Differential expression of microRNA miR-150-5p in IgA nephropathy as a potential mediator and marker of disease progression.

Understanding why certain patients with IgA nephropathy progress to kidney failure while others maintain normal kidney function remains a major unanswered question. To help answer this, we performed miRNome profiling by next generation sequencing of kidney biopsies in order to identify microRNAs specifically associated with the risk of IgA nephropathy progression. Following sequencing and validation in independent cohorts, four microRNAs (-150-5p, -155-5p, -146b-5p, -135a-5p) were found to be differentially expressed in IgA nephropathy progressors compared to non-progressors, and patients with thin membrane nephropathy, lupus nephritis and membranous nephropathy, and correlated with estimated glomerular filtration rate, proteinuria, and the Oxford MEST-C scores (five histological features that are independent predictors of clinical outcome). Each individual microRNA increased the discrimination score of the International IgAN Prediction Tool, although due to the small number of samples the results did not reach statistical significance. miR-150-5p exhibited the largest amplitude of expression between cohorts and displayed the best discrimination between IgA nephropathy progressors and non-progressors by receiver operating curve analysis (AUC: 0.8). However, expression was similarly upregulated in kidneys with established fibrosis and low estimated glomerular filtration rates at the time of biopsy. Consistent with a more generic role in kidney fibrosis, in situ hybridization revealed that miR-150-5p was found in lymphoid infiltrates, and areas of proliferation and fibrosis consistent with the known drivers of progression. Thus, miR-150-5p may be a potential functional mediator of kidney fibrosis that may add value in predicting risk of progression in IgA nephropathy and other kidney diseases.

The Metalloproteinase ADAMTS5 Is Expressed by Interstitial Inflammatory Cells in IgA Nephropathy and Is Proteolytically Active on the Kidney Matrix.

In IgA nephropathy (IgAN), IgA immune complexes are deposited in the mesangium and drive inflammation and extracellular matrix (ECM) remodelling. The functional links between IgA deposition, inflammation, and matrix remodelling are not well characterized. We recently performed urine liquid chromatography-tandem mass spectrometry proteomics and identified multiple ECM glycoproteins whose expression and function in IgAN is unclear. None of the urine glycoproteins was regulated in IgAN transcriptomics, indicating that tissue remodelling rather than increased expression might contribute to their presence in urine. To investigate this, we examined the IgAN expression profile of metalloproteinases, enzymes involved in the remodelling of ECM proteins, and noted that the proteoglycanase ADAMTS5 was upregulated in IgAN kidneys. ADAMTS5 accumulated in areas of inflammation, and ADAMTS5+ cells were seen in the tubulointerstitium and glomeruli. The enzyme was expressed by CD64+ cells and its expression was increased by IL-1 and LPS. Analysis of myeloid cell transcriptomics revealed that ADAMTS5 is enriched in human classical monocytes. ADAMTS5+ cells were present in areas of matrix remodelling and associated with ECM proteins lumican, versican, and collagen-4. Liquid chromatography-tandem mass spectrometry proteomics of kidney explants digested with ADAMTS5, identified multiple kidney proteins affected by ADAMTS5 and revealed specific proteolysis of complement C3 and fibronectin associated with IgA on immune complexes. ADAMTS5 processing of immune complex proteins reduced binding to cultured mesangial cells. ADAMTS5 is associated with interstitial inflammatory cells in IgAN and other kidney lesions and fragments relevant extracellular proteins. The proteolytic enzyme might be a new translational target relevant to inflammation and scarring in kidney disease.

COVID-19 Hyperinflammation: What about Neutrophils?

