GILZ regulates Th17 responses and restrains IL-17-mediated skin inflammation.

Patients with inflammatory autoimmune diseases are routinely treated with synthetic glucocorticoids to suppress immunopathology. A crucial outcome of glucocorticoid exposure is induction of glucocorticoid-induced leucine zipper (GILZ), a protein with multiple functions that include inhibition of key immune cell signalling pathways. Here we report that GILZ maintains a threshold for activation of Th17 responses and IL-17-dependent pathology. GILZ expression was deficient in lesional skin of psoriasis patients and was negatively correlated with the pro-inflammatory cytokines IL-23, IL-17A and IL-22, and with STAT3 expression. Deficiency of GILZ in mice resulted in excessive inflammation and pro-inflammatory cytokine expression in the imiquimod model of psoriasis, and dendritic cells lacking GILZ produced greater IL-1, IL-23 and IL-6 in response to imiquimod stimulation in vitro. These cytokines stimulate Th17 cell differentiation, and we found unchallenged GILZ-deficient mice to have spontaneous production of IL-17A and IL-22 in vivo. We also identified a T cell-intrinsic role for GILZ in limiting Th17 cell formation in vitro in response to Th17-promoting cytokines IL-1ß and IL-23. Addition of IL-6 under these conditions suppressed GILZ, allowing T cell proliferation and expression of Th17 genes, whereas exogenous delivery of GILZ using a cell-permeable fusion protein restored regulation of Th17 cell proliferation. Thus, GILZ has a non-redundant function to constrain pathogenic Th17 responses, with clinical implications for psoriasis.

Macrophage migration inhibitory factor is essential for osteoclastogenic mechanisms in vitro and in vivo mouse model of arthritis.

Macrophage migration inhibitory factor (MIF) enhances activation of leukocytes, endothelial cells and fibroblast-like synoviocytes (FLS), thereby contributing to the pathogenesis of rheumatoid arthritis (RA). A MIF promoter polymorphism in RA patients resulted in higher serum MIF concentration and worsens bone erosion; controversially current literature reported an inhibitory role of MIF in osteoclast formation. The controversial suggested that the precise role of MIF and its putative receptor CD74 in osteoclastogenesis and RA bone erosion, mediated by locally formed osteoclasts in response to receptor activator of NF-κB ligand (RANKL), is unclear. We reported that in an in vivo K/BxN serum transfer arthritis, reduced clinical and histological arthritis in MIF(-/-) and CD74(-/-) mice were accompanied by a virtual absence of osteoclasts at the synovium-bone interface and reduced osteoclast-related gene expression. Furthermore, in vitro osteoclast formation and osteoclast-related gene expression were significantly reduced in MIF(-/-) cells via decreasing RANKL-induced phosphorylation of NF-κB-p65 and ERK1/2. This was supported by a similar reduction of osteoclastogenesis observed in CD74(-/-) cells. Furthermore, a MIF blockade reduced RANKL-induced osteoclastogenesis via deregulating RANKL-mediated NF-κB and NFATc1 transcription factor activation. These data indicate that MIF and CD74 facilitate RANKL-induced osteoclastogenesis, and suggest that MIF contributes directly to bone erosion, as well as inflammation, in RA.

GILZ overexpression inhibits endothelial cell adhesive function through regulation of NF-κB and MAPK activity.

Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory protein first identified in T lymphocytes. We recently observed that GILZ is highly expressed in synovial endothelial cells in rheumatoid arthritis. However, the function of GILZ in endothelial cells is unknown. To investigate the actions of GILZ in this cell type, we induced GILZ expression in HUVECs via transient transfection. GILZ overexpression significantly reduced the capacity of TNF-stimulated HUVECs to support leukocyte rolling, adhesion, and transmigration. These effects were associated with decreased expression of E-selectin, ICAM-1, CCL2, CXCL8, and IL-6. Experiments in a human microvascular endothelial cell line demonstrated that TNF-inducible NF-κB activity was significantly inhibited by overexpression of GILZ. Exogenous GILZ inhibited TNF-induced NF-κB p65 DNA binding, although this occurred in the absence of an effect on p65 nuclear translocation, indicating that the mechanism of action of exogenous GILZ in endothelial cells differs from that reported in other cell types. GILZ overexpression also inhibited TNF-induced activation of p38, ERK, and JNK MAPKs, as well as increased expression of the MAPK inhibitory phosphatase, MKP-1. In contrast, silencing endogenous GILZ in glucocorticoid-treated HUVECs did not alter their capacity to support leukocyte interactions. These data demonstrate that exogenous GILZ exerts inhibitory effects on endothelial cell adhesive function via a novel pathway involving modulation of NF-κB p65 DNA binding and MAPK activity. Induction of GILZ expression in endothelial cells may represent a novel therapeutic modality with the potential to inhibit inflammatory leukocyte recruitment.

