The severity of experimental arthritis is independent of IL-36 receptor signaling

Introduction

Interleukin (IL)-36 refers to three related IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, that bind to the IL-36 receptor (IL-36R). IL-36 exerts proinflammatory effects in skin and lung and stimulates T cell responses. In the present study, we examined the expression and function of IL-36R and its ligands in experimental arthritis.

Methods

Collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and K/BxN serum transfer-induced arthritis were induced according to standard protocols. Messenger RNA levels for IL-36R and its ligands in the joints of mice with CIA were determined by RT-qPCR. Mice with CIA were injected with a blocking monoclonal anti-IL-36R, a blocking anti-IL-1RI, or their isotype-matched control antibodies at the time of arthritis onset. Anti-IL-36R or control antibodies were also injected at the time of AIA induction. Finally, IL-36R-deficient mice were examined in AIA and serum transfer-induced arthritis. The development and severity of arthritis were assessed by clinical and histological scoring.

Results

IL-36R, IL-36Ra and IL-36γ mRNA were detected in the joints of mice with CIA, but their levels did not correlate with arthritis severity. As opposed to anti-IL-1RI antibody treatment, the injection of an anti-IL-36R antibody was devoid of effect on the development and severity of CIA. The severity of joint inflammation and structural damage in AIA was also unaltered by anti-IL-36R antibody treatment. Finally, the severity of AIA and K/BxN serum transfer-induced arthritis was similar in IL-36R-deficient and wild-type mice.

Conclusions

The development and severity of experimental arthritis are independent of IL-36R signaling.     

Ric-8B is a GTP-dependent G protein alphas guanine nucleotide exchange factor

ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including Gα(q) and Gα(s) regulation of neurotransmission, Gα(i)-dependent mitotic spindle positioning during (asymmetric) cell division, and Gα(olf)-dependent odorant receptor signaling. Mammalian Ric-8 activities are partitioned between two genes, ric-8A and ric-8B. Ric-8A is a guanine nucleotide exchange factor (GEF) for Gα(i)/α(q)/α(12/13) subunits. Ric-8B potentiated G(s) signaling presumably as a Gα(s)-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be Gα subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed Gα(s) and lesser amounts of Gα(q) and Gα(13). Ric-8BFL stimulated guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding to these subunits and Gα(olf), whereas the Ric-8BΔ9 isoform stimulated Gα(s short) GTPγS binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V(max) of Gα(s) steady state GTP hydrolysis and the apparent K(m) values of GTP binding to Gα(s) from ～385 nm to an estimated value of ～42 μM. Directionality of the Ric-8BFL-catalyzed Gα(s) exchange reaction was GTP-dependent. At sub-K(m) GTP, Ric-BFL was inhibitory to exchange despite being a rapid GDP release accelerator. Ric-8BFL binds nucleotide-free Gα(s) tightly, and near-K(m) GTP levels were required to dissociate the Ric-8B·Gα nucleotide-free intermediate to release free Ric-8B and Gα-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce Gα-GTP in cells.     

Synovial phenotypes in rheumatoid arthritis correlate with response to biologic therapeutics

Introduction

Rheumatoid arthritis (RA) is a complex and clinically heterogeneous autoimmune disease. Currently, the relationship between pathogenic molecular drivers of disease in RA and therapeutic response is poorly understood.

Methods

We analyzed synovial tissue samples from two RA cohorts of 49 and 20 patients using a combination of global gene expression, histologic and cellular analyses, and analysis of gene expression data from two further publicly available RA cohorts. To identify candidate serum biomarkers that correspond to differential synovial biology and clinical response to targeted therapies, we performed pre-treatment biomarker analysis compared with therapeutic outcome at week 24 in serum samples from 198 patients from the ADACTA (ADalimumab ACTemrA) phase 4 trial of tocilizumab (anti-IL-6R) monotherapy versus adalimumab (anti-TNFα) monotherapy.

Results

We documented evidence for four major phenotypes of RA synovium – lymphoid, myeloid, low inflammatory, and fibroid - each with distinct underlying gene expression signatures. We observed that baseline synovial myeloid, but not lymphoid, gene signature expression was higher in patients with good compared with poor European league against rheumatism (EULAR) clinical response to anti-TNFα therapy at week 16 (P =0.011). We observed that high baseline serum soluble intercellular adhesion molecule 1 (sICAM1), associated with the myeloid phenotype, and high serum C-X-C motif chemokine 13 (CXCL13), associated with the lymphoid phenotype, had differential relationships with clinical response to anti-TNFα compared with anti-IL6R treatment. sICAM1-high/CXCL13-low patients showed the highest week 24 American College of Rheumatology (ACR) 50 response rate to anti-TNFα treatment as compared with sICAM1-low/CXCL13-high patients (42% versus 13%, respectively, P =0.05) while anti-IL-6R patients showed the opposite relationship with these biomarker subgroups (ACR50 20% versus 69%, P =0.004).

