Manipulating keratinocyte stem cell proliferation and differentiation using RNA interference

ABSTRACT : The epidermis, the outermost compartment of the skin, is a stratified and squamous epithelium that constantly self-renews. Keratinocytes, which represent the main epidermal population, are responsible for its cohesion and barrier function. Epidermal renewal necessitates a fine equilibrium between keratinocyte proliferation and differentiation. The keratinocyte stem cell, located in the basal cell layer, is responsible for epidermal homeostasis and regeneration during the wound healing process. The transcription factor p63 structurally belongs to the p53 superfamily. It is expressed in the basal and supra-basal cell layers of stratified epithelia and is thought to be important for the renewal or the differentiation of keratinocyte stem cells (Yang et al., 1999; Mills et al., 1999). In order to better understand its function, we established an in vitro model of p63 deficient human keratinocyte stem cells using a shp63 mediated RNA interference. Knockdown of endogenous p63 induces downregulation of cell-adhesion genes as previously described (Carroll et al., 2006). Interestingly, the replating of attached p63-knockdown keratinocytes on a feeder layer results in a loss of attachment and proliferation. They are no longer clonogenic. However, if the same population are replated in a fibrin matrix, extended fibrinolysis is reported, a common process in wound healing, suggesting that p63 regulates the fibrinolytic pathway. This result was confirmed by Q-PCR and shows that the urokinase pathway, which mediates fibrinolysis, is upregulated. Altogether, these findings suggest a mechanism in which the fine tuning of p63 expression promotes attachment or release of the keratinocyte stem cell from the basement membrane by inducing genes of adhesion and/or of fibrinolysis. This mechanism may be important for epidermal self-renewal, differentiation as well as wound healing. Its misregulation may be partly responsible for the p63 knockout phenotype. The downregulation of p63 also induces a decrease in LEKTI expression. LEKTI (lymphoepithelial Kazal-type serine protease inhibitor) is a serine protease inhibitor encoded by the Spink5 gene. It is expressed and secreted in the uppermost differentiated layers of stratified epithelia and plays a role in the desquamation process. When this gene is disrupted, humans develop the Netherton syndrome (Chavanas et al., 2000b). It is a dermatosis characterized by hair dysplasias, ichtyosiform erythroderma and impairment in epidermal barrier function promoting inflammation similarly as in psoriasis with inflammatory infiltrate in excess. TNFα (tumor necrosis factor alpha) and EDA1 (ectodysplasin A1) are two transmembraneprecursors that belong to the TNF superfamily, which is involved in immune and inflammation regulation (Smahi et al., 2002). We suggest that the secreted serine protease inhibitor LEKTI plays a role in the regulation of TNFα and EDA1 precursor cleavage and absence of LEKTI induces excess of inflammation. To investigate this hypothesis, we induced downregulation of Spink5 expression in rat keratinocyte stem cells by using a shSpink5 mediated RNA interference approach. Interestingly, expression of TNFα and EDA1 is modified after knockdown of Spink5 by Q-PCR. Moreover, downregulation of Spink5 induces loss of cohesiveness between keratinocytes and colonies adopt a scattered phenotype. Altogether, these preliminary data suggest that downregulation of LEKTI may play a role in the inflammatory response in Netherton syndrome patients, by regulating TNFα expression.

Immune system's role in viral encephalitis.

Viral infections can be a major thread for the central nervous system (CNS), therefore, the immune system must be able to mount a highly proportionate immune response, not too weak, which would allow the virus to proliferate, but not too strong either, to avoid collateral damages. Here, we aim at reviewing the immunological mechanisms involved in the host defense in viral CNS infections. First, we review the specificities of the innate as well as the adaptive immune responses in the CNS, using several examples of various viral encephalitis. Then, we focus on three different modes of interactions between viruses and immune responses, namely human Herpes virus-1 encephalitis with the defect in innate immune response which favors this disease; JC virus-caused progressive multifocal leukoencephalopathy and the crucial role of adaptive immune response in this example; and finally, HIV infection with the accompanying low grade chronic inflammation in the CNS in some patients, which may be an explanation for the presence of cognitive disorders, even in some well-treated HIV-infected patients. We also emphasize that, although the immune response is generally associated with viral replication control and limited cellular death, an exaggerated inflammatory reaction can lead to tissue damage and can be detrimental for the host, a feature of the immune reconstitution inflammatory syndrome (IRIS). We will briefly address the indication of steroids in this situation.

