL-18 expression was confirmed using curcumin and PDTC, two inhibitors of NF-kappaB. Our results show that UVB and curcumin or PDTC co-treatment led to a down-regulation of IL-18 expression associated with an inhibition of NF-kappaB DNA binding. Hence, our results demonstrated that this transcription factor is implicated in biologically active IL-18 production by human keratinocytes irradiated by UVB.

Basal cells of the human adult airway surface epithelium retain transit-amplifying cell properties.

In numerous airway diseases, such as cystic fibrosis, the epithelium is severely damaged and must regenerate to restore its defense functions. Although the human airway epithelial stem cells have not been identified yet, we have suggested recently that epithelial stem/progenitor cells exist among both human fetal basal and suprabasal cell subsets in the tracheal epithelium. In this study, we analyzed the capacity of human adult basal cells isolated from human adult airway tissues to restore a well-differentiated and functional airway epithelium. To this end, we used the human-specific basal cell markers tetraspanin CD151 and tissue factor (TF) to separate positive basal cells from negative columnar cells with a FACSAria cell sorter. Sorted epithelial cells were seeded into epithelium-denuded rat tracheae that were grafted subcutaneously in nude mice and on collagen-coated porous membranes, where they were grown at the air-liquid interface. Sorted basal and columnar populations were also analyzed for their telomerase activity, a specific transit-amplifying cell marker, by the telomeric repeat amplification protocol assay. After cell sorting, the pure and viable CD151/TF-positive basal cell population proliferated on plastic and adhered on epithelium-denuded rat tracheae, as well as on collagen-coated porous membranes, where it was able to restore a fully differentiated mucociliary and functional airway epithelium, whereas viable columnar negative cells did not. Telomerase activity was detected in the CD151/TF-positive basal cell population, but not in CD151/TF-negative columnar cells. These results demonstrate that human adult basal cells are at least airway surface transit-amplifying epithelial cells.