The mechanistic basis for psoriasis immunopathogenesis: translating genotype to phenotype. Report of a workshop, Venice, 2012.

The International Psoriasis Council, a global nonprofit organization dedicated to advancing psoriasis research and treatment, led an initiative to better define the pathogenic mechanisms that constitute psoriasis. In September 2012, a workshop was held at the 42nd Annual European Society for Dermatological Research in Venice, Italy. By assembling a panel of global dermatology and immunology experts, the objective was to evaluate the current status of the science explaining the mechanism of disease in psoriasis, e.g. dysregulation of the skin immune system and perturbations of epidermal homeostasis. The workshop consisted of four oral presentations, which addressed key topics in psoriasis, delivered by Hervé Bachelez (Paris, France), Antonio Costanzo (Rome, Italy), Michelle Lowes (New York, NY, U.S.A.) and Frank Nestle (London, U.K.). A global expert panel was assembled to stimulate dialogue and debate: Kevin Cooper (Cleveland, OH, U.S.A.), Michel Gilliet (Lausanne, Switzerland), Joerg Prinz (Munich, Germany), Martin Röcken (Tubingen, Germany), Jens Schroeder (Kiel, Germany), Manuelle Viguier (Paris, France), Mayte Suárez-Fariñas (New York, NY, U.S.A.) and Cristina Zielinski (Berlin, Germany). Collectively, the presentations demonstrated the significant advances in understanding immune regulation that have occurred over the past decade by virtue of the study of psoriasis subtypes, phenotypic manifestations and genetic associations. Elucidating the pathogenic and genetic basis of psoriasis holds the promise of a complete understanding of disease mechanisms, predictors of treatment response, novel drug development strategies and customized therapeutic regimens for the individual patient.

Exploring the association between cardiovascular and other disease-related risk factors in the psoriasis population: the need for increased understanding across the medical community.

There is abundant and accumulating evidence on the classification of psoriasis as a systemic disease that exhibits a host of co-morbidities. As a consequence, the second Interdisciplinary Conference on Co-morbidities and Lifestyle Modification, convened by the International Psoriasis Council, has concluded that specialist physicians, primary care physicians and dermatologists are faced with an opportunity to impact, not just psoriasis disease understanding and management, but overall patient well-being. The conference panel was represented by the disciplines of dermatology, cardiology, rheumatology, epidemiology, endocrinology, hepatology and gastroenterology, and medical specialists with particular expertise in obesity, diabetes mellitus, inflammation and genetics. The multiple co-morbidities associated with psoriasis were reviewed with a view to identify possible mechanisms linking psoriatic disease with obesity, metabolic syndrome, diabetes, cardiovascular disease and non-alcoholic fatty liver disease. Consensus was established on the association of psoriasis with other co-morbidities and disease states. Consequently, there is a significant opportunity for specialist and primary care physicians to collaborate with dermatologists in the management of the overall health of psoriasis patients. First, there is an important need for physicians to routinely screen psoriasis patients for the multiple susceptibility risk factors and co-morbidities associated with psoriasis. Second, the design and implementation of lifestyle modification plans including exercise, diet and the limitation of alcohol and tobacco intake, will not only benefit their general medical health but also their psoriasis.

Adaptive immune responses of patients with asthma to the attachment (G) glycoprotein of respiratory synctial virus.

A history of acute bronchiolitis in infancy caused by respiratory syncytial virus is a risk factor for recurrent wheezing in early childhood. Because the attachment (G) protein sensitizes mice for pulmonary eosinophilia and because Th2 cells are central in the pathogenesis of asthma, plasma and peripheral blood mononuclear cells (PBMC) from donors with asthma and from healthy donors were evaluated for anti-G protein responses. A significant trend connecting severity of asthma with anti-G protein IgG1 and IgG2 titers was observed. The correlation between anti-F protein IgG3 titers and asthma severity approached significance. Peptide mapping studies revealed that more positive recall responses (interferon-gamma and interleukin-10 secretion) occurred after PBMC from donors with asthma were stimulated with peptides representing the nonglycosylated domain of G protein. The same peptides elicited more positive recall responses (proliferation and interferon-gamma secretion) in the PBMC of healthy donors. These data suggest that a mechanism may exist whereby adaptive immune responses against G protein contribute to wheezing.

