Correlation of the levels of DNA-binding inhibitor Id3 and regulatory T cells with SLE disease severity.

E proteins, a subset of basic helix-loop-helix (bHLH) proteins, are transcription activators and their functions are inhibited by DNA-binding inhibitor (Id) 1-4. Studies have shown that Treg levels are decreased in Id3 knockout mice. Mice over-expressing Id1 in CD4 T cells possessed a greater number of regulatory T cells (Treg) and exhibited attenuated experimental autoimmune encephalomyelitis (EAE). The significance of Id proteins in human systemic lupus erythematosus (SLE) remains unclear. In this study, we systematically analyzed Id transcription in naïve, memory CD4 cells and regulatory T cells in peripheral blood mononuclear cells (PBMCs) in patients with active or inactive SLE. In parallel, Treg subsets in PBMCs were analyzed using different strategies. Id expression levels were correlated with Treg numbers as well as clinical indicators. We found that Id genes expressed in human peripheral CD4 cells were mainly Id2 and Id3. Id3 levels were significantly elevated in CD4+CD25hi T cells of patients with active SLE. Likewise, Id3 levels were positively correlated with increased CD4+FoxP3+ and CD4+Helios+FoxP3+ Treg cells in these patients. Id3 levels were found to be positively correlated with erythrocyte sedimentation rate (ESR), lupus anticoagulant (LAC), ribosomal antibody and SLE Disease Activity Index (SLEDAI) in patients with active SLE. Mice overexpressing Id1 in CD4+ T cells possessed significantly higher Treg levels in spleen and lower autoantibody concentrations in serum. Our results suggest that during the pathogenesis of SLE, up-regulation of Id3 can promote Treg differentiation to play an inhibitory effect on autoimmune responses.

Positional identification of RT1-B (HLA-DQ) as susceptibility locus for autoimmune arthritis.

Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA.

Secondary metabolites in plants: transport and self-tolerance mechanisms.

Plants produce a host of secondary metabolites with a wide range of biological activities, including potential toxicity to eukaryotic cells. Plants generally manage these compounds by transport to the apoplast or specific organelles such as the vacuole, or other self-tolerance mechanisms. For efficient production of such bioactive compounds in plants or microbes, transport and self-tolerance mechanisms should function cooperatively with the corresponding biosynthetic enzymes. Intensive studies have identified and characterized the proteins responsible for transport and self-tolerance. In particular, many transporters have been isolated and their physiological functions have been proposed. This review describes recent progress in studies of transport and self-tolerance and provides an updated inventory of transporters according to their substrates. Application of such knowledge to synthetic biology might enable efficient production of valuable secondary metabolites in the future.

Vγ9Vδ2 TCR-activation by phosphorylated antigens requires butyrophilin 3 A1 (BTN3A1) and additional genes on human chromosome 6.

Pyrophosphorylated metabolites of isoprenoid-biosynthesis (phosphoantigens, PAgs) activate Vγ9Vδ2 T cells during infections and trigger antitumor activity. This activation depends on expression of butyrophilin 3 A1 (BTN3A1) by antigen-presenting cells. This report defines the minimal genetic requirements for activation of Vγ9Vδ2 T cells by PAgs and mAb 20.1. We compared PAg-presentation by BTN3A1-transduced CHO hamster cells with that of CHO cells containing the complete human chromosome 6 (Chr6). BTN3A1 expression alone was sufficient for activation of Vγ9Vδ2 T-cell receptor transductants by mAb 20.1., while activation by PAgs also required the presence of Chr6. We take this finding as evidence that gene(s) on Chr6 in addition to BTN3A1 are mandatory for PAg-mediated activation of Vγ9Vδ2 T cells. This observation is important for the design of animal models for PAg-mediated immune responses and provokes speculations about the analogy between genes controlling PAg presentation and MHC-localized genes controlling peptide-antigen presentation.

Harnessing the power of Vδ2 cells in cancer immunotherapy.

γδ T cells are a subset of T lymphocytes that have been implicated in immunosurveillance against infections and tumours. In the peripheral blood of humans the γδ T cell pool is made up predominantly of Vδ2 cells, which can detect both foreign and self-metabolites of the isoprenoid biosynthesis pathway. This unique axis of antigen recognition enables Vδ2 cells to respond to a range of pathogenic infections as well as perturbations in endogenous isoprenoid biosynthesis that can occur during cell stress and malignant transformation. There has been growing interest in Vδ2 cells as a potential avenue for cancer immunotherapy, and a number of strategies have been utilized in an attempt to boost the anti-tumour response of Vδ2 cells in patients. In this review we discuss critically the evidence that Vδ2 cells contribute to the cytotoxic response against tumours and evaluate current immunotherapeutic approaches that target these cells in cancer patients, with specific focus on their shortcomings and how they may be improved.

