Feraheme (Ferumoxytol) Is Recognized by Proinflammatory and Anti-inflammatory Macrophages via Scavenger Receptor Type AI/II.

Feraheme (ferumoxytol), a negatively charged, carboxymethyl dextran-coated ultrasmall superparamagnetic iron oxide nanoparticle (USPIO, 30 nm, -16 mV), is clinically approved as an iron supplement and is used off-label for magnetic resonance imaging (MRI) of macrophage-rich lesions, but the mechanism of recognition is not known. We investigated mechanisms of uptake of Feraheme by various types of macrophages in vitro and in vivo. The uptake by mouse peritoneal macrophages was not inhibited in complement-deficient serum. In contrast, the uptake of larger and less charged SPIO nanoworms (60 nm, -5 mV; 120 nm, -5 mV, respectively) was completely inhibited in complement deficient serum, which could be attributed to more C3 molecules bound per nanoparticle than Feraheme. The uptake of Feraheme in vitro was blocked by scavenger receptor (SR) inhibitor polyinosinic acid (PIA) and by antibody against scavenger receptor type A I/II (SR-AI/II). Antibodies against other SRs including MARCO, CD14, SR-BI, and CD11b had no effect on Feraheme uptake. Intraperitoneally administered PIA inhibited the peritoneal macrophage uptake of Feraheme in vivo. Nonmacrophage cells transfected with SR-AI plasmid efficiently internalized Feraheme but not noncharged ultrasmall SPIO of the same size (26 nm, -6 mV), suggesting that the anionic carboxymethyl groups of Feraheme are responsible for the SR-AI recognition. The uptake by nondifferentiated bone marrow derived macrophages (BMDM) and by BMDM differentiated into M1 (proinflammatory) and M2 (anti-inflammatory) types was efficiently inhibited by PIA and anti-SR-AI/II antibody. Interestingly, all BMDM types expressed similar levels of SR-AI/II. In conclusion, Feraheme is efficiently recognized via SR-AI/II but not via complement by different macrophage types. The recognition by the common phagocytic receptor has implications for specificity of imaging of macrophage subtypes.

Evaluation of Targeting Efficiency of Joints with Anticollagen II Antibodies.

Diseases of the joints affect over 10% of the world's population, resulting in significant morbidity. There is an unmet need in strategies for specific delivery of therapeutics to the joints. Collagen type II is synthesized by chondrocytes and is mainly restricted to the cartilage and tendons. Arthrogen-CIA is a commercially available anticollagen II antibody cocktail that reacts with 5 different epitopes on human, bovine, and mouse collagen II. Arthrogen has been used for induction of experimental rheumatoid arthritis (RA) in mice because of high complement activation on the cartilage surface. Native collagen II might serve as a useful target for potential delivery of therapeutics to the joint. To evaluate the efficiency and specificity of targeting collagen II, Arthrogen was labeled with near-infrared (NIR) dye IRDye 800 or IRDye 680. Using ex vivo NIR imaging, we demonstrate that Arthrogen efficiently and specifically accumulated in the limb joints regardless of the label dye or injection route (intravenous and subcutaneous). After subcutaneous injection, the mean fluorescence of the hind limb joints was 19 times higher than that of the heart, 8.7 times higher than that of the liver, and 3.7 times higher than that of the kidney. Control mouse IgG did not show appreciable accumulation. Microscopically, the antibody accumulated on the cartilage surface of joints and on endosteal surfaces. A monoclonal antibody against a single epitope of collagen II showed similar binding affinity and elimination half-life, but about three times lower targeting efficiency than Arthrogen in vitro and ex vivo, and about two times lower targeting efficiency in vivo. We suggest that an antibody against multiple epitopes of collagen II could be developed into a highly effective and specific targeting strategy for diseases of the joints or spine.

Deconstructing the Lectin Pathway in the Pathogenesis of Experimental Inflammatory Arthritis: Essential Role of the Lectin Ficolin B and Mannose-Binding Protein-Associated Serine Protease 2.