Effective mucosal immunization against respiratory syncytial virus using purified F protein and a genetically detoxified cholera holotoxin, CT-E29H.

We exploited the powerful adjuvant properties of cholera holotoxin (CT) to create a mucosally administered subunit vaccine against respiratory syncytial virus (RSV). A genetically detoxified mutant CT with an E to H substitution at amino acid 29 of the CT-A1 subunit (CT-E29H) was compared to wild type CT for toxicity and potential use as an intranasal (IN) adjuvant for the natural fusion (F) protein of RSV. When compared to CT the results demonstrated that: (1) CT-E29H binding to GM1 ganglioside was equivalent, (2) ADP-ribosylation of agmatine was 11.7%, and (3) toxicity was attenuated in both Y-1 adrenal (1.2%) and patent mouse gut weight assays. IN vaccination with F protein formulated with CT-E29H induced serum anti-CT and anti-F protein antibodies that were comparable to those obtained after vaccination with equivalent doses of CT. Vaccinations containing CT-E29H at doses of 0.1 microg were statistically equivalent to 1.0 microg in enhancing responses to F protein. Antigen-specific mucosal IgA and anti-RSV neutralizing antibodies were detected in nasal washes and sera, respectively, of mice that had received F protein and 0.1 or 1.0 microg of CT-E29H. Anti-F protein IgA was not detected in the nasal washes from mice IN vaccinated with 0.01 microg CT-E29H or IM with F protein adsorbed to AlOH adjuvant. In addition, the formulation of purified F protein and CT-E29H (0.1 and 1.0 microg) facilitated protection of both mouse lung and nose from live RSV challenge. Collectively, the data have important implications for vaccine strategies that use genetically detoxified mutant cholera holotoxins for the mucosal delivery of highly purified RSV antigens.

A novel and effective intranasal immunization strategy for respiratory syncytial virus.

In designing subunit vaccination strategies for respiratory syncytial virus (RSV), immunization by mucosal routes may present a realistic alternative to parenteral administration for inducing protective immune responses. To this end, we have utilized the BALB/c mouse model and an adjuvant formulation containing caprylic/capric glycerides (CCG) and polyoxyethylene-20-sorbitan monolaurate (PS). The intranasal (i.n.) delivery of purified natural F protein (3 microg per vaccine) formulated with CCG-PS resulted in the generation of statistically heightened serum anti-F protein immunoglobulin G (IgG), IgG1, IgG2b, and IgA antibodies. In addition, the presence of locally produced anti-F protein IgA was demonstrated in both vaginal and nasal washes of vaccinated mice. That production of specific serum and mucosal immunoglobulins resulted in functional immune responses was shown in neutralizing antibody assays and protection of mouse lungs against subsequent live virus challenge. Consequently, we propose a novel vaccine formulation composed of purified natural RSV F protein in CCG-PS as a viable intranasal immunogen to stimulate anti-RSV immune responses in humans.

Atypical pulmonary eosinophilia is mediated by a specific amino acid sequence of the attachment (G) protein of respiratory syncytial virus.

We analyzed the immune responses evoked by a series of overlapping peptides to better understand the molecular basis for respiratory syncytial virus (RSV) G protein-induced eosinophilia in BALB/c mice. In vitro stimulation of spleen cells from natural G protein-primed mice showed dominant proliferative and cytokine (interferon [IFN]-gamma and interleukin [IL]-5) responses to a peptide encompassing amino acids 184-198. Mice vaccinated with peptide 184- 198 conjugated to keyhole limpet hemocyanin showed significant pulmonary eosinophilia (39.5%) after challenge with live RSV. In contrast, mice immunized with a peptide (208-222) conjugate associated with induction of IFN-gamma secreting spleen cells did not exhibit pulmonary eosinophilia after challenge. The in vivo depletion of CD4(+) cells abrogated pulmonary eosinophilia in mice vaccinated with the peptide 184-198 conjugate, whereas the depletion of CD8(+) cells had a negligible effect. Therefore, we have identified an association between peptide 184- 198 of natural G protein and the CD4(+) T cell-mediated induction of pulmonary eosinophilia after live RSV challenge. Out of 43 human donors, 6 provided peripheral blood mononuclear cells that showed reactivity to G protein from RSV A2, 3 of which responded to peptide 184- 198. The results have important implications for the development of a vaccine against RSV.