Usage tests of oak moss absolutes containing high and low levels of atranol and chloroatranol.

Atranol and chloroatranol are strong contact allergens in oak moss absolute, a lichen extract used in perfumery. Fifteen subjects with contact allergy to oak moss absolute underwent a repeated open application test (ROAT) using solutions of an untreated oak moss absolute (sample A) and an oak moss absolute with reduced content of atranol and chloroatranol (sample B). All subjects were in addition patch-tested with serial dilutions of samples A and B. Statistically significantly more subjects reacted to sample A than to sample B in the patch tests. No corresponding difference was observed in the ROAT, though there was a significant difference in the time required to elicit a positive reaction. Still, the ROAT indicates that the use of a cosmetic product containing oak moss absolute with reduced levels of atranol and chloroatranol is capable of eliciting an allergic reaction in previously sensitised individuals.

Limonene hydroperoxide analogues show specific patch test reactions.

BACKGROUND: The fragrance terpene R-limonene is a very weak sensitizer, but forms allergenic oxidation products upon contact with air. The primary oxidation products of oxidized limonene, the hydroperoxides, have an important impact on the sensitizing potency of the oxidation mixture. One analogue, limonene-1-hydroperoxide, was experimentally shown to be a significantly more potent sensitizer than limonene-2-hydroperoxide in the local lymph node assay with non-pooled lymph nodes. OBJECTIVES: To investigate the pattern of reactivity among consecutive dermatitis patients to two structurally closely related limonene hydroperoxides, limonene-1-hydroperoxide and limonene-2-hydroperoxide. METHODS: Limonene-1-hydroperoxide, limonene-2-hydroperoxide, at 0.5% in petrolatum, and oxidized limonene 3.0% pet. were tested in 763 consecutive dermatitis patients. RESULTS: Of the tested materials, limonene-1-hydroperoxide gave most reactions, with 2.4% of the patients showing positive patch test reactions. Limonene-2-hydroperoxide and oxidized R-limonene gave 1.7% and 1.2% positive patch test reactions, respectively. Concomitant positive patch test reactions to other fragrance markers in the baseline series were frequently noted. CONCLUSIONS: The results are in accordance with the experimental studies, as limonene-1-hydroperoxide gave more positive patch test reactions in the tested patients than limonene-2-hydroperoxide. Furthermore, the results support the specificity of the allergenic activity of the limonene hydroperoxide analogues and the importance of oxidized limonene as a cause of contact allergy.

An immune response study of oakmoss absolute and its constituents atranol and chloroatranol.

BACKGROUND: Atranol and chloroatranol are the main allergens of oakmoss absolute. However, the immune responses induced by these substances are poorly characterized. OBJECTIVES: To characterize immune responses induced by atranol, chloroatranol and oakmoss absolute in mice. METHODS: Mice were sensitized and challenged with various concentrations of atranol, chloroatranol, and oakmoss absolute. The immune responses were analysed as B cell infiltration, T cell proliferation in the draining lymph nodes, and expression of interleukin (IL)-18, IL-1ß and tumour necrosis factor-α in skin. The cytotoxicity of atranol and chloroatranol against keratinocytes was determined. RESULTS: Sensitization experiments showed that atranol, chloroatranol and oakmoss induced sensitization when applied in high concentrations. Challenge experiments showed that even low concentrations of atranol and chloroatranol induced sensitization. In parallel, atranol and chloroatranol elicited challenge reactions following sensitization with oakmoss. The magnitude of the immune response to the three allergens increased in the following order: atranol, chloroatranol, and oakmoss. The expression of proinflammatory cytokines was induced by chloroatranol and oakmoss, but not by atranol. Chloroatranol was found to be more cytotoxic than atranol against keratinocytes. CONCLUSIONS: Atranol and chloroatranol can elicit both sensitization and challenge reactions, but the mixture of allergens in oakmoss absolute is more potent than atranol and chloroatranol alone.

Cross-reactivity between citral and geraniol - can it be attributed to oxidized geraniol?

