Substitution of pseudokinase domain residue Val-617 by large non-polar amino acids causes activation of JAK2

Explaining the uniqueness of the acquired somatic JAK2 V617F mutation, which is present in more than 95% of polycythemia vera patients, has been a challenge. The V617F mutation in the pseudokinase domain of JAK2 renders the unmutated kinase domain constitutively active. We have performed random mutagenesis at position 617 of JAK2 and tested each of the 20 possible amino acids for ability to induce constitutive signaling in Ba/F3 cells expressing the erythropoietin receptor. Four JAK2 mutants, V617W, V617M, V617I, and V617L, were able to induce cytokine independence and constitutive downstream signaling. Only V617W induced a level of constitutive activation comparable with V617F. Also, only V617W stabilized tyrosine-phosphorylated suppressor of cytokine signaling 3 ( SOCS3), a mechanism by which JAK2 V617F overcomes inhibition by SOCS3. The V617W mutant induced a myeloproliferative disease in mice, mainly characterized by erythrocytosis and megakaryocytic proliferation. Although JAK2 V617W would predictably be pathogenic in humans, the substitution of the Val codon, GTC, by TTG, the codon for Trp, would require three base pair changes, and thus it is unlikely to occur. We discuss how the predicted conformations of the activated JAK2 mutants can lead to better screening assays for novel small molecule inhibitors.

Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07.

PURPOSE. A spontaneously arising retinal pigment epithelial (RPE) cell line (B6-RPE07) was cloned from a primary culture of mouse RPE cells and maintained in culture for more than 18 months. Morphologic and functional properties of this cell line have been characterized.

METHODS. The morphology of the B6-RPE07 cells was examined by phase-contrast light microscopy, electron microscopy, and confocal microscopy. Barrier properties were measured by the flux of fluorescence from the apical to the basolateral compartment of culture chambers. The abilities of the cells to bind/phagocytose photoreceptor outer segments (POS) were determined by confocal microscopy, electron microscopy, and flow cytometry. Cytokine/chemokine secretion was measured by cytometric bead array. The expression of visual cycle proteins was determined by RT-PCR and Western blotting.

RESULTS. In standard culture conditions, B6-RPE07 cells display cobblestone morphology. When cultured on three-dimensional (3D) collagen gel–coated membranes, B6-RPE07 cells exhibit a monolayer epithelial polarization with apical surface microvilli. Immunohistochemistry of B6-RPE07 cultures revealed a high expression of pan-cytokeratin. B6-RPE07 cells also expressed the retinal pigment epithelium-specific marker CRALBP, but not RPE65. Cell junction proteins ZO-1 and ß-catenin, but not claudin-1/3 or occludin-1, were observed in B6-RPE07 cells. B6-RPE07 cells are able to bind, phagocytose, and digest POS. Finally, B6-RPE07 cells produce high levels of IL-6 and CCL2.

CONCLUSIONS. This is the first report of a mouse RPE cell line with morphology, phenotype, and function similar to those of in vivo mouse RPE cells. This cell line will be a valuable resource for future RPE studies, in particular for in vivo gene modification and transplantation studies.

The human prion protein residue 129 polymorphism lies within a cluster of epitopes for T cell recognition.

T cell immune responses to central nervous system-derived and other self-antigens are commonly described in both healthy and autoimmune individuals. However, in the case of the human prion protein (PrP), it has been argued that immunologic tolerance is uncommonly robust. Although development of an effective vaccine for prion disease requires breaking of tolerance to PrP, the extent of immune tolerance to PrP and the identity of immunodominant regions of the protein have not previously been determined in humans. We analyzed PrP T cell epitopes both by using a predictive algorithm and by measuring functional immune responses from healthy donors. Interestingly, clusters of epitopes were focused around the area of the polymorphic residue 129, previously identified as an indicator of susceptibility to prion disease, and in the C-terminal region. Moreover, responses were seen to PrP peptide 121-134 containing methionine at position 129, whereas PrP 121-134 [129V] was not immunogenic. The residue 129 polymorphism was also associated with distinct patterns of cytokine response: PrP 128-141 [129M] inducing IL-4 and IL-6 production, which was not seen in response to PrP 128-141 [129V]. Our data suggest that the immunogenic regions of human PrP lie between residue 107 and the C-terminus and that, like with many other central nervous system antigens, healthy individuals carry responses to PrP within the T cell repertoire and yet do not experience deleterious autoimmune reactions.