Conclusions

These data demonstrate that underlying molecular and cellular heterogeneity in RA impacts clinical outcome to therapies targeting different biological pathways, with patients with the myeloid phenotype exhibiting the most robust response to anti-TNFα. These data suggest a path to identify and validate serum biomarkers that predict response to targeted therapies in rheumatoid arthritis and possibly other autoimmune diseases.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01119859","term_id":"NCT01119859"}}NCT01119859     

Broad-spectrum antimicrobial activity of hemoglobin

While hemoglobin is one of the most well characterized proteins due to its function in oxygen transport, few additional properties of hemoglobin have been described. While screening serum samples for novel antimicrobial factors, it was found that intact hemoglobin tetramers, including that from human, exhibited considerable activity against gram-positive and gram-negative bacteria, and fungi. To further characterize this surprising activity, the antimicrobial potency of sections of human hemoglobin was tested against a panel of microorganisms. In all cases separate testing of the alpha and beta subunits provided activity at least as potent as the intact tetramer. This activity is derived from the protein portion of hemoglobin since removal of the heme prosthetic group did not lead to decreases in potency. In addition, cyanogen bromide cleavage of both subunits provided fragments that still contained substantial antimicrobial activity. It has been possible to map specific regions of the human hemoglobin molecule that are responsible for significant antimicrobial activity. The carboxyl terminal thirty amino acids of the beta subunit, which form a cationic alpha-helix based on the crystal structure of the intact tetramer, were active against Escherichia coli, Staphylococcus aureus and Candida albicans. In view of the fact that different hemoglobin-derived peptide fragments exhibit diverse antibiotic activities, it is conceivable that, in addition to its role in oxygen transport. hemoglobin functions as an important multi-defense agent against a wide range of microorganisms.     

Investigation of Caucasian rheumatoid arthritis susceptibility loci in African patients with the same disease

Introduction

The largest genetic risk to develop rheumatoid arthritis (RA) arises from a group of alleles of the HLA DRB1 locus ('shared epitope', SE). Over 30 non-HLA single nucleotide polymorphisms (SNPs) predisposing to disease have been identified in Caucasians, but they have never been investigated in West/Central Africa. We previously reported a lower prevalence of the SE in RA patients in Cameroon compared to European patients and aimed in the present study to investigate the contribution of Caucasian non-HLA RA SNPs to disease susceptibility in Black Africans.

Methods

RA cases and controls from Cameroon were genotyped for Caucasian RA susceptibility SNPs using Sequenom MassArray technology. Genotype data were also available for 5024 UK cases and 4281 UK controls and for 119 Yoruba individuals in Ibadan, Nigeria (YRI, HapMap). A Caucasian aggregate genetic-risk score (GRS) was calculated as the sum of the weighted risk-allele counts.

Results

After genotyping quality control procedures were performed, data on 28 Caucasian non-HLA susceptibility SNPs were available in 43 Cameroonian RA cases and 44 controls. The minor allele frequencies (MAF) were tightly correlated between Cameroonian controls and YRI individuals (correlation coefficient 93.8%, p = 1.7E-13), and they were pooled together. There was no correlation between MAF of UK and African controls; 13 markers differed by more than 20%. The MAF for markers at PTPN22, IL2RA, FCGR2A and IL2/IL21 was below 2% in Africans. The GRS showed a strong association with RA in the UK. However, the GRS did not predict RA in Africans (OR = 0.71, 95% CI 0.29 - 1.74, p = 0.456). Random sampling from the UK cohort showed that this difference in association is unlikely to be explained by small sample size or chance, but is statistically significant with p<0.001.

Conclusions

The MAFs of non-HLA Caucasian RA susceptibility SNPs are different between Caucasians and Africans, and several polymorphisms are barely detectable in West/Central Africa. The genetic risk of developing RA conferred by a set of 28 Caucasian susceptibility SNPs is significantly different between the UK and Africa with p<0.001. Taken together, these observations strengthen the hypothesis that the genetic architecture of RA susceptibility is different in different ethnic backgrounds.     