Complement plays an important role in the pathogenesis of rheumatoid arthritis. Although the alternative pathway (AP) is known to play a key pathogenic role in models of rheumatoid arthritis, the importance of the lectin pathway (LP) pattern recognition molecules such as ficolin (FCN) A, FCN B, and collectin (CL)-11, as well as the activating enzyme mannose-binding lectin-associated serine protease-2 (MASP-2), are less well understood. We show in this article that FCN A-/- and CL-11-/- mice are fully susceptible to collagen Ab-induced arthritis (CAIA). In contrast, FCN B-/- and MASP-2-/-/sMAp-/- mice are substantially protected, with clinical disease activity decreased significantly (p < 0.05) by 47 and 70%, respectively. Histopathology scores, C3, factor D, FCN B deposition, and infiltration of synovial macrophages and neutrophils were similarly decreased in FCN B-/- and MASP-2-/-/sMAp-/- mice. Our data support that FCN B plays an important role in the development of CAIA, likely through ligand recognition in the joint and MASP activation, and that MASP-2 also contributes to the development of CAIA, likely in a C4-independent manner. Decreased AP activity in the sera from FCN B-/- and MASP-2-/-/sMAp-/- mice with arthritis on adherent anti-collagen Abs also support the hypothesis that pathogenic Abs, as well as additional inflammation-related ligands, are recognized by the LP and operate in vivo to activate complement. Finally, we also speculate that the residual disease seen in our studies is driven by the AP and/or the C2/C4 bypass pathway via the direct cleavage of C3 through an LP-dependent mechanism.

Mannan-Binding Lectin-Associated Serine Protease 1/3 Cleavage of Pro-Factor D into Factor D In Vivo and Attenuation of Collagen Antibody-Induced Arthritis through Their Targeted Inhibition by RNA Interference-Mediated Gene Silencing.

The complement system is proposed to play an important role in the pathogenesis of rheumatoid arthritis (RA). The complement system mannan-binding lectin-associated serine proteases (MASP)-1/3 cleave pro-factor D (proDf; inactive) into Df (active), but it is unknown where this cleavage occurs and whether inhibition of MASP-1/3 is a relevant therapeutic strategy for RA. In the present study, we show that the cleavage of proDf into Df by MASP-1/3 can occur in the circulation and that inhibition of MASP-1/3 by gene silencing is sufficient to ameliorate collagen Ab-induced arthritis in mice. Specifically, to examine the cleavage of proDf into Df, MASP-1/3-producing Df-/- liver tissue (donor) was transplanted under the kidney capsule of MASP-1/3-/- (recipient) mice. Five weeks after the liver transplantation, cleaved Df was present in the circulation of MASP-1/3-/- mice. To determine the individual effects of MASP-1/3 and Df gene silencing on collagen Ab-induced arthritis, mice were injected with scrambled, MASP-1/3-targeted, or Df-targeted small interfering RNAs (siRNAs). The mRNA levels for MASP-1 and -3 decreased in the liver to 62 and 58%, respectively, in mice injected with MASP-1/3 siRNAs, and Df mRNA decreased to 53% in the adipose tissue of mice injected with Df siRNAs; additionally, circulating MASP-1/3 and Df protein levels were decreased. In mice injected with both siRNAs the clinical disease activity, histopathologic injury scores, C3 deposition, and synovial macrophage/neutrophil infiltration were significantly decreased. Thus, MASP-1/3 represent a new therapeutic target for the treatment of RA, likely through both direct effects on the lectin pathway and indirectly through the alternative pathway.

A New Approach for the Treatment of Arthritis in Mice with a Novel Conjugate of an Anti-C5aR1 Antibody and C5 Small Interfering RNA.

