Antagonistic effect of the inflammasome on thymic stromal lymphopoietin expression in the skin.

BACKGROUND: Innate immune sensors control key cytokines that regulate T-cell priming and T-cell fate. This is particularly evident in allergic reactions, which represent ideal systems to study the interplay of innate and adaptive immunity. In patients with contact dermatitis, inflammasome-mediated IL-1 activation is responsible for a TH1 immune response. Surprisingly, the IL-1 signaling pathway was also proposed to control the activation of thymic stromal lymphopoietin (TSLP), a cytokine implicated in development of the T(H)2 response in patients with atopic dermatitis (AD) and asthma. OBJECTIVES: We sought to assess the effect of the inflammasome on TSLP expression levels and the development of AD. METHODS: We studied the effect of the inflammasome activator 2,4-dinitrofluorobenzene, and IL-1ß on TSLP mRNA expression levels in mouse and human cell lines (in vitro assays), as well as in live mice and on human skin transplants. We also assessed the effect of 2,4-dinitrofluorobenzene on TSLP and the TH2 response in mice in which the inflammasome and IL-1 signaling pathways were blocked, either genetically or pharmacologically, in 2 models of AD. RESULTS: We provide in vitro and in vivo evidence that inflammasome activation has an inhibitory role on TSLP mRNA expression and T(H)2 cell fate in the skin. We also show that solvents influence the activation of TSLP and IL-1ß and direct the T-cell fate to a given hapten. CONCLUSION: Our observations strongly suggest that the TH1 versus TH2 cell fate decision is regulated at multiple levels and starts with innate immune events occurring within peripheral epithelial tissues.

TNF-α and IFN-γ are potential inducers of Fas-mediated keratinocyte apoptosis through activation of inducible nitric oxide synthase in toxic epidermal necrolysis.

Toxic epidermal necrolysis (TEN) is a severe immune-mediated adverse cutaneous drug eruption characterized by rapid and extensive epithelial cell death in the epidermis and mucosae. The molecular events leading to this often fatal condition are only partially understood, but evidence suggests a dual mechanism implicating a "drug"-specific immune response on one side and the onset of target cell death by proapoptotic molecules including FasL on the other side. Herein, we describe a potential molecular bridge between these two events that involves inducible nitric oxide synthase (iNOS), which is highly upregulated in the skin of TEN patients. We show that activated T cells secrete high amounts of tumor necrosis factor-α (TNF-α) and IFN-γ, and that both cytokines lead to increased expression and activity of keratinocyte iNOS. A similar observation has been made with drug-specific T lymphocytes from a TEN patient exposed to the culprit drug. The resulting increase in nitric oxide significantly upregulates keratinocyte FasL expression, resulting in Fas- and caspase-8-mediated keratinocyte cell death. Taken together, our data suggest that T-lymphocyte activation by drugs in TEN patients may indirectly lead to FasL-mediated keratinocyte apoptosis, via a molecular bridge involving TNF-α, IFN-γ, and iNOS.

Danger signaling through the inflammasome acts as a master switch between tolerance and sensitization.

Efficient priming of adaptive immunity depends on danger signals provided by innate immune pathways. As an example, inflammasome-mediated activation of caspase-1 and IL-1beta is crucial for the development of reactive T cells targeting sensitizers like dinitrofluorobenzene (DNFB). Surprisingly, DNFB and dinitrothiocyanobenzene provide cross-reactive Ags yet drive opposing, sensitizing vs tolerizing, T cell responses. In this study, we show that, in mice, inflammasome-signaling levels can be modulated to turn dinitrothiocyanobenzene into a sensitizer and DNFB into a tolerizer, and that it correlates with the IL-6 and IL-12 secretion levels, affecting Th1, Th17, and regulatory T cell development. Hence, our data provide the first evidence that the inflammasome can define the type of adaptive immune response elicited by an Ag, and hint at new strategies to modulate T cell responses in vivo.

Resistance to FasL and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in Sezary syndrome T-cells associated with impaired death receptor and FLICE-inhibitory protein expression.

Because of the low proliferative potential of tumor cells in patients with Sézary syndrome (SzS), their accumulation has been suggested to be due to defective regulation of apoptosis. We analyzed the sensitivity to soluble Fas-ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), 2 members of the TNF superfamily in peripheral blood leukocytes (PBL) from patients with SzS. Compared with healthy donors, CD4(+) cells from patients with SzS were completely resistant to FasL in 9 of 16 cases. Of these 9 FasL-resistant cases, 4 revealed a loss in Fas (CD95) expression, whereas the remaining 5 exhibited normal or enhanced Fas expression. In the latter 5 cases, the apoptosis inhibitor cFLIP was overexpressed in CD4(+)/CD26(-) tumor cells compared with CD4(+)/CD26(-) cells from Fas-expressing FasL-sensitive patients and healthy donors. Furthermore, resistance to TRAIL and tumor cell-restricted loss of TRAIL-receptor 2 were observed in 16 of 16 SzS PBLs. It is noteworthy that resistance to FasL could be overcome by the use of a hexameric FasL or upon exposure of SzS cells to interferon-alpha (IFN-alpha) or IFN-gamma, the latter by an increase of Fas expression. Our data on primary SzS lymphocytes reveal frequent resistance to apoptosis induced by FasL and TRAIL, which may contribute to their accumulation in patients with SzS and be relevant at a therapeutic level.

Activation of the IL-1beta-processing inflammasome is involved in contact hypersensitivity.