COVID-19 is often related to hyperinflammation that drives lung or multiorgan injury. The immunopathological mechanisms that cause excessive inflammation are under investigation and constantly updated. Here, a gene network approach was used on recently published data sets to identify possible COVID-19 inflammatory mechanisms and bioactive genes. First, network analysis of putative SARS-CoV-2 cellular receptors led to the mining of a neutrophil-response signature and relevant inflammatory genes. Second, analysis of RNA-seq data sets of lung cells infected with SARS-CoV-2 revealed that infected cells expressed neutrophil-attracting chemokines. Third, analysis of RNA-seq data sets of bronchoalveolar lavage fluid cells from COVID-19 patients identified upregulation of neutrophil genes and chemokines. Different inflammatory genes mined here, including TNFR, IL-8, CXCR1, CXCR2, ADAM10, GPR84, MME, ANPEP, and LAP3, might be druggable targets in efforts to limit SARS-CoV-2 inflammation in severe clinical cases. The possible role of neutrophils in COVID-19 inflammation needs to be studied further.

From expression footprints to causal pathways: contextualizing large signaling networks with CARNIVAL.

While gene expression profiling is commonly used to gain an overview of cellular processes, the identification of upstream processes that drive expression changes remains a challenge. To address this issue, we introduce CARNIVAL, a causal network contextualization tool which derives network architectures from gene expression footprints. CARNIVAL (CAusal Reasoning pipeline for Network identification using Integer VALue programming) integrates different sources of prior knowledge including signed and directed protein-protein interactions, transcription factor targets, and pathway signatures. The use of prior knowledge in CARNIVAL enables capturing a broad set of upstream cellular processes and regulators, leading to a higher accuracy when benchmarked against related tools. Implementation as an integer linear programming (ILP) problem guarantees efficient computation. As a case study, we applied CARNIVAL to contextualize signaling networks from gene expression data in IgA nephropathy (IgAN), a condition that can lead to chronic kidney disease. CARNIVAL identified specific signaling pathways and associated mediators dysregulated in IgAN including Wnt and TGF-ß, which we subsequently validated experimentally. These results demonstrated how CARNIVAL generates hypotheses on potential upstream alterations that propagate through signaling networks, providing insights into diseases.

Phosphatidylethanolamine binding protein-4 (PEBP4) is increased in IgA nephropathy and is associated with IgA-positive B-cells in affected kidneys.

IgA nephropathy (IgAN) is the most common glomerulonephritis worldwide and a major cause of chronic kidney disease and failure. IgAN is driven by an autoimmune reaction against galactose-deficient IgA1 that results in the generation of autoantibodies and large IgG-IgA immune complexes. Immune complexes accumulate in the glomerular mesangium causing chronic inflammation and renal scarring. A significant proportion of IgAN patients develop end-stage kidney disease and require dialysis or transplantation. Currently, there are no approved specific therapies that can ameliorate the systemic autoimmune reaction in IgAN and no biomarkers that can predict renal inflammation and scarring. In this study, we used shotgun LC-MS/MS proteomics to compare small volumes of urine from healthy subjects and IgAN patients. We identified multiple urine proteins with unknown renal or IgAN function. Our attention was captured by the increase of phosphatidylethanolamine binding protein-4 (PEBP4) in IgAN urine. The function of PEBP4 in IgAN or renal disease is unknown. Increased levels of urine and serum PEBP4 were subsequently validated in different cohorts of IgAN patients and PEBP4 was linked to declining kidney function in IgAN. Strong PEBP4 staining was sporadically seen in IgAN kidney biopsies, colocalising with IgA in glomeruli and in the lumen of kidney tubules. In a small number of IgAN biopsies, PEBP4 colocalised with IgA and CD19 while the increased excretion of PEBP4 in IgAN urine was accompanied by increased excretion of classic B-cell factors BAFF, BCMA and TACI as well as IgA and IgG. PEBP4 is a new IgAN-related protein with unknown function and a likely renal disease marker in urine and serum.

Comparative Transcriptomics of Rat and Axolotl After Spinal Cord Injury Dissects Differences and Similarities in Inflammatory and Matrix Remodeling Gene Expression Patterns.