Rheumatoid arthritis (RA) is an inflammatory autoimmune joint disease in which the complement system plays an important role. Of the several components of complement, current evidence points to C5 as the most important inducer of inflammation. Several groups generated Abs or small interfering RNAs (siRNAs) or small molecule inhibitors against C5 and C5aR1 (CD88) that have showed some efficacy in RA in animal models. However, none of these candidate therapeutics has moved from bench to bedside. In this study, we test in collagen Ab-induced arthritis (CAIA) a new therapeutic strategy using a novel anti-C5ab-C5 siRNA conjugate. We first demonstrate that although C5aR2 or C5L2 (GPR77) plays no role in CAIA, C5aR1 contributes to pathogenesis. We demonstrate that injection of siRNAs blocking C5, C5aR1, or the combination decreased clinical disease activity in mice with CAIA by 45%, 51%, and 58%, respectively. Anti-C5 Ab (BB5.1) has only limited efficacy, but significantly reduced arthritis up to 66%. We then generated a novel anti-C5aR1 Ab-protamine-C5 siRNA conjugate. To our knowledge, we show for the first time that whereas unconjugated Ab plus siRNAs reduce arthritis by 19%, our anti-C5aR1 Ab-protamine-C5 siRNA conjugate was effective in reducing arthritis by 83% along with a parallel decrease in histopathology, C3 deposition, neutrophils, and macrophages in the joints of mice with CAIA. These data suggest that by targeting anti-C5 siRNAs to the receptor for its C5a activation fragment (C5aR1), a striking clinical effect can be realized.

Inhibition of acute complement responses towards bolus-injected nanoparticles using targeted short-circulating regulatory proteins.

Effective inhibition of the complement system is needed to prevent the accelerated clearance of nanomaterials by complement cascade and inflammatory responses. Here we show that a fusion construct consisting of human complement receptor 2 (CR2) (which recognizes nanosurface-deposited complement 3 (C3)) and complement receptor 1 (CR1) (which blocks C3 convertases) inhibits complement activation with picomolar to low nanomolar efficacy on many types of nanomaterial. We demonstrate that only a small percentage of nanoparticles are randomly opsonized with C3 both in vitro and in vivo, and CR2-CR1 immediately homes in on this subpopulation. Despite rapid in vivo clearance, the co-injection of CR2-CR1 in rats, or its mouse orthologue CR2-Crry in mice, with superparamagnetic iron oxide nanoparticles nearly completely blocks complement opsonization and unwanted granulocyte/monocyte uptake. Furthermore, the inhibitor completely prevents lethargy caused by bolus-injected nanoparticles, without inducing long-lasting complement suppression. These findings suggest the potential of the targeted complement regulators for clinical evaluation.

Functional differences in the mu opioid receptor SNP 118A>G are dependent on receptor splice-variant and agonist-specific recruitment of ß-arrestin.

The OPRM1 gene codes for the mu opioid receptor (MOR) and polymorphisms are associated with complex patient clinical responses. The most studied single nucleotide polymorphism (SNP) in OPRM1 is adenine (A) substituted by guanine (G) at position 118 (118A>G, rs1799971) leading to a substitution of asparagine (Asn) for aspartic acid (Asp) at position 40 in the N terminus of the resulting protein. To date, no structural explanation for the associated clinical responses resulting from the 118A>G polymorphism has been proposed. We utilized computational modeling paired with functional cellular assays to predict unstructured N- and C-terminal regions of MOR-1. Using molecular docking and post-docking energy minimizations with morphine, we show that the extracellular substitution of Asn at position 40 alters the cytoplasmic C-terminal conformation, while leaving the G-protein binding interface unaffected. A real-time BRET assay measuring G-protein and ß-arrestin association with MOR r generated data that tested this prediction. Consistent with this in silico prediction, we show changes in morphine-mediated ß-arrestin association with receptor variants with little change in morphine-mediated G-protein association comparing MOR-1 wild type (WT) to MOR-1118A>G. We tested the system with different opioid agonists, the OPRM1 118A>G SNP, and different MOR splice variants (MOR-1 and MOR-1O). These results are consistent with the observation that patients with the 118A>G OPRM1 allele respond more readily to fentanyl than to morphine. In conclusion, the 118A>G substitution alters receptor responses to opioids through variable C-terminal domain movements that are agonist and splice variant dependent.

