The deficiency in IL-1R1 apparently protected the mice against LPSinduced mortality in d/d mice. P.aeruginosa-LPS challenges were responsible for leucocyte recruitment, including neutrophils. Despite increased cell infiltration in the lung of d/d mice, collagen deposition and mucus production were only slightly increased after multiple LPS challenges. Repeated LPS challenges did not cause epithelial damage and chronic inflammation, which we explain by tolerance induction, that is the reason to switch to P. aeruginosa infection. No spontaneous inflammation was found in young adult F508del CFTR mutant on C57BL/6 genetic background under our conditions. Whereas signs of inflammation and tissue remodeling develop early in most CF patients, in murine models are variable. These data are consistent with published data on the absence of spontaneous pathology such as mucus plugging, neutrophil accumulation or bronchiectasis, in young mice deficient or mutated for the Cftr gene, but neutrophilic inflammation has been reported in absence of infection. Spontaneous lung pathology development seems to be dependent on the age of mice, genetic background and animal facility health status. However, in CF mutant mice challenged with pro-inflammatory agents enhanced injury and inflammation compared to wild type was reported in several independent studies. Therefore, to get closer to a model of lung infections that occur in CF patients, we used an acute P. aeruginosa infection model, which is the most current pathogen found in CF patient, in F508del CFTR mice. Twenty hours after P. aeruginosa infection, d/d mice displayed increased neutrophil recruitment in BALF as compared with WT mice. They also showed an increase in bacterial load and cytokine production, such as IL-1b and TNF-a, in BALF. These results suggest a higher sensitivity to infection in d/d mice, consistent with previous studies in CFTR mutant mice, showing an augmentation of bacterial load and production of pro-inflammatory cytokines after P. aeruginosa infection. The enhanced production of IL-1b compared to WT after infection was associated with increased epithelial damage, cell infiltration and CFU. Then, we analyzed the effect of IL-1b neutralization at steady state in the lung. We found that in d/d as well as in WT mice, IL-1b antibody had no significant effect on Palbociclib CDK inhibitor survival. Furthermore d/d and WT mice displayed no difference in basal Il1b mRNA expression, and repeated anti-IL-1b antibody administration had no significant effect on basal inflammatory parameters and cytokine production. However, our data suggest that IL-1b may cause an excessive and pathologic inflammation in challenged CF mutant lungs. IL-1b over-expression in d/d mice infected with P. aeruginosa was accompanied by a higher bacterial load. Indeed, excessive production of inflammatory cytokines has been associated with bacterial persistence in other studies as well. High concentrations of cytokine, such as IL1b, IL-6 or TNF-a, enhance intracellular and extracellular bacterial growth of P. aeruginosa, or S. aureus in the presence of monocytes.

Furthermore, Arason outlined a deficiency of complement-dependent prevention of immune precipitation in SSc and Sprott et al. documented presence of the C5b-9 complex and C5a receptor in microvessels of SSc skin sections both in early and in late phases of the disease. It is conceivable that activation of complement system in SSc might be due to immune complexes, but inadequate protection of the EC surface might also be involved. In fact, ECs located at the interface between blood and tissues are natural targets of complement attack. The classical functions of complement, such as opsonization, recruitment of inflammatory cells, target cell lysis, immune complex clearance, and its capability to influence many other pathways, such as coagulation cascade and angiogenesis, seem to be pivotal for the integrity of ECs. In normal conditions, complement attack is tightly regulated by fluid-phase and surface-bound regulatory proteins which allow adequate immune surveillance while CT99021 GSK-3 inhibitor ensuring protection of host cells. In different vascular diseases, overtly activated or poorly controlled complement activation not only promotes EC damage and apoptosis, but also enhances the expression of vascular cell adhesion molecules and amplifies the local immune response. Factor H is the main fluid-phase regulator of the alternative complement pathway. It acts on C3, the central component of the complement cascade by accelerating decay of C3 convertase and acting as a cofactor of factor I in the inactivation of C3b. This plasma regulator also contributes to human tissue protection allowing complement activation only to foreign targets or altered self cells. In our previous study, we documented high FH levels in sera of SSc and Sclerodermatous Graft Versus Host Disease patients, but only in SSc subjects we found a defective capacity of FH to protect cellular surface from complement mediated damage in in vitro experiments. On human ECs, other complement regulators participate in cell protection from activation of both AP and classical complement pathway. The group of membrane-bound complement regulators include the membrane cofactor protein, which is a cofactor of FI in the proteolytic inactivation of C3b and C4b, and the decay accelerating factor, which accelerates the breakdown of C3- and C5-convertases. Recently, Venneker et al. demonstrated an impaired expression of MCP and DAF in endothelium of the lesional and non-lesional skin of SSc patients and in the skin of patients with morphea, in comparison to healthy controls and subjects affected by other autoimmune diseases, suggesting that a defective endothelial protection might be mediated by reduced expression of the complement regulatory proteins. Since the mechanisms involved in SSc pathogenesis are still under investigation, we focused our attention on local complement activation and regulation, using skin biopsies as an observational window of the EC damage related to SSc. Here, we propose the endothelium-bound membrane attack complex of complement as a promising marker of active vascular damage in SSc.