Divergent effects of endogenous and exogenous glucocorticoid-induced leucine zipper in animal models of inflammation and arthritis.

OBJECTIVE: Glucocorticoid-induced leucine zipper (GILZ) has effects on inflammatory pathways that suggest it to be a key inhibitory regulator of the immune system, and its expression is exquisitely sensitive to induction by glucocorticoids. We undertook this study to test our hypothesis that GILZ deficiency would exacerbate experimental immune-mediated inflammation and impair the effects of glucocorticoids on inflammation and, correspondingly, that exogenous GILZ would inhibit these events. METHODS: GILZ(-/-) mice were generated using the Cre/loxP system, and responses were studied in delayed-type hypersensitivity (DTH), antigen-induced arthritis (AIA), K/BxN serum-transfer arthritis, and lipopolysaccharide (LPS)-induced cytokinemia. Therapeutic expression of GILZ via administration of recombinant adeno-associated virus expressing the GILZ gene (GILZ-rAAV) was compared to the effects of glucocorticoid in collagen-induced arthritis (CIA). RESULTS: Increased T cell proliferation and DTH were observed in GILZ(-/-) mice, but neither AIA nor K/BxN serum-transfer arthritis was affected, and GILZ deficiency did not affect LPS-induced cytokinemia. Deletion of GILZ did not impair the effects of exogenous glucocorticoids on CIA or cytokinemia. In contrast, overexpression of GILZ in joints significantly inhibited CIA, with an effect similar to that of dexamethasone. CONCLUSION: Despite effects on T cell activation, GILZ deficiency had no effect on effector pathways of arthritis and was unexpectedly redundant with effects of glucocorticoids. These findings do not support the hypothesis that GILZ is central to the actions of glucocorticoids, but the efficacy of exogenous GILZ in CIA suggests that further evaluation of GILZ in inflammatory disease is required.

Macrophage migration inhibitory factor and CD74 regulate macrophage chemotactic responses via MAPK and Rho GTPase.

Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment to sites of inflammation. However, whether this stems from a direct effect on leukocyte migration is unknown. Furthermore, the role of the MIF-binding protein CD74 in this response has not been investigated. Therefore, the aim of this study was to examine the contributions of MIF and CD74 to chemokine-induced macrophage recruitment. Intravital microscopy studies demonstrated that CCL2-induced leukocyte adhesion and transmigration were reduced in MIF(-/-) and CD74(-/-) mice. MIF(-/-) and CD74(-/-) macrophages also exhibited reduced chemotaxis in vitro, although CD74(-/-) macrophages showed increased chemokinesis. Reduced CCL2-induced migration was associated with attenuated MAPK phosphorylation, RhoA GTPase activity, and actin polymerization in MIF(-/-) and CD74(-/-) macrophages. Furthermore, in MIF(-/-) macrophages, MAPK phosphatase-1 was expressed at elevated levels, providing a potential mechanism for the reduction in MAPK phosphorylation in MIF-deficient cells. No increase in MAPK phosphatase-1 expression was observed in CD74(-/-) macrophages. In in vivo experiments assessing the link between MIF and CD74, combined administration of MIF and CCL2 increased leukocyte adhesion in both MIF(-/-) and CD74(-/-) mice, showing that CD74 was not required for this MIF-induced response. Additionally, although leukocyte recruitment induced by administration of MIF alone was reduced in CD74(-/-) mice, consistent with a role for CD74 in leukocyte recruitment induced by MIF, MIF-treated CD74(-/-) mice displayed residual leukocyte recruitment. These data demonstrate that MIF and CD74 play previously unappreciated roles in CCL2-induced macrophage adhesion and migration, and they indicate that MIF and CD74 mediate this effect via both common and independent mechanisms.