A new polycythaemia vera-associated SOCS3 SH2 mutant (SOCS3(F136L)) cannot regulate erythropoietin responses

Recently several different JAK2 exon12 mutations have been identified in V617F negative polycythaemia vera (PV) or idiopathic erythrocytosis (IE) patients. The patients present with erythrocytosis, ligand-independent cell growth and low serum erythropoietin (EPO) levels. Within this group, a deletion of amino acids 542-543 (N542-E543del) of JAK2 is most prevalent. We have previously shown that in the presence of JAK2(V617F), suppressor of cytokine signalling 3 (SOCS3) is unable to negatively regulate EPO signalling and proliferation of V617F-expressing cells. Here we report a PV patient heterozygous for the somatic JAK2(N542-E543del) mutation and a previously unreported germline mutation within the SH2 domain of SOCS3 (F136L). Interestingly, the SOCS3(F136L) mutation was detected in a Japanese myeloproliferative disorder patient cohort at double the frequency of healthy controls. Cells expressing SOCS3(F136L) had markedly elevated EPO-induced proliferation and extended EPO-induced JAK2 phosphorylation. Additionally, compared to wild-type SOCS3, mutant SOCS3 had an extended half-life in the presence of JAK2 and JAK2(N542-E543del). Our findings suggest that this loss-of-function SOCS3 mutation may have contributed to disease onset by causing deregulated JAK2 signalling in the presence of a constitutively active JAK2(N542-E543del) mutant.

Multi-Study Assessment of Vaginal Cytokines and Microflora in the Safety Evaluation of Intravaginal Rings in Pig-Tailed Macaques

BACKGROUND: HIV microbicide trials have emphasized the need to evaluate the safety of topical microbicides and delivery platforms in an animal model prior to conducting clinical efficacy trials. An ideal delivery device should provide sustainable and sufficient concentrations of effective products to prevent HIV transmission while not increasing transmission risk by either local mucosal inflammation and/or disruption of the normal vaginal microflora.

METHODS: Safety analyses of macaque-sized elastomeric silicone and polyurethane intravaginal rings (IVRs) loaded with candidate antiretroviral (ARV) drugs were tested in four studies ranging in duration from 49 to 73 days with retention of the IVR being 28 days in each study. Macaques were assigned to 3 groups; blank IVR, ARV-loaded IVR, and naïve. In sequential studies, the same macaques were used but rotated into different groups. Mucosal and systemic levels of cytokines were measured from vaginal fluids and plasma, respectively, using multiplex technology. Changes in vaginal microflora were also monitored. Statistical analysis (Mann-Whitney test) was used to compare data between two groups of unpaired samples (with and without IVR, and IVR with and without ARV) for the groups collectively, and also for individual macaques.

RESULTS: There were few statistically significant differences in mucosal and systemic cytokine levels measured longitudinally when the ring was present or absent, with or without ARVs. Of the 8 proinflammatory cytokines assayed a significant increase (p = 0.015) was only observed for IL8 in plasma with the blank and ARV loaded IVR (median of 9.2 vs. 5.7 pg/ml in the absence of IVR). There were no significant differences in the prevalence of H2O2-producing lactobacilli or viridans streptococci, or other microorganisms indicative of healthy vaginal microflora. However, there was an increase in the number of anaerobic gram negative rods in the presence of the IVR (p= < 0.0001).

CONCLUSIONS: IVRs with or without ARVs neither significantly induce the majority of potentially harmful proinflammatory cytokines locally or systemically, nor alter the lactobacillus or G. vaginalis levels. The increase in anaerobic gram negative rods alone suggests minimal disruption of normal vaginal microflora. The use of IVRs as a long-term sustained delivery device for ARVs is promising and preclinical studies to demonstrate the prevention of transmission in the HIV/SHIV nonhuman primate model should continue.