The role of chemokines in cancer immune surveillance by the adaptive immune system.

Chemokines are key molecules involved in the migration and homeostasis of immune cells. However, also tumor cells use chemokine signals for different processes such as tumor progression and metastasis. It is thus unclear whether chemokines, through their immunostimulatory roles, contribute to the repression of tumor cells by tumor immunosurveillance or whether chemokines act primarily as growth factors and chemoattractants for primary and metastatizing tumors, respectively. Research of recent years, using gene knockout mice, recombinant chemokines, and agents able to block chemokine actions, has provided further insight into the diverse functions of chemokines. Here, we review the current knowledge on the complex actions of chemokines at the interface of the immune system and the tumor.

Les hépatopathies auto-immunes et leurs traitements [Auto-immune liver diseases and their treatment]

There are three main types of auto-immune liver disease, auto-immune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis. In the case of auto-immune hepatitis, prednisone therapy, with or without azathioprine, can improve quality of life and halt progression to cirrhosis. If there is no response or if the therapy is poorly tolerated, mycophenolate mofetil or cyclosporin should be considered. Ursodeoxycholic acid (UDCA), at a dosage of 13 to 15 mg/kg/day slows the progression of fibrosis in patients with primary biliary cirrhosis. Pruritus may be treated with cholestyramine, rifampicin or opiate antagonists. Ursodeoxycholic acid at a dosage of 20 to 30 mg/kg/day will slow the evolution of fibrosis.

Activin enhances skin tumourigenesis and malignant progression by inducing a pro-tumourigenic immune cell response.

Activin is an important orchestrator of wound repair, but its potential role in skin carcinogenesis has not been addressed. Here we show using different types of genetically modified mice that enhanced levels of activin in the skin promote skin tumour formation and their malignant progression through induction of a pro-tumourigenic microenvironment. This includes accumulation of tumour-promoting Langerhans cells and regulatory T cells in the epidermis. Furthermore, activin inhibits proliferation of tumour-suppressive epidermal γδ T cells, resulting in their progressive loss during tumour promotion. An increase in activin expression was also found in human cutaneous basal and squamous cell carcinomas when compared with control tissue. These findings highlight the parallels between wound healing and cancer, and suggest inhibition of activin action as a promising strategy for the treatment of cancers overexpressing this factor.

Macrophage migration inhibitory factor and host innate immune defenses against bacterial sepsis.

Macrophages are essential effector cells of innate immunity that play a pivotal role in the recognition and elimination of invasive microorganisms. Mediators released by activated macrophages orchestrate innate and adaptive immune host responses. The cytokine macrophage migration inhibitory factor (MIF) is an integral mediator of the innate immune system. Monocytes and macrophages constitutively express large amounts of MIF, which is rapidly released after exposure to bacterial toxins and cytokines. MIF exerts potent proinflammatory activities and is an important cytokine of septic shock. Recent investigations of the mechanisms by which MIF regulates innate immune responses to endotoxin and gram-negative bacteria indicate that MIF acts by modulating the expression of Toll-like receptor 4, the signal-transducing molecule of the lipopolysaccharide receptor complex. Given its role in innate immune responses to bacterial infections, MIF is a novel target for therapeutic intervention in patients with septic shock.

Abrupt decrease in serum testosterone levels after an oral glucose load in men: implications for screening for hypogonadism.

OBJECTIVE: This study examines the physiological impact of a glucose load on serum testosterone (T) levels in men with varying glucose tolerance (GT). DESIGN: Cross-sectional study. PATIENTS AND METHODS: 74 men (19-74 years, mean 51·4 ± 1·4 years) underwent a standard 75-g oral glucose tolerance test with blood sampling at 0, 30, 60, 90 and 120 min. Fasting serum glucose, insulin, total T (and calculated free T), LH, SHBG, leptin and cortisol were measured. RESULTS: 57% of the men had normal GT, 30% had impaired GT and 13% had newly diagnosed type 2 diabetes. Glucose ingestion was associated with a 25% decrease in mean T levels (delta = -4·2 ± 0·3 nm, P < 0·0001). T levels remained suppressed at 120 min compared with baseline (13·7 ± 0·6 vs 16·5 ± 0·7 nm, P < 0·0001) and did not differ across GT or BMI. Of the 66 men with normal T levels at baseline, 10 (15%) had levels that decreased to the hypogonadal range (<9·7 nm) at one or more time points. SHBG, LH and cortisol levels were unchanged. Leptin levels decreased from baseline at all time points (P < 0·0001). CONCLUSIONS: Glucose ingestion induces a significant reduction in total and free T levels in men, which is similar across the spectrum of glucose tolerance. This decrease in T appears to be because of a direct testicular defect, but the absence of compensatory changes in LH suggests an additional central component. Men found to have low nonfasting T levels should be re-evaluated in the fasting state.