Decay-Accelerating Factor Differentially Associates With Complement-Mediated Damage in Synovium After Meniscus Tear as Compared to Anterior Cruciate Ligament Injury.

We have reported that anterior cruciate ligament (ACL) injury leads to the differential dysregulation of the complement system in the synovium as compared to meniscus tear (MT) and proposed this as a mechanism for a greater post-injury prevalence of post traumatic osteoarthritis (PTOA). To explore additional roles of complement proteins and regulators, we determined the presence of decay-accelerating factor (DAF), C5b, and membrane attack complexes (MACs, C5b-9) in discarded surgical synovial tissue (DSST) collected during arthroscopic ACL reconstructive surgery, MT-related meniscectomy, osteoarthritis (OA)-related knee replacement surgery and normal controls. Multiplexed immunohistochemistry was used to detect and quantify complement proteins. To explore the involvement of body mass index (BMI), after these 2 injuries, we examined correlations among DAF, C5b, MAC and BMI. Using these approaches, we found that synovial cells after ACL injury expressed a significantly lower level of DAF as compared to MT (p<0.049). In contrast, C5b staining synovial cells were significantly higher after ACL injury (p<0.0009) and in OA DSST (p<0.039) compared to MT. Interestingly, there were significantly positive correlations between DAF & C5b (r=0.75, p<0.018) and DAF & C5b (r=0.64 p<0.022) after ACL injury and MT, respectively. The data support that DAF, which should normally dampen C5b deposition due to its regulatory activities on C3/C5 convertases, does not appear to exhibit that function in inflamed synovia following either ACL injury or MT. Ineffective DAF regulation may be an additional mechanism by which relatively uncontrolled complement activation damages tissue in these injury states.

Pigmented-MDCK (P-MDCK) cell line with tunable melanin expression: an in vitro model for the outer blood-retinal barrier.

Retinal pigment epithelium, which forms the outer blood-retinal barrier, is a critical barrier for transport of drugs to the retina. The purpose of this study was to develop a pigmented MDCK (P-MDCK) cell line as a rapidly established in vitro model for the outer blood-retinal barrier to assess the influence of melanin pigment on solute permeability. A melanin synthesizing P-MDCK cell line was developed by lentiviral transduction of human tyrosinase and p-protein genes in MDCK (NBL-2) cells. Melanin content, tyrosinase activity (conversion of L-dopa to dopachrome), and transepithelial electrical resistance (TEER) were measured. Expression of tyrosinase protein and p-protein in P-MDCK cells was confirmed by confocal microscopy. Effect of l-tyrosine (0 to 2 mM) in culture medium on melanin synthesis in P-MDCK cells was evaluated. Cell uptake and transepithelial transport of pigment-binding chloroquine (Log D = 1.59) and a negative control salicylic acid (Log D = -1.14) were investigated. P-MDCK cells expressed tyrosinase and p-protein. Tyrosinase activity was 4.5-fold higher in P-MDCK cells compared to wild type MDCK cells. The transepithelial electrical resistance stabilized by day 4 in both cell types, with the TEER being 958 ± 33 and 964 ± 58 Ω·cm(2) for P-MDCK and wild type cells, respectively. Melanin content in P-MDCK cells depended on the concentration of l-tyrosine in culture medium, and increased from 3 to 54 µg/mg protein with an increase in l-tyrosine content from 0 to 2 mM. When the cells were grown in 2 mM l-tyrosine, uptake of chloroquine was 2.3-fold higher and the transepithelial transport was 2.2-fold lower in P-MDCK cells when compared to wild type MDCK cells. No significant difference was observed for both cell uptake and transport of salicylic acid. We developed a P-MDCK cell line with tunable melanin synthesis as a rapidly developing surrogate for retinal pigment epithelium.

Antibody-Dependent Complement Responses toward SARS-CoV-2 Receptor-Binding Domain Immobilized on "Pseudovirus-like" Nanoparticles.