BACKGROUND: The fragrance compound geraniol is susceptible to autoxidation when in contact with air, and to cutaneous metabolism. In both processes, the isomeric aldehydes geranial and neral are formed. Citral consists of geranial and neral. Among patients with positive reactions to citral, we have previously detected concomitant reactions to geraniol in 85% of cases and to oxidized geraniol in 73% of cases. OBJECTIVE: To study the pattern of concomitant reactions to geraniol and citral and its isomers geranial and neral, and to determine whether these isomers are important sensitizers in contact allergy to geraniol and oxidized geraniol. PATIENTS AND METHODS: The irritancy of geranial and citral was studied. Six hundred and fifty-five patients were patch tested with geranial, neral and citral at 3.5% pet., pure geraniol at 6.0% and 11.0% pet., and oxidized geraniol at 6.0% pet. RESULTS: Twenty-six per cent of citral-positive patients reacted to oxidized geraniol, and 10.5% reacted to pure geraniol. Citral and/or its isomers gave positive reactions in 25% of the patients who reacted to pure geraniol. CONCLUSIONS: There is little cross-reactivity between pure geraniol and citral; however, concomitant reactions to citral and oxidized geraniol were common, owing to geranial. Geranial was also the main sensitizer in the mixture citral.

Identification of two new arthritis severity loci that regulate levels of autoantibodies, interleukin-1ß, and joint damage in pristane- and collagen-induced arthritis.

OBJECTIVE: Cia3 is a locus on rat chromosome 4 that regulates severity and joint damage in collagen- and pristane-induced arthritis (CIA and PIA). This study was undertaken to refine the Cia3 gene-containing interval toward gene identification and obtain insights into its mode of action. METHODS: Five DA.F344(Cia3) subcongenic rat strains were generated and studied using the PIA and CIA models. Levels of antibodies against type II collagen (both allo- and autoantibodies) were measured. Joints and synovial tissue were collected 32 days after the induction of PIA (chronic stage) for histologic and quantitative polymerase chain reaction analysis of interleukin-1ß (IL-1ß) and matrix metalloproteinase (MMP) levels. RESULTS: Three subcongenic strains sharing the centromeric Cia3d interval were protected and 2 subcongenic strains sharing the telomeric Cia3g interval, which did not overlap with Cia3d, were also protected, developing significantly less severe CIA and PIA. Normal joint architecture was preserved in DA.F344(Cia3) and DA.F344(Cia3d) congenic rats with PIA, while DA rats had pronounced synovial hyperplasia, angiogenesis, inflammatory infiltration, and bone or cartilage erosions. The DA.F344(Cia3d) and DA.F344(Cia3g) strains had significantly lower synovial levels of IL-1ß (5-fold and nearly 2-fold, respectively [the latter not reaching statistical significance]), MMP-1 (expressed predominantly in DA rats), MMP-3 (79-fold and 8-fold, respectively), and MMP-14 (21-fold and 1.4-fold, respectively) and reduced levels of pathogenic autoantibodies against type II collagen, compared with DA rats. CONCLUSION: We have identified 2 new arthritis severity and articular damage loci within Cia3. These loci regulate pathogenic processes in 2 different models of rheumatoid arthritis, and the identification of these genes has the potential to generate new targets for therapies aimed at reducing disease severity and articular damage, and may additionally have prognostic value.

An international multicentre study on the allergenic activity of air-oxidized R-limonene.

BACKGROUND: Limonene is a common fragrance terpene that, in its pure form, is not allergenic or is a very weak allergen. However, limonene autoxidizes on air exposure, and the oxidation products can cause contact allergy. Oxidized R-limonene has previously been patch tested in multicentre studies, giving 2-3% positive patch test reactions in consecutive patients. OBJECTIVES: To investigate whether oxidized R-limonene 3.0% in petrolatum, with a stable concentration of the main haptens, limonene hydroperoxides (Lim-OOHs), could be a useful tool for the detection of contact allergy in an international setting. METHODS: Oxidized R-limonene 3.0% (Lim-OOHs 0.33%) pet. was tested in 2900 consecutive dermatitis patients in Denmark, the United Kingdom, Singapore, Spain, Sweden, and Australia. RESULTS: Overall, 5.2% (range 2.3-12.1%) of the patients showed a positive patch test reaction to oxidized R-limonene. Doubtful reactions were found in 7.0% of the patients (range 0-24%). Few irritant reactions were seen. CONCLUSIONS: Oxidized R-limonene at 3.0% pet. with a specified content of Lim-OOHs 0.33% is a standardized and useful tool for the detection of contact allergy in dermatitis patients. Many patients showing positive patch test reactions to oxidized R-limonene would not be informed of their fragrance allergy if this specific test had not been performed.