Macrophage migration inhibitory factor regulates neutrophil chemotactic responses in inflammatory arthritis in mice.

OBJECTIVE: Macrophage migration inhibitory factor (MIF) facilitates multiple aspects of inflammatory arthritis, the pathogenesis of which has been significantly linked to the activity of neutrophils. The effects of MIF on neutrophil recruitment are unknown. This study was undertaken to investigate the contribution of MIF to the regulation of neutrophil chemotactic responses. METHODS: K/BxN serum-transfer arthritis was induced in wild-type (WT), MIF(-/-) , and monocyte chemotactic protein 1 (MCP-1; CCL2)-deficient mice as well as in WT mice treated with monoclonal antibodies to cytokine-induced neutrophil chemoattractant (anti-KC). Leukocyte trafficking in vivo was examined using intravital microscopy, and neutrophil function in vitro was examined using migration chambers and assessment of MAP kinase activation. RESULTS: K/BxN serum-transfer arthritis was markedly attenuated in MIF(-/-) mice, with reductions in the clinical and histologic severity of arthritis and the synovial expression of KC and interleukin-1. Arthritis was also reduced by anti-KC antibody treatment, but not in MCP-1-deficient mice. In vivo, neutrophil recruitment responses to KC were reduced in MIF(-/-) mice. Similarly, MIF(-/-) mouse neutrophils exhibited reduced chemotactic responses to KC in vitro, despite displaying unaltered chemokine receptor expression. Reduced chemotactic responses of MIF(-/-) mouse neutrophils were associated with reduced phosphorylation of p38 and ERK MAP kinases. CONCLUSION: These findings suggest that MIF promotes neutrophil trafficking in inflammatory arthritis via facilitation of chemokine-induced migratory responses and MAP kinase activation. Therapeutic MIF inhibition could limit synovial neutrophil recruitment.

Rare variants in non-coding regulatory regions of the genome that affect gene expression in systemic lupus erythematosus.

Personalized medicine approaches are increasingly sought for diseases with a heritable component. Systemic lupus erythematosus (SLE) is the prototypic autoimmune disease resulting from loss of immunologic tolerance, but the genetic basis of SLE remains incompletely understood. Genome wide association studies (GWAS) identify regions associated with disease, based on common single nucleotide polymorphisms (SNPs) within them, but these SNPs may simply be markers in linkage disequilibrium with other, causative mutations. Here we use an hierarchical screening approach for prediction and testing of true functional variants within regions identified in GWAS; this involved bioinformatic identification of putative regulatory elements within close proximity to SLE SNPs, screening those regions for potentially causative mutations by high resolution melt analysis, and functional validation using reporter assays. Using this approach, we screened 15 SLE associated loci in 143 SLE patients, identifying 7 new variants including 5 SNPs and 2 insertions. Reporter assays revealed that the 5 SNPs were functional, altering enhancer activity. One novel variant was linked to the relatively well characterized rs9888739 SNP at the ITGAM locus, and may explain some of the SLE heritability at this site. Our study demonstrates that non-coding regulatory elements can contain private sequence variants affecting gene expression, which may explain part of the heritability of SLE.

Decorin derived antiangiogenic peptide LRR5 inhibits endothelial cell migration by interfering with VEGF-stimulated NO release.

Excessive angiogenesis plays critical roles in many human diseases including cancer. We have previously shown that human decorin derived 26 amino acids peptide Leucine Rich Repeat 5 (LRR5) inhibits multiple aspects of angiogenesis including vascular endothelial growth factor (VEGF) stimulated migration of endothelial cells (ECs). In this study, we have characterized the molecular mechanism of LRR5 which reveals that its anti-migratory effect on ECs is mediated by inhibiting VEGF-stimulated endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) release. LRR5 carried out this function through signaling pathways that involves PI3 kinase and Akt, but not ERK. This anti-NO release effect is mediated by the C-terminal 13 amino acids of LRR5, correlating with the anti-migratory function of this region.