Many aspects of innate immune responses to SARS viruses remain unclear. Of particular interest is the role of emerging neutralizing antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 in complement activation and opsonization. To overcome challenges with purified virions, here we introduce "pseudovirus-like" nanoparticles with ∼70 copies of functional recombinant RBD to map complement responses. Nanoparticles fix complement in an RBD-dependent manner in sera of all vaccinated, convalescent, and naïve donors, but vaccinated and convalescent donors with the highest levels of anti-RBD antibodies show significantly higher IgG binding and higher deposition of the third complement protein (C3). The opsonization via anti-RBD antibodies is not an efficient process: on average, each bound antibody promotes binding of less than one C3 molecule. C3 deposition is exclusively through the alternative pathway. C3 molecules bind to protein deposits, but not IgG, on the nanoparticle surface. Lastly, "pseudovirus-like" nanoparticles promote complement-dependent uptake by granulocytes and monocytes in the blood of vaccinated donors with high anti-RBD titers. Using nanoparticles displaying SARS-CoV-2 proteins, we demonstrate subject-dependent differences in complement opsonization and immune recognition.

Pazopanib, a multitargeted tyrosine kinase inhibitor, reduces diabetic retinal vascular leukostasis and leakage.

OBJECTIVE: To determine the efficacy of pazopanib eye drops in the streptozotocin induced diabetic retinopathy rat model. METHODS: A 0.5% w/v pazopanib suspension was prepared in phosphate buffered saline (PBS, pH 7.4) in the presence of 0.5% w/v sodium carboxymethyl cellulose. Brown Norway rats were divided into three groups (n=4) - (1) healthy, (2) diabetic, and (3) diabetic with treatment. The drug suspension was administered twice daily as eye drops to group 3 for 30 days. Efficacy parameters including the number of adherent leukocytes in the retinal vasculature (leukostasis), blood-retinal FITC-dextran leakage, and vitreous-to-plasma protein ratio were measured. RESULTS: Pazopanib suspension in the form of eye drops significantly reduced leukostasis (32%), FITC-dextran leakage (39%), and the vitreous-to-plasma protein ratio (64%) in diabetic animals compared to untreated diabetic group. CONCLUSION: Pazopanib eye drops can alleviate retinal complications of diabetic retinopathy.

Key Components of the Complement Lectin Pathway Are Not Only Required for the Development of Inflammatory Arthritis but Also Regulate the Transcription of Factor D.

The complement system plays an important role in the pathogenesis of rheumatoid arthritis (RA). Besides driving lectin pathway (LP) activation, the mannan-binding lectin (MBL)-associated serine proteases (MASPs) also play a key role in regulating the alternative pathway (AP). We evaluated the effects of N-acetylgalactosamine (GalNAc)-conjugated MASP-1 and MASP-2 duplexes in vitro and in mice with and without arthritis to examine whether knockdown of MASP-1 and MASP-2 expression affects the development of arthritis. GalNAc-siRNAs for MASP-1 and MASP-2 demonstrated robust silencing of MASP-1 or MASP-2 at pM concentrations in vitro. To evaluate the impact of silencing in arthritic mice, we used the collagen antibody-induced arthritis (CAIA) mouse model of RA. Mice were injected a 10 mg/kg dose of GalNAc-siRNAs 3x s.q. prior to the induction of CAIA. Liver gene expression was examined using qRT-PCR, and protein levels were confirmed in the circulation by sandwich immunoassays and Western blot. At day 10, CAIA mice separately treated with MASP-1 and MASP-2 duplexes had a specific reduction in expression of liver MASP-1 (70-95%, p < 0.05) and MASP-2 (90%, p < 0.05) mRNA, respectively. MASP-1-siRNA treatment resulted in a 95% reduction in levels of MASP-1 protein in circulation with no effect on MASP-2 levels and clinical disease activity (CDA). In mice injected with MASP-2 duplex, there was a significant (p < 0.05) 90% decrease in ex vivo C4b deposition on mannan, with nearly complete elimination of MASP-2 in the circulation. MASP-2 silencing initially significantly decreased CDA by 60% but subsequently changed to a 40% decrease vs. control. Unexpectedly, GalNAc-siRNA-mediated knockdown of MASP-1 and MASP-2 revealed a marked effect of these proteins on the transcription of FD under normal physiological conditions, whereas LPS-induced inflammatory conditions reversed this effect on FD levels. LPS is recognized by Toll-like receptor 4 (TLR4), we found MBL not only binds to TLR4 an interaction with a Kd of 907 nM but also upregulated FD expression in differentiated adipocytes. We show that MASP-2 knockdown impairs the development of RA and that the interrelationship between proteins of the LP and the AP may extend to the transcriptional modulation of the FD gene.