Adult neural stem cells expressing IL-10 confer potent immunomodulation and remyelination in experimental autoimmune encephalitis

Adult neural stem cells (aNSCs) derived from the subventricular zone of the brain show therapeutic effects in EAE, an animal model of the chronic inflammatory neurodegenerative disease MS; however, the beneficial effects are modest. One critical weakness of aNSC therapy may be an insufficient antiinflammatory effect. Here, we demonstrate that i.v. or i.c.v. injection of aNSCs engineered to secrete IL-10 (IL-10–aNSCs), a potent immunoregulatory cytokine, induced more profound functional and pathological recovery from ongoing EAE than that with control aNSCs. IL-10–aNSCs exhibited enhanced antiinflammatory effects in the periphery and inflammatory foci in the CNS compared with control aNSCs, more effectively reducing myelin damage, a hallmark of MS. When compared with mice treated with control aNSCs, those treated with IL-10–aNSCs demonstrated differentiation of transplanted cells into greater numbers of oligodendrocytes and neurons but fewer astrocytes, thus enhancing exogenous remyelination and neuron/axonal growth. Finally, IL-10–aNSCs converted a hostile environment to one supportive of neurons/oligodendrocytes, thereby promoting endogenous remyelination. Thus, aNSCs engineered to express IL-10 show enhanced ability to induce immune suppression, remyelination, and neuronal repair and may represent a novel approach that can substantially improve the efficacy of neural stem cell–based therapy in EAE/MS.

Differential Effect of IL-27 on Developing versus Committed Th17 Cells

IIL-27 counters the effect of TGF-beta+IL-6 on naive CD4(+) T cells, resulting in near complete inhibition of de novo Th17 development. In contrast, little is known about the effect of IL-27 on already differentiated Th17 cells. A better understanding of how IL-27 regulates these cells is needed to evaluate the therapeutic potential of IL-27 in Th17 cells-associated diseases. In this study, we show that IL-27 had surprisingly little effect on committed Th17 cells, despite its expression of a functional IL-27R. Contrary to de novo differentiation of Th17 cells, IL-27 did not suppress expression of retinoid-related orphan receptor (ROR)gammat or RORalpha in committed Th17 cells. Consistent with this finding, the frequency of committed Th17 cells and their cytokine secretion remained unaffected by IL-27. Both memory Th17 cells (CD4(+)CD25(-)CD62L(low)) that developed in vivo and encephalitogenic Th17 cells infiltrating the CNS of mice developing experimental autoimmune encephalomyelitis produced similar amounts of IL-17A when reactivated with IL-23 in the absence and presence of exogenous IL-27. Finally, IL-27 failed to suppress encephalitogenicity of Th17 cells in an adoptive transfer of experimental autoimmune encephalomyelitis. Analysis ex vivo of transferred Th17 cells in the spleen and CNS of recipient mice showed that cells retained similar phenotype irrespective of whether cells were treated or not with IL-27. Our data demonstrate that in contrast to inhibition of de novo differentiation of Th17 cells, IL-27 has little or no effect on committed Th17 cells. These findings indicate that therapeutic applications of IL-27 might have a limited efficacy in inflammatory conditions where aggressive Th17 responses have already developed.

Functional Interleukin-17 Receptor A is Expressed in Central Nervous System Glia and Upregulated in Experimental Autoimmune Encephalomyelitis

Background: Interleukin-17A (IL-17A) is the founding member of a novel family of inflammatory cytokines that plays a critical role in the pathogenesis of many autoimmune diseases, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). IL-17A signals through its receptor, IL-17RA, which is expressed in many peripheral tissues; however, expression of IL-17RA in the central nervous system (CNS) and its role in CNS inflammation are not well understood. Methods: EAE was induced in C57Bl/6 mice by immunization with myelin oligodendroglial glycoprotein. IL-17RA expression in the CNS was compared between control and EAE mice using RT-PCR, in situ hybridization, and immunohistochemistry. Cell-type specific expression was examined in isolated astrocytic and microglial cell cultures. Cytokine and chemokine production was measured in IL-17A treated cultures to evaluate the functional status of IL-17RA. Results: Here we report increased IL-17RA expression in the CNS of mice with EAE, and constitutive expression of functional IL-17RA in mouse CNS tissue. Specifically, astrocytes and microglia express IL-17RA in vitro, and IL-17A treatment induces biological responses in these cells, including significant upregulation of MCP-1, MCP-5, MIP-2 and KC chemokine secretion. Exogenous IL-17A does not significantly alter the expression of IL-17RA in glial cells, suggesting that upregulation of chemokines by glial cells is due to IL-17A signaling through constitutively expressed IL-17RA. Conclusion: IL-17RA expression is significantly increased in the CNS of mice with EAE compared to healthy mice, suggesting that IL-17RA signaling in glial cells can play an important role in autoimmune inflammation of the CNS and may be a potential pathway to target for therapeutic interventions. © 2009 Sarma et al; licensee BioMed Central Ltd.

