Biglycan, a novel trigger of Th1 and Th17 cell recruitment into the kidney.

Th1 and Th17 cells, T helper (Th) subtypes, are key inducers of renal fibrosis. The molecular mechanisms of their recruitment into the kidney, however, are not well understood. Here, we show that biglycan, a proteoglycan of the extracellular matrix, acting in its soluble form as a danger signal, stimulates autonomously the production of Th1 and Th17 chemoattractants CXCL10 and CCL20 in macrophages. In the presence of IFNγ, biglycan synergistically stimulates CXCL9. In macrophages deficient for TLR2, TLR4, and their adaptor molecules MyD88 or TRIF, we identified highly selective mechanisms of biglycan-dependent Th1/17 chemoattraction. Thus, the expression of CXCL9 and CXCL10, common chemoattractants for CXCR3-positive Th1 and Th17 cells, is triggered in a biglycan-TLR4/TRIF-dependent manner. By contrast, biglycan induces CCL20 chemokine production, responsible for CCR6-positive Th17 cell recruitment, in a TLR2/4/MyD88-dependent manner. Importantly, at the onset of diabetes mellitus and lupus nephritis we provide evidence for biglycan-dependent recruitment of Th1 and Th17 cells, IFNγ and IL-17 production, and development of albuminuria in mice lacking or overexpressing soluble biglycan. Furthermore, by genetic ablation of Cxcl10 we showed in vivo involvement of this chemokine in biglycan-dependent recruitment of Th1 and Th17 cells into the kidney. Finally, a positive correlation of biglycan and CXCL10/CXCL9 levels was detected in plasma from patients with diabetic nephropathy and lupus nephritis. Taken together, we identified biglycan as a novel trigger of Th1 and Th17 cell recruitment into the kidney and we postulate that interfering with biglycan/TLR/TRIF/MyD88-signaling might provide novel therapeutic avenues for renal fibrosis.

An Essential Role for SHARPIN in the Regulation of Caspase 1 Activity in Sepsis.

Sepsis is burdened by high mortality due to uncontrolled inflammatory response to pathogens. Increased caspase 1 activation causing maturation of IL1ß/18 remains a therapeutic challenge in sepsis. SHARPIN (shank-associated regulator of G-protein signaling homology domain-interacting protein), a component of the LUBAC (linear ubiquitin chain-assembly complex), regulates inflammation, with unknown effects on caspase 1 activation. Mice lacking Casp1, Casp11, or both in a Sharpin-deficient background were generated, exposed to lipopolysaccharide-induced endotoxemia, and injected with caspase 1 inhibitor. We monitored survival, Il1ß/18, and caspase 1/11 levels in plasma and organs and deciphered mechanisms of SHARPIN-dependent caspase 1 inhibition. A correlation between LUBAC and active caspase 1 was found in blood mononuclear cells from septic patients. SHARPIN bound caspase 1 and disrupted p20/p10 dimer formation, the last step of caspase 1 processing, thereby inhibiting enzyme activation and maturation of IL1ß/18 in a LUBAC-independent manner. In septic patients, LUBAC-independent decline in SHARPIN correlated with enhancement of active caspase 1 in circulating mononuclear cells. Septic Sharpin-deficient mice displayed enrichment in mature Il1ß/18 and active caspase 1, and shortened survival. Inhibition of caspase 1 reduced levels of Il1ß/18 and splenic cell death, and prolonged survival in septic Sharpin-deficient mice. Our findings identify SHARPIN as a potent in vivo caspase 1 inhibitor and propose the caspase 1-SHARPIN interaction as a target in sepsis.

Effect of M3 muscarinic acetylcholine receptor deficiency on collagen antibody-induced arthritis.