The first but not the second thrombospondin type 1 repeat of ADAMTS5 functions as an angiogenesis inhibitor.

Angiogenesis is critical for tumour growth and metastasis where factors that regulate this process are potential targets for development of anti-cancer drugs. In this study, we show that the first TSR domain of the extracellular matrix protease ADAMTS5, unlike the second TSR, has anti-angiogenic activities where it inhibits endothelial cell tube formation on Matrigel, reduces cell proliferation and attachment, while promoting cell apoptosis and migration, all in a dose-dependent manner. Furthermore, it influences the architecture of endothelial cells by disrupting actin stress fibres and reducing focal adhesions, likely via suppressing RhoA activation. TSR1 of ADAMTS5 is therefore a novel anti-angiogenic peptide and could serve as a prototype for future development into anti-cancer drugs.

Formyl peptide receptor activation inhibits the expansion of effector T cells and synovial fibroblasts and attenuates joint injury in models of rheumatoid arthritis.

The effects of formyl peptide receptors (FPRs) on effector T cells and inflammation-causing tissue-resident cells are not well known. Here, we explored the effect of FPR activation on efferent T cell responses in models of rheumatoid arthritis (RA) and on the expansion of fibroblast-like synoviocytes (FLS). Compound 43 (Cpd43; FPR1/2 agonist) was administered to mice with collagen-induced arthritis (CIA) or antigen-induced arthritis (AIA) after disease onset. Joint inflammation/damage and immunity were assessed. FLS were cultured with Cpd43 to test its effects on cell apoptosis and proliferation. To explore the effects of endogenous FPR2 ligands on FLS proliferation, FLS FPR2 was blocked or Annexin A1 (AnxA1) expression silenced. Cpd43 reduced arthritis severity in both models. In CIA, Cpd43 decreased CD4 T cell proliferation and survival and increased the production of the protective cytokine, IFNγ, in lymph nodes. In AIA, Cpd43 increased CD4 apoptosis and production of the anti-inflammatory IL-4, while augmenting the proportion of splenic regulatory T cells and their expression of IL-2Rα. In both models, Cpd43 increased CD4 IL-17A production, without affecting humoral immunity. FPR2 inhibitors reversed Cpd43-mediated effects on AIA and T cell immunity. Cpd43 decreased TNF-induced FLS proliferation and augmented FLS apoptosis in association with intracellular FPR2 accumulation, while endogenous AnxA1 and FPR2 reduced FLS proliferation via the ERK and NFκB pathways. Overall, FPR activation inhibits the expansion of arthritogenic effector CD4 T cells and FLS, and reduces joint injury in experimental arthritis. This suggests the therapeutic potential of FPR ligation for the treatment of RA.

Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation.

Macrophage migration inhibitory factor (MIF) exerts multiple effects on immune cells, as well as having functions outside the immune system. MIF can promote inflammation through the induction of other cytokines, including TNF, IL-6, and IL-1 family cytokines. Here, we show that inhibition of MIF regulates the release of IL-1α, IL-1ß, and IL-18, not by affecting transcription or translation of these cytokines, but via activation of the NLRP3 inflammasome. MIF is required for the interaction between NLRP3 and the intermediate filament protein vimentin, which is critical for NLRP3 activation. Further, we demonstrate that MIF interacts with NLRP3, indicating a role for MIF in inflammasome activation independent of its role as a cytokine. These data advance our understanding of how MIF regulates inflammation and identify it as a factor critical for NLRP3 inflammasome activation.

Loss of autophagy enhances MIF/macrophage migration inhibitory factor release by macrophages.