The inflammasome is a cytosolic protein complex regulating the activation of caspase-1, which cleaves the pro-inflammatory cytokines IL-1beta and IL-18 into their active form. The inflammasome is composed of a NACHT-, LRR- and pyrin (NALP) family member that acts as a sensor for danger signals and the adaptor protein apoptosis-associated speck-like protein containing a CARD domain (ASC), which allows the recruitment of caspase-1 in the complex. In the skin, exposure to contact sensitizers (CS) such as trinitro-chlorobenzene causes an immune response called contact hypersensitivity (CHS) or eczema. In this delayed-type hypersensitivity response, efficient priming of the adaptive immunity depends on the concomitant activation of the innate immune system, including IL-1beta/IL-18 activation in the skin. To determine if the inflammasome contributes to CHS, we have analyzed its capacity to react to CS in vitro and in vivo. We show here that key components of the inflammasome are present in human keratinocytes and that CS like trinitro-chlorobenzene induce caspase-1/ASC dependent IL-1beta and IL-18 processing and secretion. We also show that ASC- and NALP3-deficient mice display an impaired response to CS. These findings suggest that CS act as danger signals that activate the inflammasome in the skin, and reveal a new role of NALP3 and ASC as regulators of innate immunity in CHS.

Assessment of optimal transduction of primary human skin keratinocytes by viral vectors.

BACKGROUND: Genetically modified keratinocytes generate transplantable self-renewing epithelia suitable for delivery of therapeutic polypeptides. However, the variety of viral vectors and experimental conditions currently used make fragmented or contradictory the information on the transduction efficiency of the human primary keratinocytes. To compare the suitability of the most currently used viral vectors for efficient gene transfer to human keratinocytes, we have performed a comparative study using a panel of recombinant constructs. METHODS: For each vector, the transduction efficiency and the persistence of the transgene expression were quantified by fluorescence microscopy and flow cytometry analysis of the infected cells. RESULTS: We show that: (1) canine and human adenoviral vectors achieve a highly efficient but transient transduction of both primary and immortalized keratinocytes; (2) the adenovirus-associated virus (AAV) vectors transduce immortalized keratinocytes, albeit with a short-lived gene expression (<4 days), but fail to infect primary keratinocytes; and (3) under appropriate conditions, the oncoretroviral and lentiviral vectors can permanently transduce up to 100% of primary keratinocytes, but the highly clonogenic keratinocytes are more efficiently targeted by lentiviral vectors. CONCLUSIONS: Therefore, AAV vectors are unsuitable to transduce primary keratinocytes, while human and canine adenoviral vectors appears to be appropriate to achieve short-term delivery of therapeutic products. Recombinant retroviruses provide sustained expression of the transgene, but the lentiviral vectors are the most suitable for ex vivo gene therapy because of their ability to transduce clonogenic primary keratinocytes.

Transdifferentiation of preadipose cells into smooth muscle-like cells: role of aortic carboxypeptidase-like protein.

Adipocyte differentiation involves dramatic cell shape alterations that are accompanied by changes in the expression of cytoskeletal and extracellular matrix (ECM) proteins. Aortic carboxypeptidase-like protein (ACLP) is a secreted protein associated with the extracellular matrix whose expression is induced during smooth muscle (SM) differentiation. We analyzed the expression of ACLP gene during adipocyte differentiation of 3T3-F442A, 3T3-L1, and Ob1771 preadipocytes. Our results show that ACLP mRNA and protein are expressed in growing cells and after commitment. Thereafter, their expression levels decrease, as opposed to that of aP2 and PPARgamma2. Consistent with these observations, ACLP mRNA is expressed in the stromal-vascular fraction of adipose tissue but not in the adipocyte fraction. Overexpression of ACLP in 3T3-F442A preadipocytes inhibits adipocyte differentiation at both morphological and molecular level. However, ACLP overexpression promotes transdifferentiation of preadipocytes into smooth muscle-like cells, which express specific markers such as SM22alpha, SM alpha-actin, SM-MHC, and caldesmon. These findings demonstrate that overexpression of a single extracellular matrix protein is sufficient to induce transdifferentiation and that ACLP may modulate the commitment of mesodermal cells into different lineages depending upon its pattern of expression.

Rapid decay of alpha6 integrin caused by a mis-sense mutation in the propeller domain results in severe junctional epidermolysis bullosa with pyloric atresia.

Genetic mutations in alpha6beta4 integrin cause junctional epidermolysis bullosa with pyloric atresia, a genodermatosis characterized by blistering of the skin and pyloric occlusion. The lethal form of junctional epidermolysis bullosa with pyloric atresia has been mainly associated with the presence of premature termination codons in the mRNA encoding either the alpha6 or beta4 subunit causing rapid decay of the mutated transcript and absence of alpha6beta4 integrin. In this study, we disclose the genetic background of lethal junctional epidermolysis bullosa with pyloric atresia in a patient presenting absent expression of alpha6 integrin despite normal steady-state level of the alpha6beta4 mRNA. Screening for mutation in the alpha6 gene detected a homozygous base pair substitution (286 C-to-T), which results in the substitution of a serine with a leucine residue (S47L). The amino acid substitution S47L localizes in the first beta-strand of the seven-bladed beta-propeller structure of the extracellular head of alpha6 integrin, and triggers a rapid proteolysis of the aberrant polypeptides involving the lysosomal degradation pathway. This study provides new insight into the pathogenic effect of a mis-sense mutation affecting a functional domain of a protein, and identifies a critical peptide sequence of the beta-propeller domain conserved among the alpha integrin cell receptors.