Oxidative and chemical stress mimic insulin by selectively inhibiting the expression of phosphoenolpyruvate carboxykinase in hepatoma cells.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the rate-limiting step in hepatic gluconeogenesis. Glucagon (via the second messenger cAMP), retinoic acid, and glucocorticoids stimulate transcription of the PEPCK gene, whereas insulin and phorbol esters have a dominant inhibitory effect. We now show that oxidative and chemical stress (hydrogen peroxide and sodium meta-arsenite, respectively) also produce a dominant inhibitory effect, both on the endogenous PEPCK gene and on a stably transfected PEPCK-chloramphenicol acetyl transferase (CAT) fusion gene. Wortmannin, an inhibitor of 1-phosphatidylinositol 3-kinase (PI 3-kinase), blocks the inhibition of glucocorticoid and cAMP-induced PEPCK gene transcription by insulin; however, it has no effect on the inhibition elicited by oxidative or chemical stress. Thus, the mechanism(s) used by hydrogen peroxide and sodium meta-arsenite to regulate PEPCK gene expression are PI 3-kinase independent. This suggests that these agents operate by a pathway distinct from that used by insulin or that the pathways converge at a point downstream of PI 3-kinase. The reactivating kinase (RK, also known as p38 mitogen activated protein kinase) is induced by insulin, hydrogen peroxide, or sodium meta-arsenite in hepatoma cells, and these effects are blocked by SB203580, a selective inhibitor of RK. However, SB203580 has no effect on the ability of any of these agents to regulate PEPCK-CAT fusion gene expression. Thus, although RK has a role in the regulation of lymphokine gene expression in monocytes, it is not required for the regulation of PEPCK expression by either insulin or oxidative and chemical stress in hepatoma cells.

The role of the TATA box in the hormonal regulation of phosphoenolpyruvate carboxykinase gene expression.

In this study, we analyzed the role of the TATA box in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression by dexamethasone (DEX), retinoic acid (RA), glucagon (via cAMP) and insulin (INS). The PEPCK TATA box (TATTTAAA) was absolutely required for both basal promoter activity and hormone-mediated transactivation. However, the relative induction of PEPCK gene expression by DEX, RA and cAMP, and its repression by INS, remained unaltered despite the substitution of the PEPCK TATA box with TATA elements from the herpes simplex virus-thymidine kinase gene, gene 33 or a consensus TATA box sequence, TATAAA. The results indicate that the TATA box serves a permissive, but not defining, function in the response of the PEPCK gene to hormones, and that this function can be equally facilitated by heterologous TATA box elements.

Lipid hydroperoxide-induced apoptosis: lack of inhibition by Bcl-2 over-expression.

Increased membrane lipid peroxidation has recently been implicated as being associated with apoptosis. In the present study the addition of 15-hydroperoxyeicosatetraenoic acid (15-HPETE) or 13-hydroperoxydodecadienoic acid (13-HPODE) to A3.01 T cells is shown to induce marked chromatin condensation coincident with DNA fragmentation, indicative of apoptosis. 15-HPETE also evoked an immediate and sustained rise in cytoplasmic calcium which was required for the induction of apoptosis. A3.01 cells transfected with the bcl-2 proto-oncogene were 6- to 8-fold more resistant to apoptotic killing by tumor necrosis factor-alpha, but only 0.4-fold more resistant to 15-HPETE. Thus, Bcl-2 is not capable of protecting cells from undergoing apoptosis following the direct addition of lipid hydroperoxides.

Induction of stearoyl-CoA desaturase 2 gene expression correlates with fatty acid changes in phosphatidylcholine.

Chronic exposure of BW5147 cells to arachidonic acid results in a complete inhibition of stearoyl-CoA desaturase 2 gene expression. Following removal of the fatty acid, gene transcription increased with a kinetic profile similar to the observed increases in both stearoyl-CoA desaturase 2 mRNA and unsaturated fatty acid synthesis. During the time period when stearoyl-CoA desaturase 2 transcription and unsaturated fatty acid synthesis increased (6-12 h), there was only a minimal depletion (approximately 2%) of the total phospholipid arachidonic acid-content. By contrast, during the same period there was a specific depletion of arachidonic acid from phosphatidylcholine, but the proportion of arachidonic acid in other phospholipid species did not change. Thus, changes in the fatty acid content of phosphatidylcholine may be of particular importance in the genetic regulation of endogenous lipid synthesis.