BACKGROUND: There is increasing evidence that the non-neuronal cholinergic system might be of importance for the pathology of rheumatoid arthritis. The role of M3 muscarinic acetylcholine receptor (M3R) in this regard has, however, not been investigated to date. Thus, in the present study we analyzed if M3R deficiency might have a protective effect on experimentally induced arthritis. METHODS: Collagen antibody-induced arthritis (CAIA) was evoked in M3R-deficient (M3R(-/-)) mice and wild-type (WT) littermates. Severity of arthritis was assessed by scoring of paw swelling. The joints of arthritic and nonarthritic animals were analyzed for histopathological changes regarding synovial tissue, cartilage degradation and bone destruction. Further, gene expression analysis of respective markers was performed. Systemic and local inflammatory response was determined by flow cytometry and immunohistochemistry for leukocytes as well as mRNA and protein measurements for pro-inflammatory cytokines and chemokines. RESULTS: In arthritic M3R(-/-) mice the number of leukocytes, specifically neutrophils, was enhanced even though clinical arthritis score was not significantly different between WT and M3R(-/-) mice with CAIA. In M3R(-/-) mice, levels of neutrophil chemoattractant chemokine C-X-C-motif ligand 2 (CXCL2) as well as the pro-inflammatory cytokine interleukin-6 were already strongly increased in mice with low arthritis score, whereas WT mice only showed prominent expression of these markers when reaching high arthritis scores. Furthermore, arthritic M3R(-/-) mice displayed a stronger degradation of collagen II in the articular cartilage and, most strikingly, histopathological evaluation revealed more severe bone destruction in arthritic mice with M3R deficiency compared to WT littermates. Moreover, in M3R(-/-) mice, gene expression of markers for bone degradation (matrix metalloproteinase 13, cathepsin K and receptor activator of nuclear factor-κB ligand) was already increased in mice with low arthritis score. CONCLUSIONS: Taken together, the present study shows that while M3R(-/-) mice were not protected from CAIA, they had a tendency toward a higher inflammatory response after arthritis induction than WT mice. Further, arthritis-induced joint destruction was significantly stronger in mice with M3R deficiency, indicating that stimulation of M3R might have protective effects on arthritis.

Expression of choline and acetylcholine transporters in synovial tissue and cartilage of patients with rheumatoid arthritis and osteoarthritis.

Increasing evidence is showing that the non-neuronal cholinergic system plays an important role in the pathology of rheumatoid arthritis (RA). Choline transport into the cell is the rate-limiting step for the synthesis of acetylcholine (ACh), which can be released directly or in vesicles from the cell. However, in the human joint little is known about choline import or the release of ACh from the cell. Thus, we analyze the expression of members of the organic cation transporter (OCT), of the newly discovered choline transporter-like (CTL) family and of classical neuronal components such as the high-affinity choline transporter (CHT1) and the vesicular ACh transporter (VAChT) in the synovium and cartilage of the human hip joint from patients with osteoarthritis (OA) and RA. OCT1, OCT3 and OCTN1 and all members of the CTL family were expressed in synovial and cartilage samples. The expression of CTL1 and CTL2 was localized in synovial macrophages and fibroblasts. CHT1 mRNA expression was detectable only in the synovium, whereas VAChT was completely absent in all samples. Therefore, in the human joint, choline transport into the cell and the release of ACh seems to be mediated mainly by members of the OCT and CTL family. Expression of transporters appears not to be influenced by the pathological state, as no differences have been detected between joints from OA or RA patients. Importantly, however, all necessary components for choline import and the release of non-neuronal ACh are present in the human joint.

De novo expression of circulating biglycan evokes an innate inflammatory tissue response via MyD88/TRIF pathways.

Matrix-bound constituents, such as the small leucine-rich proteoglycan biglycan, can act as powerful signaling molecules when released by limited proteolysis of the extracellular matrix or de novo synthesized by macrophages in the circulation and body fluids. Specifically, biglycan acts as an endogenous ligand of innate immunity by directly engaging the Toll-like receptor (TLR)-2 and -4. In this study, we generated a transient transgenic mouse model where biglycan was de novo overproduced by hepatocytes driven by the albumin promoter. Transgenic biglycan was rapidly and abundantly synthesized by hepatocytes and released into the bloodstream. Notably, we found that circulating biglycan accumulated in the kidneys where it caused recruitment of leukocytes infiltrating the renal parenchyma concurrent with abnormal renal levels of chemoattractants CXCL1, CXCL2, CCL2 and CCL5. Using mice deficient in either TLR adapter proteins MyD88 or TRIF we discovered that MyD88 deficiency drastically reduced neutrophil and macrophage infiltration in the kidney, whereas TRIF deficiency decreased T cell infiltrates. Production of CXCL1, CXCL2 and CCL2 required MyD88, whereas the levels of T cell and macrophage attractant CCL5 required TRIF. Thus, we provide robust genetic evidence for circulating biglycan as a powerful pro-inflammatory mediator targeting the renal parenchyma. Furthermore, our results provide the first evidence that biglycan differentially triggers chemoattraction of leukocytes via two independent pathways, both under the control of TLR2/4, utilizing either MyD88 or TRIF adaptor proteins. As aberrant expression of biglycan occurs in several inflammatory diseases, this transient transgenic mouse model could serve as a valuable research tool in investigating the effects of increased biglycan expression in vivo and for the development of therapeutic strategies in the treatment of inflammatory diseases.