Following spinal cord injury in mammals, maladaptive inflammation, and matrix deposition drive tissue scarring and permanent loss of function. In contrast, axolotls regenerate their spinal cord after severe injury fully and without scarring. To explore previously unappreciated molecules and pathways that drive tissue responses after spinal cord injury, we performed a 4-way intersection of rat and axolotl transcriptomics datasets and isolated shared genes with similar or differential expression at days 1, 3, and 7 after spinal cord injury in both species. Systems-wide differences and similarities between the two species are described in detail using public-domain computational tools and key differentially regulated genes are highlighted. Amongst persistent differential expression in matching neuronal genes (upregulated in axolotls but downregulated in rats) and nucleic acid metabolism genes (downregulated in axolotls but upregulated in rats), we found multiple extracellular matrix genes that were upregulated in both species after spinal cord injury and all time-points (days 1, 3, and 7), indicating the importance of extracellular matrix remodeling in wound healing. Moreover, the archetypal transcription factor SP1, which was consistently upregulated in rats but was unchanged in axolotls, was predicted as a potential transcriptional regulator of classic inflammatory response genes in rats most of which were not regulated in regenerating axolotls. This analysis offers an extensive comparative platform between a non-regenerating mammal and a regenerating urodele after spinal cord injury. To better understand regeneration vs. scarring mechanisms it is important to understand consistent molecular differences as well as similarities after experimental spinal cord injury.

Connective tissue growth factor contributes to joint homeostasis and osteoarthritis severity by controlling the matrix sequestration and activation of latent TGFß.

OBJECTIVES: One mechanism by which cartilage responds to mechanical load is by releasing heparin-bound growth factors from the pericellular matrix (PCM). By proteomic analysis of the PCM, we identified connective tissue growth factor (CTGF) and here investigate its function and mechanism of action. METHODS: Recombinant CTGF (rCTGF) was used to stimulate human chondrocytes for microarray analysis. Endogenous CTGF was investigated by in vitro binding assays and confocal microscopy. Its release from cut cartilage (injury CM) was analysed by Western blot under reducing and non-reducing conditions. A postnatal, conditional CtgfcKO mouse was generated for cartilage injury experiments and to explore the course of osteoarthritis (OA) by destabilisation of the medial meniscus. siRNA knockdown was performed on isolated human chondrocytes. RESULTS: The biological responses of rCTGF were TGFß dependent. CTGF displaced latent TGFß from cartilage and both were released on cartilage injury. CTGF and latent TGFß migrated as a single high molecular weight band under non-reducing conditions, suggesting that they were in a covalent (disulfide) complex. This was confirmed by immunoprecipitation. Using CtgfcKO mice, CTGF was required for sequestration of latent TGFß in the matrix and activation of the latent complex at the cell surface through TGFßR3. In vivo deletion of CTGF increased the thickness of the articular cartilage and protected mice from OA. CONCLUSIONS: CTGF is a latent TGFß binding protein that controls the matrix sequestration and activation of TGFß in cartilage. Deletion of CTGF in vivo caused a paradoxical increase in Smad2 phosphorylation resulting in thicker cartilage that was protected from OA.

Immune-evasive gene switch enables regulated delivery of chondroitinase after spinal cord injury.