Detachment of cell surface-bound anticarcinoembryonic antigen immune complexes by phospholipase C.

CEA as well as normal cross-reacting antigens (NCA) are fixed to the cell membrane via phosphatidylinositol (PI). To find out whether these antigens are internalized after antibody contact, acid pH desorption was compared to phospholipase C (PLC)-mediated cleavage of the antigen anchor. With the former procedure, marked differences in the desorbability of individual MAbs were noted, while PLC was able to cleave off surface-bound immune complexes irrespective of the MAb involved. From this it is concluded that internalization of MAb complexes of CEA/NCA, if occurring at all, is a low efficiency process.

Allogeneic beta-islet cells correct diabetes and resist immune rejection.

Allogeneic MHC-incompatible organ or cell grafts are usually promptly rejected by immunocompetent hosts. Here we tested allogeneic beta-islet cell graft acceptance by immune or naive C57BL/6 mice rendered diabetic with streptozotocin (STZ). Fully MHC-mismatched insulin-producing growth-regulated beta-islet cells were transplanted under the kidney capsule or s.c. Although previously or simultaneously primed mice rejected grafts, STZ-treated diabetic mice accepted islet cell grafts, and hyperglycemia was corrected within 2-4 weeks in absence of conventional immunosuppression. Allogeneic grafts that controlled hyperglycemia expressed MHC antigens, were not rejected for &gt;100 days, and resisted a challenge by allogeneic skin grafts or multiple injections of allogeneic cells. Importantly, the skin grafts were rejected in a primary fashion by the grafted and corrected host, indicating neither tolerization nor priming. Such strictly extralymphatic cell grafts that are immunologically largely ignored should be applicable clinically.

Exploration of immune competencies of viral-specific CD8+T cells in MS patients using tetramers and functional CTL assays

Introduction: Epstein-Barr Virus(EBV) has been repeatedly associatedwith multiple sclerosis (MS). Wehave previously shown that there is ahigh peripheral as well as intrathecalactivation of EBV-, but not cytomegalovirus(CMV)-specific CD8+ Tcells, early in the course of MS,strengthening the link between EBVand MS. However, the trigger of thisincreased EBV-specific CD8+ T cellresponse remains obscure. It could resultfrom a higher EBV viral load. Alternatively,it could be due to an intrinsicallydeficient EBV-specificCTL response, cytotoxic granulesmediated.Thus, we performed anin-depth study of the phenotype of exvivo EBV- and CMV-specific CD8+T cells in MS patients and control patients,assessing their cytotoxic activity.Methods:We analyzed the profileof cytotoxic granules in EBV- andCMV-specific CD8+ T cells in a cohortof 13 early MS patients, 20 lateMS, 30 other neurological diseases(OND) patients and 7 healthy controlsubjects. Ex vivo analysis of EBV- orCMV-specific CD8+ T cells was performedusing HLA class I/tetramercomplexes coupled to CCR7 andCD57 markers in conjunction withperforin, granzymes A, BandKstaining.In a sub-cohort of MS patientsand controls, cytotoxic activity ofEBV- and CMV-specific CD8+ Tcells was investigated using a functionalCFSE CTL assay. Results: UsingHLA Class I tetramers for EBVand CMV, we found that the frequencyof EBV- or CMV-specificCD8+ T cells were similar in all studysubjects. Most of EBV- and CMVspecificCD8+Tcells were highly differentiated(CCR7-) and a variousproportion expressed the exhaustionmarker CD57. MS and OND patientshad increased perforin expression inEBV-specific CD8+ T cells. Most importantly,we found that MS patientswith longer disease duration tended tohave lower CTL cytotoxicity as comparedto earlyMSpatients or controls.Conclusions: Effector EBV-specificCD8+ T cells are increased in earlyMS, however their cytotoxic granuleprofile does not seem to be fully alteredand the cytotoxic activity ofthese cells is normal. However, thecytotoxic activity of CTL decreasedin late MS patients suggesting an exhaustionof EBV-specific CD8+ Tcells possibly due to EBV reactivation.This work was supported by theSwiss National Foundation PP00B3-124893, the Swiss Society for MS,and the Biaggi Foundation to RADP.