C2 IgM Natural Antibody Enhances Inflammation and Its Use in the Recombinant Single Chain Antibody-Fused Complement Inhibitor C2-Crry to Target Therapeutics to Joints Attenuates Arthritis in Mice.

Natural IgM antibodies (NAbs) have been shown to recognize injury-associated neoepitopes and to initiate pathogenic complement activation. The NAb termed C2 binds to a subset of phospholipids displayed on injured cells, and its role(s) in arthritis, as well as the potential therapeutic benefit of a C2 NAb-derived ScFv-containing protein fused to a complement inhibitor, complement receptor-related y (Crry), on joint inflammation are unknown. Our first objective was to functionally test mAb C2 binding to apoptotic cells from the joint and also evaluate its inflammation enhancing capacity in collagen antibody-induced arthritis (CAIA). The second objective was to generate and test the complement inhibitory capacity of C2-Crry fusion protein in the collagen-induced arthritis (CIA) model. The third objective was to demonstrate in vivo targeting of C2-Crry to damaged joints in mice with arthritis. The effect of C2-NAb on CAIA in C57BL/6 mice was examined by inducing a suboptimal disease. The inhibitory effect of C2-Crry in DBA/1J mice with CIA was determined by injecting 2x per week with a single dose of 0.250 mg/mouse. Clinical disease activity (CDA) was examined, and knee joints were fixed for analysis of histopathology, C3 deposition, and macrophage infiltration. In mice with suboptimal CAIA, at day 10 there was a significant (p < 0.017) 74% increase in the CDA in mice treated with C2 NAb, compared to mice treated with F632 control NAb. In mice with CIA, at day 35 there was a significant 39% (p < 0.042) decrease in the CDA in mice treated with C2-Crry. Total scores for histopathology were also 50% decreased (p < 0.0005) in CIA mice treated with C2-Crry. C3 deposition was significantly decreased in the synovium (44%; p < 0.026) and on the surface of cartilage (42%; p < 0.008) in mice treated with C2-Crry compared with PBS treated CIA mice. Furthermore, C2-Crry specifically bound to apoptotic fibroblast-like synoviocytes in vitro, and also localized in the knee joints of arthritic mice as analyzed by in vivo imaging. In summary, NAb C2 enhanced arthritis-related injury, and targeted delivery of C2-Crry to inflamed joints demonstrated disease modifying activity in a mouse model of human inflammatory arthritis.

Sodium butyrate alters erythropoietin glycosylation via multiple mechanisms.

Recombinant human erythropoietin (rHuEPO) produced in a human kidney fibrosarcoma cell line, HT1080, was used as a model to study the effects of sodium butyrate (SB) on protein glycosylation. Treatment with 2 mM SB resulted in complex changes with respect to sugar nucleotide pools including an increase in UDP-Gal and a decrease in UDP-GlcNac. In addition, polylactosamine structures present on rHuEPO increased after SB treatment. To determine if these phenotypic changes correlated with changes in mRNA abundance, we profiled mRNA levels over a 24-h period in the presence or absence of SB using oligonucleotide microarrays. By filtering our data through a functional glycomics gene list associated with the processes of glycan degradation, glycan synthesis, and sugar nucleotide synthesis and transport we identified 26 genes with significantly altered mRNA levels. We were able to correlate the changes in message in six of these genes with measurable phenotypic changes within our system including: neu1, b3gnt6, siat4b, b3gnt1, slc17a5, and galt. Interestingly, for the two genes: cmas and gale, our measurable phenotypic changes did not correlate with changes in mRNA expression. These data demonstrate both the utility and pit falls of coupling biochemical analysis with high throughput oligonucleotide microarrays to predict how changes in cell culture environments will impact glycoprotein oligosaccharide content.