Finding the optimal patch test material and test concentration to detect contact allergy to geraniol.

BACKGROUND: Geraniol is a commonly used fragrance terpene, and is tested in the baseline series in fragrance mix I. Geraniol is a pro-hapten and a pre-hapten, and sensitizers are formed in the autoxidation and skin metabolism of geraniol. Previous patch testing with air-exposed (oxidized) geraniol has suggested that oxidized geraniol could be a better marker for contact allergy to geraniol than pure geraniol. OBJECTIVES: To find the optimal patch test substance and concentration for detecting contact allergy to geraniol. PATIENTS AND METHODS: Six hundred and fifty-five patients were patch tested with pure and oxidized geraniol at 4.0%, 6.0% and 11.0% in petrolatum. Before patch testing, the irritant properties of pure and oxidized geraniol were studied in 27 patients at 2.5%, 5.0%, 10.0% and 20.0% pet. RESULTS: Pure geraniol detected positive reactions in 0.15-1.1% of the patients, and oxidized geraniol detected positive reactions in 0.92-4.6% of the patients. Reactions to pure geraniol in patients not reacting to oxidized geraniol indicated metabolic activation of geraniol. Neither pure nor oxidized geraniol gave significant irritant reactions. CONCLUSIONS: Increasing the test concentrations of pure and oxidized geraniol enables the detection of more cases of contact allergy. Oxidized geraniol detects more patients than pure geraniol, but patch testing with only oxidized geraniol does not detect all cases of contact allergy to geraniol.

Induction of autoimmunity by pristane and other naturally occurring hydrocarbons.

Tetramethylpentadecane (TMPD, or commonly known as pristane)-induced lupus is a murine model of systemic lupus erythematosus (SLE). Renal disease and autoantibody production strictly depend on signaling through the interferon (IFN)-I receptor. The major source of IFN-I is immature monocytes bearing high levels of the surface marker Ly6C. Interferon production is mediated exclusively by signaling through TLR7 and the adapter protein MyD88. It is likely that endogenous TLR7 ligands such as components of small nuclear ribonucleoprotein complexes are involved in triggering disease. Lupus autoantibodies are produced in ectopic lymphoid tissue developing in response to TMPD. This model is well suited for examining links between dysregulated IFN-I production and the pathogenesis of human SLE, which like TMPD-lupus, is associated with high levels of IFN-I.

Defect in mevalonate pathway induces pyroptosis in Raw 264.7 murine monocytes.

The inhibition of mevalonate pathway by the aminobisphosphonate alendronate (ALD) has been previously associated with an augmented lipopolysaccharide-induced interleukin-1beta (IL-1ß) secretion in monocytes, as demonstrated in an auto-inflammatory disease known as mevalonate kinase deficiency (MKD). In this study we investigated the effect of ALD + LPS on monocyte cell line (Raw 264.7) death. ALD strongly augmented LPS-induced programmed cell death (PCD) as well as IL-1ß secretion in Raw murine monocytes, whereas necrosis was rather unaffected. ALD + LPS induced caspase-3 activation. Inhibition of IL-1ß stimulation partially restored cell viability. These findings suggest that the inhibition of mevalonate pathway, together with a bacterial stimulus, induce a PCD partly sustained by the caspase-3-related apoptosis and partly by caspase-1-associated pyroptosis. The involvement of pyroptosis is a novel hit in our cell model and opens discussions about its role in inflammatory cells with chemical or genetic inhibition of mevalonate pathway.

Phenylacetonitrile from the giant knotweed, Fallopia sachalinensis, infested by the Japanese beetle, Popillia japonica, is induced by exogenous methyl jasmonate.

Phenylacetonitrile, (E)-ß-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene and (E,E)-α-farnesene were identified as Japanese beetle, Popillia japonica, feeding-induced volatiles from the leaves of the giant knotweed, Fallopia sachalinensis, but not by mechanical damage. Volatile emission was also induced by treatment with a cellular signaling molecule, methyl jasmonate. These results suggest that volatiles will be synthesized de novo by a biotic elicitor from P. japonica oral secretion.