The non-neuronal cholinergic system in health and disease.

Acetylcholine (ACh) is not only a neurotransmitter but is an ancient molecule that can be released by and act on non-neuronal cells. In these cells the system of ACh-synthesizing enzymes, transporters, receptors and degrading enzymes is termed the non-neuronal cholinergic system (NNCS). There is increasing evidence that the NNCS is dysregulated in various diseases and can have an influence on their pathology. However, for many organ systems not much is known about the expression and function of the NNCS. Thus, this review focusses on the role of the NNCS in different organ systems in health and disease. Dysregulation of ACh synthesis and release, mutations or polymorphisms in genes encoding NNCS components, and auto-antibodies against NNCS components are common factors influencing disease progression. Pharmacological agents targeting the NNCS are already successfully in clinical use for some disorders, indicating that interfering with this system is very promising and more research is needed to elucidate the role of the NNCS in different tissues and pathological states.

Role of Nox4 in murine models of kidney disease.

Nox4 is a hydrogen peroxide-producing NADPH oxidase highly expressed in the kidney which has been linked to epithelial cell injury and diabetic-induced cellular dysfunction in cultured cells. The role of the enzyme for renal pathology in vivo, however, is unclear. To address this, three experimental animal models of renal injury (streptozotocin diabetes I, unilateral ureteral ligation (UUO), and 5/6 nephrectomy (5/6Nx)) were studied in either Nox4-inducible (Nox4(*/*)) or constitutive knockout (Nox4(-/-)) mice. Nox4 contributed more than 80% of diphenylene iodonium-sensitive H(2)O(2) formation of freshly isolated tubules determined by Amplex Red assay. In streptozotocin diabetes, acute deletion of Nox4 by tamoxifen-activated cre-recombinase increased albuminuria, whereas matrix deposition was similar between WT and Nox4(*/*) mice. Interestingly, renal Nox4 expression, mainly localized to tubular cells, decreased in the course of diabetes and this was not associated with a compensatory upregulation of Nox1 or Nox2. In the UUO model, renal expression of ICAM1, connective tissue growth factor, and fibronectin were higher in kidneys of Nox4(*/*) than control mice. Also in this model, levels of Nox4 decreased in the course of the disease. In the 5/6Nx model, which was performed in SV129 and SV129-Nox4(-/-) mice, no difference in the development of hypertension and albuminuria was found between the strains. Collectively, the first in vivo data of the kidney do not support the view that Nox4 is a main driver of renal disease. It rather appears that under specific conditions Nox4 may even slightly limit injury and disease progression.

Expression of the non-neuronal cholinergic system in human knee synovial tissue from patients with rheumatoid arthritis and osteoarthritis.

AIMS: As the stimulation of the α7-nicotinic acetylcholine receptor (nAChR), which is present in the synovium of patients with rheumatoid arthritis (RA), leads to a decrease in pro-inflammatory cytokines, the α7-nAChR is being discussed as a new therapeutic target. On this background we addressed the question whether α7-nAChR mRNA was differentially expressed in RA compared to osteoarthritis (OA) synovial samples and whether other components of the non-neuronal cholinergic system were also present and differentially expressed in the synovium of patients with RA in comparison to OA. MAIN METHODS: The expression of nicotinic and muscarinic acetylcholine receptors (mAChRs), choline and acetylcholine transporters, synthesising and degrading enzymes was determined in human samples of synovial tissue from patients with RA and OA using RT-PCR and immunofluorescence labelling. KEY FINDINGS: Compared to OA, patients with RA showed increased expression of nAChR subunit ß4 while a decline in subunits α2 and α4 as well as in mAChR M1R was observed. For all other nAChR subunits and mAChRs however there was no significant difference between RA and OA patients. With regard to the ACh transporters and enzymes no expressional changes were observed between OA and RA patients, except for the choline acetyltransferase (ChAT) which was only detected in OA but not in RA synovium. SIGNIFICANCE: Our results indicate that besides α7-nAChR other components of the non-neuronal cholinergic system are present and differentially expressed in the synovium of RA and OA patients, which makes them interesting alternative targets in the development of new strategies for RA therapy.