Loss of the LAT adaptor converts antigen-responsive T cells into pathogenic effectors that function independently of the T cell receptor.

Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the adaptor linker for activation of T cells (LAT) was constitutively mutated (Lat(Y136F) mice) accumulate CD4(+) T cells that trigger autoimmunity and inflammation. Here we show that equipping postthymic CD4(+) T cells with LATY136F molecules or rendering them deficient in LAT molecules triggers a lymphoproliferative disorder dependent on prior TCR engagement. Therefore, such disorders required neither faulty thymic T cell maturation nor LATY136F molecules. Unexpectedly, in CD4(+) T cells recently deprived of LAT, the proximal triggering module of the TCR induced a spectrum of protein tyrosine phosphorylation that largely overlapped the one observed in the presence of LAT. The fact that such LAT-independent signals result in lymphoproliferative disorders with excessive cytokine production demonstrates that LAT constitutes a key negative regulator of the triggering module and of the LAT-independent branches of the TCR signaling cassette.

Interleukin-4 and interleukin-4 soluble receptor α levels in bronchoalveolar lavage from children with asthma

Background: In asthma there is increased expression of the Th2-type cytokine interleukin-4 (IL-4). IL-4 is important in immunoglobulin isotype switching to immunoglobulin E and adhesion of eosinophils to endothelium.

Objectives: We hypothesized that levels of IL-4 in bronchoalveolar lavage (BAL) fluid would be increased in stable, atopic asthmatic children compared with controls and that levels of its physiologic inhibitor IL-4 soluble receptor α (IL-4sRα) would be correspondingly decreased.

Methods: One hundred sixteen children attending a children's hospital for elective surgery were recruited. A nonbronchoscopic BAL was performed, and IL-4 and IL-4sRα were measured in the BAL supernatants.

Results: There was no significant difference in IL-4 concentrations between atopic asthmatic children, atopic normal controls, and nonatopic normal controls [0.13 pg/mL (0.13 to 0.87) vs 0.13 pg/mL (0.13 to 0.41) vs 0.13 pg/mL (0.13 to 0.5), P = 0.65]. IL-4sRα levels were significantly increased in asthmatic patients compared with atopic controls [6.4 pg/mL (5.0 to 25.5) vs 5.0 pg/mL (5.0 to 9.9), P = 0.018], but not when compared with the nonatopic controls [5.2 pg/mL (5.0 to 10.6), P = 0.19].

Conclusions: Contrary to expectation, IL-4sRα levels are increased in BAL from stable asthmatic children compared with nonatopic controls, and we speculate that IL-4sRα is released by inflammatory cells in the airways to limit the proinflammatory effects of IL-4.

STAT3, p38 MAP Kinase and NF-{kappa}B Drive Unopposed Monocyte-dependent Fibroblast MMP-1 Secretion in Tuberculosis.