Hence, it is expected that several of the protein disulphide bonds are disrupted upon UV illumination, inducing conformational changes that can affect the functionality of the biomolecule. It is well known that the microtubule-associated protein tau is expressed in the central and peripheral nervous system. However, tau is also found in many other tissues such as SP600125 citations kidney, lung, muscle, and breast. Tau is able to bind to both the outer and inner surfaces of microtubules, and plays an important role in promoting tubulin polymerization and stabilizing microtubules. Tau has been divided into four regions: an amino-terminal projection region, a proline-rich domain, a microtubule-binding domain, and an acidic carboxyl-terminal region. In adult human brain, six developmentally modulated isoforms are generated by alternative splicing around the amino-terminal region and MTB. The tau isoforms are distinct from each other by containing the different numbers of microtubule binding repeat sequences and amino-terminal exons. Numerous studies have indicated that abnormally hyperphosphorylated tau was involved in the formation of neurofibrillary tangles, a pathological hallmark of Alzheimer’s disease. Many serine and threonine residues on tau are phosphorylated by various kinases, which include glycogen synthase kinase 3, cyclic-AMP-dependent kinase, and microtubule-affinity regulating kinase. Dephosphorylation mediated by protein phosphatases counterbalances the effects of protein kinases; therefore, an imbalance of between protein kinases and protein phosphatases was considered as underlying mechanism of tau hyperphosphorylation in AD. The abnormal alterations of tau were also observed in many neurodegenerative disorders, and thus those diseases were referred to as tauopathies. Previous works have shown that estrogen was involved in improvement of neuronal survival and cognitive function. Ferreira et al reported that estrogen-enhanced neurite growth was mediated by promoting the levels of tau protein in dissociated cultures of hypothalamic neurons. In agreement with such study, overexpression of tau was able to promote neurite extension in cell culture. Moreover, tau was able to obstruct the binding of taxane and paclitaxel to the inner surface of microtubules, and thus reduced the sensitivity of breast cancer tissues with expression of estrogen receptors to these drugs, showing a promoting role of tau in tumor growth. These results implied that tau was capable to improve cell growth under some pathophysiological conditions. Ion channels have an essential role in cell proliferation and the development of cancer. On the other hand, cancer cells are, on average, more depolarized than healthy cells at the same histological origin. Kv channels, which contained six transmembrane segments and one pore-forming region, show a wide range of voltage dependence and kinetic properties. These channels are widely expressed in different kinds of excitable and non-excitable tissues, and have a crucial role in tuning action potential and neuronal excitability.