Loss of a gimap/ian gene leads to activation of NF-kappaB through a MAPK-dependent pathway.

The diabetes-prone biobreeding (BB-DP) rat contains the lyp mutation which results in lymphopenia and promotes the progression of a T cell-mediated autoimmune attack of the pancreas in certain rat strains. This mutation has been mapped to a gene which bears homology to human Gimap5/Ian5 and results in the truncation and loss of activity of this protein. The lymphopenic state induced by the loss of this protein has led to the proposal that Gimap5 has an anti-apoptotic function. Previously we described an additional phenotype of incomplete activation mediated by the loss of Gimap5 function. Here we further characterize this incomplete activation phenotype and map a potential signal transduction pathway leading to activation. We show that CD5 expression on peripheral T cells is elevated in Gimap5 animals, while thymocyte expression remains similar between the two strains. Additionally, we show that NF-kappaB but not NFAT is activated in unstimulated Gimap5 mutant T cells as compared to unstimulated wild type T cells. Mapping this activation to its upstream source we show that activation of NF-kappaB is correlated with an activation of IKK. Using a variety of kinase inhibitors we further map this increase in IKK to an increase in MEK activation. Finally, to counter the possibility that activation is an indirect consequence of the lymphopenic environment, we created bone marrow chimeras in which Gimap5 mutant T cells developed in a normal environment and show that these cells retain their activated phenotype. Together, we interpret these data as demonstrating that the activation caused by loss of Gimap5 is a cell intrinsic phenomenon caused, in part, by a MEK-dependent activation of IKK. This, in turn, would suggest that Gimap5 functions to promote both T cell survival and quiescence and that these pathways are biochemically linked.

Targeting of Liver Mannan-Binding Lectin-Associated Serine Protease-3 with RNA Interference Ameliorates Disease in a Mouse Model of Rheumatoid Arthritis.

Mannan-binding lectin-associated serine protease 3 (MASP-3) regulates the alternative pathway of complement and is predominantly synthesized in the liver. The role of liver-derived MASP-3 in the pathogenesis of rheumatoid arthritis (RA) is unknown. We hypothesized that liver-derived MASP-3 is essential for the development of joint damage and that targeted inhibition of MASP-3 in the liver can attenuate arthritis. We used MASP-3-specific small interfering RNAs (siRNAs) conjugated to N-acetylgalactosamine (GalNAc) to specifically target the liver via asialoglycoprotein receptors. Active GalNAc-MASP3-siRNA conjugates were identified, and in vivo silencing of liver MASP-3 mRNA was demonstrated in healthy mice. The s.c. treatment with GalNAc-MASP-3-siRNAs specifically decreased the expression of MASP-3 in the liver and the level of MASP-3 protein in circulation of mice without affecting the levels of the other spliced products. In mice with collagen Ab-induced arthritis, s.c. administration of GalNAc-MASP-3-siRNA decreased the clinical disease activity score to 50% of controls, with decrease in histopathology scores and MASP-3 deposition. To confirm the ability to perform MASP-3 gene silencing in human cells, we generated a lentivirus expressing a short hairpin RNA specific for human MASP-3 mRNA. This procedure not only eliminated the short-term (at day 15) expression of MASP-3 in HepG2 and T98G cell lines but also diminished the long-term (at day 60) synthesis of MASP-3 protein in T98G cells. Our study demonstrates that isoform-specific silencing of MASP-3 in vivo modifies disease activity in a mouse model of RA and suggests that liver-directed MASP3 silencing may be a therapeutic approach in human RA.