Tissue destruction characterizes infection with Mycobacterium tuberculosis (Mtb). Type I collagen provides the lung's tensile strength, is extremely resistant to degradation, but is cleaved by matrix metalloproteinase (MMP)-1. Fibroblasts potentially secrete quantitatively more MMP-1 than other lung cells. We investigated mechanisms regulating Mtb-induced collagenolytic activity in fibroblasts in vitro and in patients. Lung fibroblasts were stimulated with conditioned media from Mtb-infected monocytes (CoMTb). CoMTb induced sustained increased MMP-1 (74 versus 16 ng/ml) and decreased tissue inhibitor of metalloproteinase (TIMP)-1 (8.6 versus 22.3 ng/ml) protein secretion. CoMTb induced a 2.7-fold increase in MMP-1 promoter activation and a 2.5-fold reduction in TIMP-1 promoter activation at 24 hours (P = 0.01). Consistent with this, TIMP-1 did not co-localize with fibroblasts in patient granulomas. MMP-1 up-regulation and TIMP-1 down-regulation were p38 (but not extracellular signal–regulated kinase or c-Jun N-terminal kinase) mitogen-activated protein kinase–dependent. STAT3 phosphorylation was detected in fibroblasts in vitro and in tuberculous granulomas.STAT3 inhibition reduced fibroblast MMP-1 secretion by 60% (P = 0.046). Deletion of the MMP-1 promoter NF-B–binding site abrogated promoter induction in response to CoMTb. TNF-, IL-1ß, or Oncostatin M inhibition in CoMTb decreased MMP-1 secretion by 65, 63, and 25%, respectively. This cytokine cocktail activated the same signaling pathways in fibroblasts and induced MMP-1 secretion similar to that induced by CoMTb. This study demonstrates in a cellular model and in patients with tuberculosis that in addition to p38 and NF-B, STAT3 has a key role in driving fibroblast-dependent unopposed MMP-1 production that may be key in tissue destruction in patients.

A Paradoxical Effect of Systemic IL-23 in EAE – Limitation of Autoimmune Inflammatory Demyelination

The heterodimeric cytokine IL-23 plays a non-redundant function in the development of cell-mediated, organspecific autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE). To further characterize the mechanisms of action of IL-23 in autoimmune inflammation, we administered IL-23 systemically at different time points during both relapsing and chronic EAE. Surprisingly, we found suppression of disease in all treatment protocols. We observed a reduction in the number of activated macrophages and microglia in the CNS, while T cell infiltration was not significantly affected. Disease suppression correlated with reduced expansion of myelin-reactive T cells, loss of T-bet expression, loss of lymphoid structures, and increased production of IL-6 and IL-4. Here we describe an unexpected function of exogenous IL-23 in limiting the scope and extent of organ-specific autoimmunity.

Impaired Immune Tolerance to Porphyromonas gingivalis Lipopolysaccharide Promotes Neutrophil Migration and Decreased Apoptosis.

Periodontitis, a chronic inflammatory disease of the tissues supporting the teeth, is characterized by an exaggerated host immune and inflammatory response to periopathogenic bacteria. Toll-like receptor activation, cytokine network induction, and accumulation of neutrophils at the site of inflammation are important in the host defense against infection. At the same time, induction of immune tolerance and the clearance of neutrophils from the site of infection are essential in the control of the immune response, resolution of inflammation, and prevention of tissue destruction. Using a human monocytic cell line, we demonstrate that Porphyromonas gingivalis lipopolysaccharide (LPS), which is a major etiological factor in periodontal disease, induces only partial immune tolerance, with continued high production of interleukin-8 (IL-8) but diminished secretion of tumor necrosis factor alpha (TNF-) after repeated challenge. This cytokine response has functional consequences for other immune cells involved in the response to infection. Primary human neutrophils incubated with P. gingivalis LPS-treated naïve monocyte supernatant displayed a high migration index and increased apoptosis. In contrast, neutrophils treated with P. gingivalis LPS-tolerized monocyte supernatant showed a high migration index but significantly decreased apoptosis. Overall, these findings suggest that induction of an imbalanced immune tolerance in monocytes by P. gingivalis LPS, which favors continued secretion of IL-8 but decreased TNF- production, may be associated with enhanced migration of neutrophils to the site of infection but also with decreased apoptosis and may play a role in the chronic inflammatory state seen in periodontal disease.

The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluste

Background: The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A). Subsequently we have identified a number of human family members and shown that one of these (DUB-3) is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1.

Results: Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable betadefensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating.

Conclusions: The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

Inhibition of Secretion of Interleukin (IL)-12/IL-23 Family Cytokines by 4-Trifluoromethyl-celecoxib Is Coupled to Degradation via the Endoplasmic Reticulum Stress Protein HERP

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RTPCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate in appropriate inflammation in autoimmune conditions.