MIF (macrophage migration inhibitory factor [glycosylation-inhibiting factor]) is a pro-inflammatory cytokine expressed in multiple cells types, including macrophages. MIF plays a pathogenic role in a number of inflammatory diseases and has been linked to tumor progression in some cancers. Previous work has demonstrated that loss of autophagy in macrophages enhances secretion of IL1 family cytokines. Here, we demonstrate that loss of autophagy, by pharmacological inhibition or siRNA silencing of Atg5, enhances MIF secretion by monocytes and macrophages. We further demonstrate that this is dependent on mitochondrial reactive oxygen species (ROS). Induction of autophagy with MTOR inhibitors had no effect on MIF secretion, but amino acid starvation increased secretion. This was unaffected by Atg5 siRNA but was again dependent on mitochondrial ROS. Our data demonstrate that autophagic regulation of mitochondrial ROS plays a pivotal role in the regulation of inflammatory cytokine secretion in macrophages, with potential implications for the pathogenesis of inflammatory diseases and cancers.

[Archaeal diversity of Zabuye Lake in Tibet analyzed by culture-independent approach].

With culture-independent approach, microbial DNA was directly extracted from samples of Zabuye saline soda lake. Using the microbial DNA as template, archaeal 16S rDNAs were amplified by PCR. Amplified products were cloned and sequenced. 60 different cloned partial sequences, most of which were related to haloalkaliphilic archaeon, were acquired. In the phylogenetic tree, some clones of Zabuye lake belonged to Genus Natronobacterium, Natrinema, Natronococcus, Natronorubrum, Natronomonas, Halorubrum, Haloterrigena, Halorhabdus in Family Halobacteriaceae. Other clones represent some novel groups. All of them show prolific archaeal diversity of Zabuye lake.

Macrophage migration inhibitory factor inhibits the antiinflammatory effects of glucocorticoids via glucocorticoid-induced leucine zipper.

OBJECTIVE: Glucocorticoids remain a mainstay in the treatment of rheumatoid arthritis (RA). Dose-dependent adverse effects highlight the need for therapies that regulate glucocorticoid sensitivity to enable dosage reduction. Macrophage migration inhibitory factor (MIF) is a proinflammatory protein that has been implicated in the pathogenesis of RA; it impairs glucocorticoid sensitivity via MAPK phosphatase 1 (MKP-1) inhibition. The intracellular protein glucocorticoid-induced leucine zipper (GILZ) mimics the effects of glucocorticoids in models of RA, but whether it represents a target for the modulation of glucocorticoid sensitivity remains unknown. We undertook this study to investigate whether GILZ is involved in the regulation of glucocorticoid sensitivity by MIF. METHODS: GILZ expression was studied in the presence and absence of MIF, and the role of GILZ in the MIF-dependent regulation of the glucocorticoid sensitivity mediator MKP-1 was studied at the level of expression and function. RESULTS: GILZ expression was significantly inhibited by endogenous MIF, both basally and during responses to glucocorticoid treatment. The effects of MIF on GILZ were dependent on the expression and Akt-induced nuclear translocation of the transcription factor FoxO3A. GILZ was shown to regulate the expression of MKP-1 and consequent MAPK phosphorylation and cytokine release. CONCLUSION: MIF exerts its effects on MKP-1 expression and MAPK activity through inhibitory effects on GILZ. These findings suggest a previously unsuspected interaction between MIF and GILZ and identify GILZ as a potential target for the therapeutic regulation of glucocorticoid sensitivity.

Targeting the side effects of steroid therapy in autoimmune diseases: the role of GILZ.

Glucocorticoids are among the most widely prescribed drugs used for human diseases, and are especially commonly used in autoimmune diseases. Their use reflects their rapid and broad spectrum actions on immune cells, which in turn reflect the multiple mechanisms of cell activation upon which glucocorticoids impact. While inhibition of pro-inflammatory gene expression is a major effect of glucocorticoids, they also induce the expression of numerous molecules that exert regulatory influences on the immune system. Among these is glucocorticoid induced leucine zipper (GILZ), a recently described, highly glucocorticoid-induced, transcriptional regulatory protein which has important inhibitory effects on immune and inflammatory cell functions. In this review, we summarize knowledge of the actions of glucocorticoids relevant to autoimmune disease, and focus on the potential for greater understanding of the function of GILZ to facilitate discovery of new therapeutic options for these diseases.