AIRE recruits multiple transcriptional components to specific genomic regions through tethering to nuclear matrix.

Thymic selection requires that diverse self antigens be presented to developing thymocytes by stromal cells. Consistent with this function, medullary thymic epithelial cells have been shown to express a large number of genes, many of which are tissue restricted. Autoimmune regulator (AIRE) is a nuclear protein, which has recently been identified as a regulator of this process, however, the mechanism by which AIRE functions is not well understood. Here we use a transrepression assay to demonstrate that AIRE interacts with multiple components of the transcription complex including a novel interaction with the UBA domain protein, GBDR1. When AIRE is expressed in cultured human thymic epithelial cells, it tightly associates with nuclear matrix, suggesting that AIRE responsive genes may be localized to specific regions. Using a mathematical approach we have re-analyzed an Affymetrix dataset identifying AIRE responsive genes and show that they tend to localize to specific regions of the genome. Together, these data suggest that AIRE regulates gene expression by recruiting components of the transcription complex to specific regions of the genome via interactions with nuclear matrix.

TRAIL and inhibitors of apoptosis are opposing determinants for NF-kappaB-dependent, genotoxin-induced apoptosis of cancer cells.

Opposing pro- and anti-apoptotic actions of TRAIL and the inhibitors of apoptosis (IAPs) contribute to the cell's decision to survive or die. We demonstrate that in H157 human lung carcinoma cells, etoposide and doxorubicin induce the NF-kappaB-dependent expression of both pro- and anti-apoptotic proteins including TRAIL and its death receptor, DR5, and IAPs. Inhibition of NF-kappaB activation in H157 cells in response to genotoxin resulted in loss of cell surface expression of TRAIL and DR5, aggressive growth and chemotherapy resistance of tumors in nude mice. Similar to the paracrine TRAIL response in H157 cells, the sensitivity of normal lung and breast epithelium and carcinomas to undergo genotoxin-induced apoptosis correlates strongly with cell surface expression of TRAIL. Suppression of TRAIL signaling by expression of the TRAIL decoy receptor, DcR1, confers chemoresistance to cancer cells. These findings demonstrate that TRAIL signaling via its death receptors is a significant contributor to genotoxin-induced apoptosis in human epithelial carcinomas.

Measurement of IKK activity in primary rat T cells: rapid activation and inactivation.

The translocation of the transcription factor NF-kappaB into the nucleus plays a critical role in many physiological events. In unstimulated cells, NF-kappaB is sequestered in the cytosol, bound to its inhibitor IkappaB. Activation primarily occurs via the IkappaB kinase (IKK) complex which phosphorylates IkappaBalpha at serines 32 and 36, creating a recognition site for IkappaB ubiquitination which then targets IkappaB for degradation. Often it is useful to measure IKK activity to assess upstream signaling events leading to NF-kappaB activation. Current methods of assessing IKK activity are limited to IKK isoforms which are recognized by available IKK antibodies. Here, we describe a procedure to qualitatively assess the overall IKK activity in a cell lysate which can be used on any IKK isoform capable of phosphorylating human IkappaBalpha. This nonradioactive assay is based on measurement of the ability of the cell lysate to phosphorylate GST-IkappaBalpha, as measured by Western blotting, using an anti-phospho-IkappaBalpha antibody. We have used this assay to observe the kinetics of TCR-mediated activation of IKK as compared to PMA/ionomycin in primary rat T cells. PMA/ionomycin induces maximal IKK activity within 1 min of stimulation and this activity remains elevated for over 20 min. In comparison, TCR ligation induces maximal IKK activity after 5 min of stimulation and this activity rapidly diminishes to background levels. These data indicate that different stimuli can activate and inactivate IKK with different kinetics and suggest that TCR-mediated activation of IKK is closely linked to the rapid phosphorylation and dephosphorylation, respectively, of TCR-associated kinases.