Chondroitinase ABC is a promising preclinical therapy that promotes functional neuroplasticity after CNS injury by degrading extracellular matrix inhibitors. Efficient delivery of chondroitinase ABC to the injured mammalian spinal cord can be achieved by viral vector transgene delivery. This approach dramatically modulates injury pathology and restores sensorimotor functions. However, clinical development of this therapy is limited by a lack of ability to exert control over chondroitinase gene expression. Prior experimental gene regulation platforms are likely to be incompatible with the non-resolving adaptive immune response known to occur following spinal cord injury. Therefore, here we apply a novel immune-evasive dual vector system, in which the chondroitinase gene is under a doxycycline inducible regulatory switch, utilizing a chimeric transactivator designed to evade T cell recognition. Using this novel vector system, we demonstrate tight temporal control of chondroitinase ABC gene expression, effectively removing treatment upon removal of doxycycline. This enables a comparison of short and long-term gene therapy paradigms in the treatment of clinically-relevant cervical level contusion injuries in adult rats. We reveal that transient treatment (2.5 weeks) is sufficient to promote improvement in sensory axon conduction and ladder walking performance. However, in tasks requiring skilled reaching and grasping, only long term treatment (8 weeks) leads to significantly improved function, with rats able to accurately grasp and retrieve sugar pellets. The late emergence of skilled hand function indicates enhanced neuroplasticity and connectivity and correlates with increased density of vGlut1+ innervation in spinal cord grey matter, particularly in lamina III-IV above and below the injury. Thus, our novel gene therapy system provides an experimental tool to study temporal effects of extracellular matrix digestion as well as an encouraging step towards generating a safer chondroitinase gene therapy strategy, longer term administration of which increases neuroplasticity and recovery of descending motor control. This preclinical study could have a significant impact for tetraplegic individuals, for whom recovery of hand function is an important determinant of independence, and supports the ongoing development of chondroitinase gene therapy towards clinical application for the treatment of spinal cord injury.

Combined Transcriptomics, Proteomics and Bioinformatics Identify Drug Targets in Spinal Cord Injury.

Spinal cord injury (SCI) causes irreversible tissue damage and severe loss of neurological function. Currently, there are no approved treatments and very few therapeutic targets are under investigation. Here, we combined 4 high-throughput transcriptomics and proteomics datasets, 7 days and 8 weeks following clinically-relevant rat SCI to identify proteins with persistent differential expression post-injury. Out of thousands of differentially regulated entities our combined analysis identified 40 significantly upregulated versus 48 significantly downregulated molecules, which were persistently altered at the mRNA and protein level, 7 days and 8 weeks post-SCI. Bioinformatics analysis was then utilized to identify currently available drugs with activity against the filtered molecules and to isolate proteins with known or unknown function in SCI. Our findings revealed multiple overlooked therapeutic candidates with important bioactivity and established druggability but with unknown expression and function in SCI including the upregulated purine nucleoside phosphorylase (PNP), cathepsins A, H, Z (CTSA, CTSH, CTSZ) and proteasome protease PSMB10, as well as the downregulated ATP citrate lyase (ACLY), malic enzyme (ME1) and sodium-potassium ATPase (ATP1A3), amongst others. This work reveals previously unappreciated therapeutic candidates for SCI and available drugs, thus providing a valuable resource for further studies and potential repurposing of existing therapeutics for SCI.

Role of ADAMTS-5 in Aortic Dilatation and Extracellular Matrix Remodeling.

OBJECTIVE: Thoracic aortic aneurysm (TAA), a degenerative disease of the aortic wall, is accompanied by changes in the structure and composition of the aortic ECM (extracellular matrix). The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases has recently been implicated in TAA formation. This study aimed to investigate the contribution of ADAMTS-5 to TAA development. APPROACH AND RESULTS: A model of aortic dilatation by AngII (angiotensin II) infusion was adopted in mice lacking the catalytic domain of ADAMTS-5 (Adamts5Δcat). Adamts5Δcat mice showed an attenuated rise in blood pressure while displaying increased dilatation of the ascending aorta (AsAo). Interestingly, a proteomic comparison of the aortic ECM from AngII-treated wild-type and Adamts5Δcat mice revealed versican as the most upregulated ECM protein in Adamts5Δcat mice. This was accompanied by a marked reduction of ADAMTS-specific versican cleavage products (versikine) and a decrease of LRP1 (low-density lipoprotein-related protein 1). Silencing LRP1 expression in human aortic smooth muscle cells reduced the expression of ADAMTS5, attenuated the generation of versikine, but increased soluble ADAMTS-1. A similar increase in ADAMTS-1 was observed in aortas of AngII-treated Adamts5Δcat mice but was not sufficient to maintain versican processing and prevent aortic dilatation. CONCLUSIONS: Our results support the emerging role of ADAMTS proteases in TAA. ADAMTS-5 rather than ADAMTS-1 is the key protease for versican regulation in murine aortas. Further studies are needed to define the ECM substrates of the different ADAMTS proteases and their contribution to TAA formation.

Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques.

BACKGROUND: The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the "vulnerable plaque," necessitating an improved approach for predicting onset of symptoms. METHODS: We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies. RESULTS: Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study. CONCLUSION: The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery. FUNDING: UK: British Heart Foundation (BHF); King's BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy's and St Thomas' NHS Foundation Trust and King's College London in partnership with King's College Hospital. Austria: Federal Ministry for Transport, Innovation and Technology (BMVIT); Federal Ministry of Science, Research and Economy (BMWFW); Wirtschaftsagentur Wien; and Standortagentur Tirol.

Manipulating the extracellular matrix: an animal model of the bladder pain syndrome.

Bladder pain syndrome (BPS) is associated with breakdown of the protective uroepithelial barrier of the urinary bladder allowing urinary constituents access to bladder sensory neurons. Although there are several animal models of cystitis, none specifically relates to BPS. Here, we aimed to create such a model using enzymatic digestion of the barrier proteoglycans (PGs) in the rat. Twenty female Wistar rats were anaesthetized and transurethrally catheterized. Ten animals were treated with 0.25IU of intravesical chondroitinase ABC and heparanase III to digest chondroitin sulphate and heparin sulphate PGs, respectively. Ten animals received saline. Following PG deglycosylation, bladders showed irregular loss of the apical uroplakin and a significant increase in neutrophils, not evident in the control group. Spinal cord sections were also collected for c-fos analysis. A large and significant increase in fos immunoreactivity in the L6/S1 segments in the treatment vs control bladders was observed. Cystometry was performed on 5 treatment and 5 control animals. Analysis revealed a significant increase in micturition reflex excitability postdeglycosylation. On a further group of 10 animals, von Frey mechanical withdrawal thresholds were tested on abdominal skin before and after PG digestions. There was a significant decrease in abdominal mechanical withdrawal threshold postdeglycosylation compared with controls. The results of this animal study suggest that many of the clinical features of BPS are seen after PG digestion from the bladder lumen. This model can be used to further understand mechanisms of pain in patients with BPS and to test new therapeutic strategies.

Rapid Activation of Transforming Growth Factor ß-Activated Kinase 1 in Chondrocytes by Phosphorylation and K63 -Linked Polyubiquitination Upon Injury to Animal Articular Cartilage.

OBJECTIVE: Mechanical injury to cartilage predisposes to osteoarthritis (OA). Wounding of the articular cartilage surface causes rapid activation of MAP kinases and NF-κB, mimicking the response to inflammatory cytokines. This study was undertaken to identify the upstream signaling mechanisms involved. METHODS: Cartilage was injured by dissecting it from the articular surface of porcine metacarpophalangeal (MCP) joints or by avulsing murine proximal femoral epiphyses. Protein phosphorylation was assayed by Western blotting of cartilage lysates. Immunolocalization of phosphorylated activating transcription factor 2 (ATF-2) and NF-κB/p65 was detected by confocal microscopy. Messenger RNA (mRNA) was measured by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Receptor associated protein 80 (RAP-80) ubiquitin interacting motif agarose was used in a pull-down assay to obtain K63 -polyubiquitinated proteins. Ubiquitin linkages on immunoprecipitated transforming growth factor ß-activated kinase 1 (TAK-1) were analyzed with deubiquitinases. RESULTS: Sharp injury to porcine cartilage caused rapid activation of JNK and NF-κB pathways and the upstream kinases MKK-4, IKK, and TAK-1. Pharmacologic inhibition of TAK-1 in porcine cartilage abolished JNK and NF-κB activation and reduced the injury-dependent inflammatory gene response. High molecular weight species of phosphorylated TAK-1 were induced by injury, indicating its ubiquitination. An overall increase in K63 -linked polyubiquitination was detected upon injury, and TAK-1 was specifically linked to K63 - but not K48 -polyubiquitin chains. In mice, avulsion of wild-type femoral epiphyses caused similar intracellular signaling that was reduced in cartilage-specific TAK-1-null mice. Epiphyseal cartilage of MyD88-null and TRAF-6-null mice responded to injury, suggesting the involvement of a ubiquitin E3 ligase other than TRAF-6. CONCLUSION: Activation of TAK-1 by phosphorylation and K63 -linked polyubiquitination by injury indicates its role in driving cell activation. Further studies are needed to identify the upstream ubiquitination mechanisms, including the E3 ligase involved.