Arachidonic acid down-regulates the insulin-dependent glucose transporter gene (GLUT4) in 3T3-L1 adipocytes by inhibiting transcription and enhancing mRNA turnover.

Chronic exposure of fully differentiated 3T3-L1 adipocytes to 50 microM arachidonic acid (AA) resulted in an inhibition (approximately 91%) in cellular GLUT4 mRNA content after a 48-h exposure, without similarly affecting the mRNA content of the ubiquitous glucose transporter, GLUT1. Subsequent investigations revealed that transcription of the GLUT4 gene was reduced by approximately 50% in response to AA treatment and the half-life of GLUT4 mRNA decreased from 8.0 to 4.6 h. By contrast, AA increased the accumulation of GLUT1 mRNA by 65%, by a mechanism that also involved regulation at both transcriptional and mRNA stability levels. Western blot analysis revealed that AA was specifically reducing the insulin-responsive glucose transporter (GLUT4) in both plasma and intracellular membranes. Subsequently, AA was observed to alter the ability of the GLUT4 transporter to respond to insulin and mediate a significant enhancement of glucose uptake. The results presented in this study indicate that AA can partially mimic the effects of both tumor necrosis factor-alpha and insulin which, when chronically supplied to 3T3-L1 adipocytes, also down-regulate GLUT4 gene expression. Therefore, these data may have relevance to the insulin-resistance associated with non-insulin-dependent diabetes mellitus.

Lipid hydroperoxides induce apoptosis in T cells displaying a HIV-associated glutathione peroxidase deficiency.

8E5 is a chronically human immunodeficiency virus (HIV)-infected human T cell line, which we have previously shown to be extremely susceptible to hydrogen peroxide (H2O2)-induced apoptosis due to a HIV-associated catalase deficiency. Here we report that HIV gene expression additionally renders 8E5 cells 10-fold more sensitive than either uninfected A3.01 cells or HIV-infected but nonexpressing 8E5L cells to killing by 15-hydroperoxyeicosatetraenoic acid (15-HPETE), as well as several other hydroperoxy fatty acids. Whereas the viability of A3.01 and 8E5L cells was relatively unaffected by exposure to 10 microM 15-HPETE, similarly treated 8E5 cells underwent apoptosis, as demonstrated by morphological changes and the presence of fragmented DNA. The unique susceptibility of 8E5 cells was attributable to their inability to convert 15-HPETE to 15-hydroxy-eicosatetraenoic acid (15-HETE) owing to a marked reduction in glutathione peroxidase activity. Since oxidized lipids have been reported to accumulate in oxidatively stressed, HIV-infected individuals, a HIV-associated glutathione peroxidase deficiency may contribute to the depletion of CD4 T cells that occurs in the acquired immune deficiency syndrome (AIDS).

Independent arachidonic acid-mediated gene regulatory pathways in lymphocytes.

In the BW5147 T cell line, we have identified two independent regulatory pathways by which arachidonic acid (20:4) can alter gene expression. The inhibitory effect of 20:4 upon stearoyl-CoA desaturase 2 (SCD2) gene expression was seen to be independent of oxidation of 20:4 by either the lipoxygenase or cyclooxygenase pathways. Moreover, oxidized metabolites of 20:4 (15-HPETE and 15-HETE) failed to diminish SCD2 mRNA accumulation whereas 20:4 itself was effective in completely suppressing SCD2 gene expression. In contrast, the transcriptional induction of the proto-oncogene c-fos was dependent upon the oxidation of 20:4 by the lipoxygenase pathway. By using the protein synthesis inhibitor, cycloheximide, we also show that the 20:4-mediated regulatory effects upon SCD2 or c-fos are completely independent of new protein synthesis. Collectively, the results identify the existence of multiple, independent, intracellular 20:4-mediated regulatory pathways operating simultaneously within this cell type.