Halalkalicoccus tibetensis gen. nov., sp. nov., representing a novel genus of haloalkaliphilic archaea.

A haloalkaliphilic archaeon (strain DS12T) isolated from Lake Zabuye, the Tibetan Plateau, China, was characterized to elucidate its taxonomy. The strain was aerobic, chemo-organotrophic, and grew optimally at 40 degrees C, pH 9.5-10.0 and 3.4 M NaCl. Cells of strain DS12T were non-motile cocci and stained Gram-variable. The major polar lipids of strain DS12T were diphytanyl and phytanyl-sesterterpanyl diether derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. No glycolipids were detected. Phylogenetic analysis revealed that the strain formed a distinct lineage within the family Halobacteriaceae. The low 16S rRNA gene sequence similarity values to its closest relatives (91.5-92.5 %) and its signature bases both suggest that the strain has no close affinity with any members of the family Halobacteriaceae with validly published names. Therefore, it is proposed that strain DS12T (=AS 1.3240T=JCM 11890T) represents the type strain of a novel species in a new genus, Halalkalicoccus tibetensis gen. nov., sp. nov.

Peptides derived from human decorin leucine-rich repeat 5 inhibit angiogenesis.

Excessive angiogenesis is involved in many human diseases, and inhibiting angiogenesis is an important area of drug development. There have been conflicting reports as to whether decorin could function as an angiogenic inhibitor when used as an extracellular soluble factor. In this study, we demonstrated that not only purified decorin but also the 26-residue leucine-rich repeat 5 (LRR5) of decorin core protein functions as angiogenesis inhibitor by inhibiting both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-induced angiogenesis. Peptide LRR5 inhibited angiogenesis through multiple mechanisms, including inhibiting VEGF-stimulated endothelial cell (EC) migration, tube formation on Matrigel, cell attachment to fibronectin, as well as induction of EC apoptosis without significantly affecting their proliferation. We further demonstrated that different subregions of LRR5 inhibited different aspects of angiogenesis, with the middle region (LRR5M, 12 residues) inhibiting endothelial cell tube formation up to 1000 times more potently than LRR5. Although the C-terminal region (LRR5C) potently inhibited VEGF-stimulated endothelial cell migration, the N-terminal region (LRR5N) is as active as LRR5 in inhibiting endothelial cell attachment to fibronectin. Although both LRR5M and LRR5N induced EC apoptosis dose-dependently similar to LRR5 through a caspase-dependent pathway, LRR5C has no such function. We further showed that the inhibition of tube formation by LRR5 and LRR5M is linked with their ability to suppress VEGF-induced focal adhesion kinase phosphorylation and the assembly of focal adhesions and actin stress fibers in ECs, but not their ability to interfere with endothelial cell attachment to the matrix. Circular dichroism studies revealed that LRR5 undergoes an inter-conversion between 3(10) helix and beta-sheet structure in solution, a characteristic potentially important for its anti-angiogenic activity. Peptide LRR5 and its derivatives are therefore novel angiogenesis inhibitors that may serve as prototypes for further development into anti-angiogenic drugs.

Halorubrum tibetense sp. nov., a novel haloalkaliphilic archaeon from Lake Zabuye in Tibet, China.

A novel haloalkaliphilic archaeon, strain 8W8T, was isolated from Lake Zabuye, on the Tibetan Plateau, China. On the basis of 16S rRNA gene sequence analysis, strain 8W8T was shown to belong to the genus Halorubrum and was related to Halorubrum vacuolatum (96.7% sequence similarity), Halorubrum saccharovorum (96.0%), Halorubrum lacusprofundi (95.4%) and Halorubrum sodomense (95.3%). The phylogenetic distance from any species within the other genera of Halobacteriales was lower than 90%. The major polar lipids of strain 8W8T were C20C20 and C20C25 derivatives of phosphatidylglycerol phosphate and phosphatidylglycerol phosphate methyl ester. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 8W8T from the eight Halorubrum species with validly published names. Therefore, strain 8W8T represents a novel species, for which the name Halorubrum tibetense sp. nov. is proposed, with the type strain 8W8T (=AS 1.3239T=JCM 11889T).