Persistent Alterations in Microglial Enhancers in a Model of Chronic Pain.

Chronic pain is a common and devastating condition that induces well-characterized changes in neurons and microglia. One major unanswered question is why these changes should persist long after the precipitating injury has healed. Here, we suggest that some of the longer-lasting consequences of nerve injury may be hidden in the epigenome. Cell sorting and sequencing techniques were used to characterize the spinal cord immune response in a mouse model of chronic neuropathic pain. Infiltration of peripheral myeloid cells was found to be absent, and RNA sequencing (RNA-seq) of central microglia revealed transient gene expression changes in response to nerve ligation. Conversely, examination of microglial enhancers revealed persistent, post-injury alterations in close proximity to transcriptionally regulated genes. Enhancers are regions of open chromatin that define a cell's transcription factor binding profile. We hypothesize that changes at enhancers may constitute a mechanism by which painful experiences are recorded at a molecular level.

The Expression of Inflammatory Mediators in Bladder Pain Syndrome.

BACKGROUND: Bladder pain syndrome (BPS) pathology is poorly understood. Treatment strategies are empirical, with limited efficacy, and affected patients have diminished quality of life. OBJECTIVE: We examined the hypothesis that inflammatory mediators within the bladder contribute to BPS pathology. DESIGN, SETTING, AND PARTICIPANTS: Fifteen women with BPS and 15 women with stress urinary incontinence without bladder pain were recruited from Cork University Maternity Hospital from October 2011 to October 2012. During cystoscopy, 5-mm bladder biopsies were taken and processed for gene expression analysis. The effect of the identified genes was tested in laboratory animals. OUTCOME MEASURES AND STATISTICAL ANALYSIS: We studied the expression of 96 inflammation-related genes in diseased and healthy bladders. We measured the correlation between genes and patient clinical profiles using the Pearson correlation coefficient. RESULTS AND LIMITATIONS: Analysis revealed 15 differentially expressed genes, confirmed in a replication study. FGF7 and CCL21 correlated significantly with clinical outcomes. Intravesical CCL21 instillation in rats caused increased bladder excitability and increased c-fos activity in spinal cord neurons. CCL21 atypical receptor knockout mice showed significantly more c-fos upon bladder stimulation with CCL21 than wild-type littermates. There was no change in FGF7-treated animals. The variability in patient samples presented as the main limitation. We used principal component analysis to identify similarities within the patient group. CONCLUSIONS: Our study identified two biologically relevant inflammatory mediators in BPS and demonstrated an increase in nociceptive signalling with CCL21. Manipulation of this ligand is a potential new therapeutic strategy for BPS. PATIENT SUMMARY: We compared gene expression in bladder biopsies of patients with bladder pain syndrome (BPS) and controls without pain and identified two genes that were increased in BPS patients and correlated with clinical profiles. We tested the effect of these genes in laboratory animals, confirming their role in bladder pain. Manipulating these genes in BPS is a potential treatment strategy.

High-throughput proteomics reveal alarmins as amplifiers of tissue pathology and inflammation after spinal cord injury.