Structural influence on radical formation and sensitizing capacity of alkylic limonene hydroperoxide analogues in allergic contact dermatitis.

Hydroperoxides are known to be strong contact allergens and a common cause of contact allergy. They are easily formed by the autoxidation of, for example, fragrance terpenes, compounds that are common in perfumes, cosmetics, and household products. A requirement of the immunological mechanisms of contact allergy is the formation of an immunogenic hapten-protein complex. For hydroperoxides, a radical mechanism is postulated for this formation. In our previous investigations of allylic limonene hydroperoxides, we found that the formation of carbon- and oxygen-centered radicals, as well as the sensitizing capacity, is influenced by the structure of the hydroperoxides. The aim of the present work was to further investigate the connection between structure, radical formation, and sensitizing capacity by studying alkylic analogues of the previously investigated allylic limonene hydroperoxides. The radical formation was studied in radical-trapping experiments employing 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride as an initiator and 1,1,3,3-tetramethylisoindolin-2-yloxyl as a radical trapper. We found that the investigated hydroperoxides initially form carbon- and oxygen-centered radicals that subsequently form alcohols and ketones. Trapped carbon-centered radicals and nonradical products were isolated and identified. Small changes in structure, like the omission of the endocyclic double bond or the addition of a methyl group, resulted in large differences in radical formation. The results indicate that alkoxyl radicals seem to be more important than carbon-centered radicals in the immunogenic complex formation. The sensitizing capacities were studied in the murine local lymph node assay (LLNA), and all hydroperoxides tested were found to be potent sensitizers. For two of the hydroperoxides investigated, the recently suggested thiol-ene reaction is a possible mechanism for the formation of immunogenic complexes. For the third investigated, fully saturated, hydroperoxide, the thiol-ene mechanism is not possible for immunogenic complex formation. This strongly indicates that several radical reaction pathways for immunogenic complex formation of limonene hydroperoxides are active in parallel.

Protein Prenylation Drives Discrete Signaling Programs for the Differentiation and Maintenance of Effector Treg Cells.

Effector regulatory T (eTreg) cells are essential for immune tolerance and depend upon T cell receptor (TCR) signals for generation. The immunometabolic signaling mechanisms that promote the differentiation and maintenance of eTreg cells remain unclear. Here, we show that isoprenoid-dependent posttranslational lipid modifications dictate eTreg cell accumulation and function by intersecting with TCR-induced intracellular signaling. We find that isoprenoids are essential for activated Treg cell suppressive activity, and Treg cell-specific deletion of the respective farnesylation- and geranylgeranylation-promoting enzymes Fntb or Pggt1b leads to the development of fatal autoimmunity, associated with reduced eTreg cell accumulation. Mechanistically, Fntb promotes eTreg cell maintenance by regulating mTORC1 activity and ICOS expression. In contrast, Pggt1b acts as a rheostat of TCR-dependent transcriptional programming and Rac-mediated signaling for establishment of eTreg cell differentiation and immune tolerance. Therefore, our results identify bidirectional metabolic signaling, specifically between immunoreceptor signaling and metabolism-mediated posttranslational lipid modifications, for the differentiation and maintenance of eTreg cells.

Gymnosperm glandular trichomes: expanded dimensions of the conifer terpenoid defense system.

Glandular trichomes (GTs) are defensive structures that produce and accumulate specialized metabolites and protect plants against herbivores, pathogens, and abiotic stress. GTs have been extensively studied in angiosperms for their roles in defense and biosynthesis of high-value metabolites. In contrast, trichomes of gymnosperms have been described in fossilized samples, but have not been studied in living plants. Here, we describe the characterization of GTs on young stems of a hybrid white spruce. Metabolite and histological analysis of spruce GTs support a glandular function with accumulation of a diverse array of mono-, sesqui- and diterpenes including diterpene methylesters. Methylated diterpenes have previously been associated with insect resistance in white spruce. Headspeace analysis of spruce GTs showed a profile of volatiles dominated by monoterpenes and a highly diverse array of sesquiterpenes. Spruce GTs appear early during shoot growth, prior to the development of a lignified bark and prior to accumulation of terpenes in needles. Spruce GTs may provide an early, terpene-based chemical defense system at a developmental stage when young shoots are particularly vulnerable to foliage and shoot feeding insects, and before the resin duct system characteristic of conifers has fully developed.