Role of defensins and cathelicidin LL37 in auto-immune and auto-inflammatory diseases.

Defensins and cathelicidins are anti-microbial peptides (AMPs) that act as natural antibiotics and are part of the innate immune defence in many species. We consider human defensins and LL37, the only human member of the cathelicidin family. In particular, we refer to the human alpha-defensins called human neutrophil peptides (HNP1 through 4), which are produced by neutrophils, HD5 and HD6, mainly expressed in Paneth cells of intestine, the human beta-defensins HBD1, HBD2 and HBD3, synthesized by epithelial cells and LL37, which is located in granulocytes, but is also produced by epithelial cells of the skin, lungs, and gut. In the last years, the study of AMPs activity and regulation has allowed to understand the important role of these peptides not only in the innate defence mechanisms against bacteria, viruses, fungi, but also in the regulation of immune cell activation and migration. Complementary studies have disclosed a role for AMPs in modulating many physiological processes that involve non-immune cells, such as activation of wound healing, angiogenesis, cartilage remodeling. Due to the pleiotropic tasks of these peptides, many of them are now being discovered to contribute to immune pathology of chronic diseases that affect skin, gut, joints; this is supported by many examples of immune-mediated pathologies in which their expression is disregulated. In this article we review the current literature that suggests a role for human defensins and LL37 in pathogenic mechanisms of several chronic diseases that are considered of auto-immune or auto-inflammatory origin.

Aging of myelinating glial cells predominantly affects lipid metabolism and immune response pathways.

Both the central and the peripheral nervous systems are prone to multiple age-dependent neurological deficits, often attributed to still unknown alterations in the function of myelinating glia. To uncover the biological processes affected in glial cells by aging, we analyzed gene expression of the Schwann cell-rich mouse sciatic nerve at 17 time points throughout life, from day of birth until senescence. By combining these data with the gene expression data of myelin mouse mutants carrying deletions of either Pmp22, SCAP, or Lpin1, we found that the majority of age-related transcripts were also affected in myelin mutants (54.4%) and were regulated during PNS development (59.5%), indicating a high level of overlap in implicated molecular pathways. The expression profiles in aging copied the direction of transcriptional changes observed in neuropathy models; however, they had the opposite direction when compared with PNS development. The most significantly altered biological processes in aging involved the inflammatory/immune response and lipid metabolism. Interestingly, both these pathways were comparably changed in the aging optic nerve, suggesting that similar biological processes are affected in aging of glia-rich parts of the central and peripheral nervous systems. Our comprehensive comparison of gene expression in three distinct biological conditions including development, aging, and myelin disease thus revealed a previously unanticipated relationship among themselves and identified lipid metabolism and inflammatory/immune response pathways as potential therapeutical targets to prevent or delay so far incurable age-related and inherited forms of neuropathies.

Persistence of EBV Antigen-Specific CD8 T Cell Clonotypes during Homeostatic Immune Reconstitution in Cancer Patients.

Persistent viruses are kept in check by specific lymphocytes. The clonal T cell receptor (TCR) repertoire against Epstein-Barr virus (EBV), once established following primary infection, exhibits a robust stability over time. However, the determinants contributing to this long-term persistence are still poorly characterized. Taking advantage of an in vivo clinical setting where lymphocyte homeostasis was transiently perturbed, we studied EBV antigen-specific CD8 T cells before and after non-myeloablative lympho-depleting chemotherapy of melanoma patients. Despite more advanced T cell differentiation, patients T cells showed clonal composition comparable to healthy individuals, sharing a preference for TRBV20 and TRBV29 gene segment usage and several co-dominant public TCR clonotypes. Moreover, our data revealed the presence of relatively few dominant EBV antigen-specific T cell clonotypes, which mostly persisted following transient lympho-depletion (TLD) and lymphocyte recovery, likely related to absence of EBV reactivation and de novo T cell priming in these patients. Interestingly, persisting clonotypes frequently co-expressed memory/homing-associated genes (CD27, IL7R, EOMES, CD62L/SELL and CCR5) supporting the notion that they are particularly important for long-lasting CD8 T cell responses. Nevertheless, the clonal composition of EBV-specific CD8 T cells was preserved over time with the presence of the same dominant clonotypes after non-myeloablative chemotherapy. The observed clonotype persistence demonstrates high robustness of CD8 T cell homeostasis and reconstitution.