In physiologic conditions, the concentration of eATP is quite low but it can be rapidly released in various amounts after cell stress, damage or death. Then, eATP exerts immunostimulatory or immunosuppressive effects depending on its extracellular concentration, on which P2 receptors are engaged on specific immune cells and on the extent of the stimulation. Roughly, murine models of inflammatory and autoimmune diseases have shown that eATP can act as a proinflammatory molecule not only by stimulating innate immune responses but also by favoring effector T-cell activation, mainly through P2X7 signaling. In our work, by using a specific gene knockout approach, we have focused on the SAR131675 effect of eATP on P2Y2R instead of using receptor specific antagonists. However, it is likely that other P2 receptors, including P2X receptors might also play a role in EAU development. In this context, two studies reported contradictory effect of P2X7 deficiency on experimental autoimmune encephalomyelitis development, either protective or deleterious. Yet, It has been shown also that the nucleotide affinity for P2Y2 receptors is significantly higher than for P2X7 receptors In humans, several in vitro studies have pointed out a more complex role of eATP, able also to inhibit the cytokine secretion, proliferation or cytotoxic activity of immune cells such as DC, macrophages, NK cells and T lymphocytes through the activation of P2Y11 receptors. Those P2Y11 receptors are however not expressed on murine cells. Another major fact to point out is that nucleotides are unstable short-lived molecules acting in an autocrine or paracrine manner. Especially, eATP is hydrolyzed into ADP/AMP and adenosine by plasma membranebound ectoenzymes, i.e. CD39 and CD73, whose expression on different immune cells or even on a same cell subset but in different location is quite variable. Therefore, a lot of studies evaluating the effect of eATP were done with ATPγS, a non-hydrolysable ATP analogue, rendering those data not comparable to ours. To our knowledge, there is no other publication on the role of the P2Y2 deficiency during the development of an experimental autoimmune disease. In order to evaluate a potential role of P2Y2R deficiency in lymphocyte migration, we similarly transferred Indium-radiolabeled TL and followed their in vivo migration with a SPECT camera. Unfortunately, this methodology appeared not sensitive enough to detect the presence of a small number of autoreactive TL in the eye. Besides, our findings are in line with several studies showing the importance of P2Y2R expression on epithelial and endothelial cells for VCAM1 expression and secondary recruitment of inflammatory cells. Altogether our results are in agreement with the present literature showing the DAMPs properties of nucleotides. Hence, Idzko M et al have shown that extracellular ATP triggers and maintains asthmatic airway Th2 inflammation and Granstein RD et al have demonstrated that ATPγS enhances cutaneous Th1 immune response. However, our data showed that the DC migration toward secondary lymphoid organs was not influenced by P2Y2R deficiency.

Among the many human mutations that lead to a pathological condition, few have a spontaneous orthologous equivalent in the mouse and vice versa. In this regard, the spf-J mouse is a unique tool for studying mild OTC deficiency. Several factors are required to trigger an autoimmune organ-specific disease. Genetic and environmental components will collaborate to cause a breakdown of the peripheral immune tolerance and activation of resident cells of affected tissues. Autoimmune uveitis illustrates these autoimmunity paradigms. Genetic susceptibility of individuals with AIU has been widely studied and the association of several polymorphisms at Perifosine different loci of the HLA system is well known. Similarly, different groups have demonstrated the presence of autoreactive T cells and the role of cytokines released by these cells in the activation of resident cells of the eye during development of AIU. Several data also attest the importance of the role of danger signals during these two key phases of pathological activation. If a central place was given to exogenous danger signals, in particular microbial, the importance of endogenous danger signals began to emerge. In this context, nucleotides are an important family of potential endogenous danger signals. In fact, normally, they are present almost exclusively within the cells. However, during cellular destruction or stress, like inflammation, nucleotides can be released in various amounts in the extracellular space and activate nucleotide receptors belonging to the P2X and P2Y families. In many cases, the activation of P2 receptors results in an increase of the inflammatory process. In agreement with the danger signal theory, we have shown that different nucleotides, ATPγS, UTP and UDP, are involved in the activation of retinal pigment epithelium and increase the basal as well as the TNFα-induced release of IL-8. Similarly, several works have demonstrated that nucleotides also profoundly influence antigen presentation and lymphocyte activation. Accordingly, Granstein et al have shown, in vivo, that extracellular nucleotides strongly increase lymphocyte activation after systemic immunization. In this work, we have thus hypothesized that nucleotides can act as danger signals during AIU and interfere with both the activation of autoreactive lymphocytes and the stimulation of blood retinal barrier cells. The development of autoimmune uveitis requires both the activation of retinal specific autoreactive lymphocyte clones, their migration to the eye and the breakdown of the blood retinal barrier. In this work, we found that P2Y2 deficiency attenuates EAU development and strongly affects the activation of IRBP-specific autoreactive lymphocytes after systemic immunization. Our results are in accordance with the danger model, which makes a link between autoreactive lymphocyte activation, immune cell migration and the release of endogenous danger signals such as extracellular nucleotides. Among them, extracellular ATP was recognized as an important modulator of immune responses through its binding to plasma membrane P2 purinergic receptors.