Signaling by the matrix proteoglycan decorin controls inflammation and cancer through PDCD4 and MicroRNA-21.

The mechanisms linking immune responses and inflammation with tumor development are not well understood. Here, we show that the soluble form of the extracellular matrix proteoglycan decorin controls inflammation and tumor growth through PDCD4 (programmed cell death 4) and miR-21 (microRNA-21) by two mechanisms. First, decorin acted as an endogenous ligand of Toll-like receptors 2 and 4 and stimulated production of proinflammatory molecules, including PDCD4, in macrophages. Second, decorin prevented translational repression of PDCD4 by decreasing the activity of transforming growth factor-ß1 and the abundance of oncogenic miR-21, a translational inhibitor of PDCD4. Moreover, increased PDCD4 abundance led to decreased release of the anti-inflammatory cytokine interleukin-10, thereby making the cytokine profile more proinflammatory. This pathway operates in both pathogen-mediated and sterile inflammation, as shown here for sepsis and growth retardation of established tumor xenografts, respectively. Decorin was an early response gene evoked by septic inflammation, and protein concentrations of decorin were increased in the plasma of septic patients and mice. In cancer, decorin reduced the abundance of anti-inflammatory molecules and increased that of proinflammatory molecules, thereby shifting the immune response to a proinflammatory state associated with reduced tumor growth. Thus, by stimulating proinflammatory PDCD4 and decreasing the abundance of miR-21, decorin signaling boosts inflammatory activity in sepsis and suppresses tumor growth.

CXCL16 and oxLDL are induced in the onset of diabetic nephropathy.

Diabetic nephropathy (DN) is a major cause of end-stage renal failure worldwide. Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN. In this study we present evidence, that CXCL16 is the main receptor in human podocytes mediating the uptake of oxLDL. In contrast, in primary tubular cells CD36 was mainly involved in the uptake of oxLDL. We further demonstrate that oxLDL down-regulated alpha(3)-integrin expression and increased the production of fibronectin in human podocytes. In addition, oxLDL uptake induced the production of reactive oxygen species (ROS) in human podocytes. Inhibition of oxLDL uptake by CXCL16 blocking antibodies abrogated the fibronectin and ROS production and restored alpha(3) integrin expression in human podocytes. Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes. Importantly, in streptozotocin-induced diabetic mice an early induction of CXCL16 was accompanied by higher levels of oxLDL. Finally immunofluorescence analysis in biopsies of patients with DN revealed increased glomerular CXCL16 expression, which was paralleled by high levels of oxLDL. In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN.

Subunit composition and pigmentation of fucoxanthin-chlorophyll proteins in diatoms: evidence for a subunit involved in diadinoxanthin and diatoxanthin binding.

Two different fucoxanthin-chlorophyll protein complexes (FCP) were purified from the centric diatom Cyclotella meneghiniana and characterized with regard to their polypeptide and pigment composition. Whereas the oligomeric FCPb complex is most probably composed of fcp5 gene products, the trimeric FCPa has subunits encoded by fcp1-3 and fcp6/7. The amount of the latter polypeptide is enhanced when FCPa is isolated from algae grown under HL conditions. This increase in Fcp6/7 polypeptides is accompanied by an increase in the pool of xanthophyll cycle pigments, diadinoxanthin and diatoxanthin, and a concomitant decrease in fucoxanthin content. In addition, the de-epoxidation ratio, i.e., the amount of diatoxanthin in relation to the pool of xanthophyll cycle pigments, is increased by a factor of 2. With regard to fluorescence yield, HL FCPa was quenched in comparison to LL FCPa. This is in accordance with the larger amount of diatoxanthin that is bound, which is supposed to act as a quencher like zeaxanthin in higher plants. Thus, we conclude that the enhanced content of diatoxanthin in FCPa plays a protective role, which is paralleled by a weakened light harvesting function due to a smaller amount of fucoxanthin.