The proinflammatory cytokine response to Chlamydia trachomatis elementary bodies in human macrophages is partly mediated by a lipoprotein, the macrophage infectivity potentiator, through TLR2/TLR1/TLR6 and CD14

Chlamydiae components and signaling pathway(s) responsible for the production of proinflammatory cytokines by human monocytes/macrophages are not clearly identified. To this aim, Chlamydia trachomatis-inactivated elementary bodies (EB) as well as the following seven individual Ags were tested for their ability to induce the production of proinflammatory cytokines by human monocytes/macrophages and THP-1 cells: purified LPS, recombinant heat shock protein (rhsp)70, rhsp60, rhsp10, recombinant polypeptide encoded by open reading frame 3 of the plasmid (rpgp3), recombinant macrophage infectivity potentiator (rMip), and recombinant outer membrane protein 2 (rOmp2). Aside from EB, rMip displayed the highest ability to induce release of IL-1beta, TNF-alpha, IL-6, and IL-8. rMip proinflammatory activity could not be attributed to Escherichia coli LPS contamination as determined by the Limulus Amoebocyte lysate assay, insensitivity to polymyxin B (50 microg/ml), and different serum requirement. We have recently demonstrated that Mip is a "classical" bacterial lipoprotein, exposed at the surface of EB. The proinflammatory activity of EB was significantly attenuated in the presence of polyclonal Ab to rMip. Native Mip was able to induce TNF-alpha and IL-8 secretion, whereas a nonlipidated C20A rMip variant was not. Proinflammatory activity of rMip was unaffected by heat or proteinase K treatments but was greatly reduced by treatment with lipases, supporting a role of lipid modification in this process. Stimulating pathways appeared to involve TLR2/TLR1/TLR6 with the help of CD14 but not TLR4. These data support a role of Mip lipoprotein in pathogenesis of C. trachomatis-induced inflammatory responses.     

Leptin signaling deficiency impairs humoral and cellular immune responses and attenuates experimental arthritis.

Leptin is produced almost exclusively by adipocytes and regulates body weight at the hypothalamic level. In addition, recent studies showed that leptin plays an important role in T lymphocyte responses. To examine the role of leptin in Ag-induced arthritis, the development of joint inflammation was assessed in immunized leptin-deficient mice (ob/ob), +/?, and wild-type mice (+/+) following the administration of methylated BSA into the knees. The results showed that ob/ob mice developed less severe arthritis compared with control mice. The levels of IL-1beta and TNF-alpha mRNA in the synovium of arthritic knees were lower in ob/ob than in +/? mice. In vitro Ag-specific T cell proliferative responses were significantly decreased in ob/ob mice with lower IFN-gamma and higher IL-10 production, suggesting a shift toward a Th2-type response in ob/ob mice. The serum levels of anti-methylated BSA Abs of any isotype were significantly decreased in arthritic ob/ob mice compared with controls. Essentially identical results were obtained in db/db mice, which lack the expression of the long isoform of leptin receptor. By RT-PCR, we observed that B lymphocytes express leptin receptor mRNA, indicating that in addition to its effect on the cellular response, leptin may exert a direct effect on B cell function. In conclusion, leptin contributes to the mechanisms of joint inflammation in Ag-induced arthritis by regulating both humoral and cell-mediated immune responses.     

Peptidomimetic fluoromethylketone rescues mice from lethal endotoxic shock.

BACKGROUND: Septic shock is a leading cause of mortality in intensive care units. No new interventions in the last 20 years have made a substantial impact on the outcome of patients with septic shock. Identification of inhibitable pathways that mediate death in shock is an important goal. MATERIALS AND METHODS: Two novel caspase inhibitors, (2-indolyl)-carbonyl-Ala-Asp-fluoromethylketone (IDN 1529) and (1-methyl-3-methyl-2-indolyl)-carbonyl-Val-Asp-fluoromethylketone (IDN 1965), were studied in a murine model of endotoxic shock. RESULTS: IDN 1529 prolonged survival when given before or up to 3 hr after high-dose LPS (p < 0.01) and increased by 2.2-fold the number of animals surviving longterm after a lower dose of LPS (p < 0.01). Despite its similar chemical structure, IDN 1965 lacked these protective effects. Both compounds inhibited caspases 1, 2, 3, 6, 8, and 9, and both afforded comparable reduction in Fas- and LPS-induced caspase 3-like activity and apoptosis. Paradoxically, administration of IDN 1529 but not IDN 1965 led to an increase in the LPS-induced elevation of serum cytokines related directly (IL-1beta, IL-18) or indirectly (IL-1alpha, IL-1Ra) to the action of caspase 1. CONCLUSIONS: A process that appears to be distinct from both apoptosis and the release of inflammatory cytokines is a late-acting requirement for lethality in endotoxic shock. Inhibition of this process can rescue mice even when therapy is initiated after LPS has made the mice severely ill.