Arachidonic acid regulates unsaturated fatty acid synthesis in lymphocytes by inhibiting stearoyl-CoA desaturase gene expression.

This work was based upon the observation that a reduction in the level of serum, provided to murine lymphocytes in culture, augmented endogenous unsaturated fatty acid (UFA) synthesis. Since the phospholipids of BW5147 cells grown in 1% serum were especially deficient in arachidonic acid (20:4), and given the findings of previous workers, we suspected that the availability of exogenous 20:4 in serum might correlate with the squelching of UFA synthesis. Indeed, after a 5 h exposure to 4-28 microM 20:4, the 20:4 content of BW5147 cell phospholipids increased from 1% to 15% of the total fatty acids with a coincident reduction in 18:1 synthesis to approx. 30% of starting values. Subsequent studies were done to define the mechanism by which 20:4 down-regulates 18:1 synthesis. The results indicated that 20:4 inhibited endogenous 18:1 synthesis by reducing stearoyl-CoA desaturase (SCD) enzyme activity. Moreover, as determined by Northern blot analyses, the inhibitory effect of 20:4 on stearoyl-CoA desaturase activity coincided with decreased stearoyl-CoA desaturase mRNA levels. Exposure of BW5147 cells to either 20:4, actinomycin D, or both, resulted in a temporal decay of stearoyl-CoA desaturase mRNAs with half-lives ranging from 4.0 h to 4.4 h. Such a similarity in decay times implied that 20:4 regulates stearoyl-CoA desaturase expression by inhibiting transcription. This was confirmed by nuclear run-on studies in which 20:4 was found to inhibit transcription of nascent stearoyl-CoA desaturase mRNA. Collectively, these findings implicate 20:4 as an important regulator of stearoyl-CoA desaturase gene expression, and hence UFA synthesis, in lymphoid cells.

Stearoyl-CoA desaturase gene expression in lymphocytes.

B lymphocytes from the spleens of normal (BALB/c) and autoimmune (MRL/lpr) strains of mice express the SCD-2 form of stearoyl-CoA desaturase as opposed to the SCD-1 form of the gene which is expressed in liver. However, whereas BALB/c T cells did not express SCD-1 or SCD-2, both BALB/c thymocytes and MRL/lpr T cells expressed SCD-2, suggesting a developmental down-regulation of SCD-2 within the T cell lineage. Northern analyses also revealed the expression of SCD-2 in the T cell lines BW5147, CTLL-2 and HT-2 and in BCL1, a B cell line. SCD-1 expression was not detected in any of the lymphoid cells tested. Finally, we show that SCD-2 gene expression is inhibited by arachidonic acid (20:4). These results demonstrate the complexity of SCD-2 regulation in lymphoid cells.

Molecular basis for the immunosuppressive action of stearic acid on T cells.

Studies were performed to determine the mechanism by which stearic acid (18:0) selectively inhibits T-dependent immune responses in vitro. Incubation of mitogen-activated B and T cells with 18:0 resulted in dissimilar patterns of incorporation of the saturated fatty acid into their membranes. High-performance liquid chromatography (HPLC) analyses of T cells showed an accumulation of disaturated [corrected] 18:0-containing phosphatidylcholine (PC) that replaced normal cellular PC. Less significant quantities of the same PC species were seen to accumulate in B-cell membranes; rather, they increased their proportion of oleic acid (18:1)-containing PC. The different lipid compositions of the lymphocyte cell membranes after exposure to 18:0 were correlated with their plasma membrane potentials. In T cells, the accumulation of disaturated [corrected], 18:0-containing PC coincided with a rapid (within 8 hr) collapse of membrane integrity, as determined by flow cytometry. The collapse of membrane integrity was found to be time and dose dependent. No such depolarization was observed in B cells which, by virtue of their desaturating ability, were able to avoid incorporating large amounts of disaturated [corrected] 18:0-containing phospholipids into their membranes. It is proposed that a lack of stearoyl-CoA desaturase in T cells precludes them from desaturating exogenously derived 18:0, thus leading to increased proportions of 18:0-containing disaturated [corrected] PC in their cell membranes. The increased abundance of this PC species may enhance membrane rigidity to an extent that plasma membrane integrity is significantly impaired, leading to a loss of membrane potential and ultimately cell function and viability.