Spinal cord injury is characterized by acute cellular and axonal damage followed by aggressive inflammation and pathological tissue remodelling. The biological mediators underlying these processes are still largely unknown. Here we apply an innovative proteomics approach targeting the enriched extracellular proteome after spinal cord injury for the first time. Proteomics revealed multiple matrix proteins not previously associated with injured spinal tissue, including small proteoglycans involved in cell-matrix adhesion and collagen fibrillogenesis. Network analysis of transcriptomics and proteomics datasets uncovered persistent overexpression of extracellular alarmins that can trigger inflammation via pattern recognition receptors. In mechanistic experiments, inhibition of toll-like receptor-4 (TLR4) and the receptor for advanced glycation end-products (RAGE) revealed the involvement of alarmins in inflammatory gene expression, which was found to be dominated by IL1 and NFκΒ signalling. Extracellular high-mobility group box-1 (HMGB1) was identified as the likely endogenous regulator of IL1 expression after injury. These data reveal a novel tissue remodelling signature and identify endogenous alarmins as amplifiers of the inflammatory response that promotes tissue pathology and impedes neuronal repair after spinal cord injury.

The role of G-protein receptor 84 in experimental neuropathic pain.

G-protein receptor 84 (GPR84) is an orphan receptor that is induced markedly in monocytes/macrophages and microglia during inflammation, but its pathophysiological function is unknown. Here, we investigate the role of GPR84 in a murine model of traumatic nerve injury. Naive GPR84 knock-out (KO) mice exhibited normal behavioral responses to acute noxious stimuli, but subsequent to partial sciatic nerve ligation (PNL), KOs did not develop mechanical or thermal hypersensitivity, in contrast to wild-type (WT) littermates. Nerve injury increased ionized calcium binding adapter molecule 1 (Iba1) and phosphorylated p38 MAPK immunoreactivity in the dorsal horn and Iba1 and cluster of differentiation 45 expression in the sciatic nerve, with no difference between genotypes. PCR array analysis revealed that Gpr84 expression was upregulated in the spinal cord and sciatic nerve of WT mice. In addition, the expression of arginase-1, a marker for anti-inflammatory macrophages, was upregulated in KO sciatic nerve. Based on this evidence, we investigated whether peripheral macrophages behave differently in the absence of GPR84. We found that lipopolysaccharide-stimulated KO macrophages exhibited attenuated expression of several proinflammatory mediators, including IL-1ß, IL-6, and TNF-α. Forskolin-stimulated KO macrophages also showed greater cAMP induction, a second messenger associated with immunosuppression. In summary, our results demonstrate that GPR84 is a proinflammatory receptor that contributes to nociceptive signaling via the modulation of macrophages, whereas in its absence the response of these cells to an inflammatory insult is impaired.

Regulation of IL-10 by chondroitinase ABC promotes a distinct immune response following spinal cord injury.

Chondroitinase ABC (ChABC) has striking effects on promoting neuronal plasticity after spinal cord injury (SCI), but little is known about its involvement in other pathological mechanisms. Recent work showed that ChABC might also modulate the immune response by promoting M2 macrophage polarization. Here we investigate in detail the immunoregulatory effects of ChABC after SCI in rats. Initially, we examined the expression profile of 16 M1/M2 macrophage polarization markers at 3 h and 7 d postinjury. ChABC treatment had a clear effect on the immune signature after SCI. More specifically, ChABC increased the expression of the anti-inflammatory cytokine IL-10, accompanied by a reduction in the proinflammatory cytokine IL-12B in injured spinal tissue. These effects were associated with a distinct, IL-10-mediated anti-inflammatory response in ChABC-treated spinal cords. Mechanistically, we show that IL-10 expression is driven by tissue injury and macrophage infiltration, while the p38 MAPK is the central regulator of IL-10 expression in vivo. These findings provide novel insights into the effects of ChABC in the injured spinal cord and explain its immunoregulatory activity.

Large-scale chondroitin sulfate proteoglycan digestion with chondroitinase gene therapy leads to reduced pathology and modulates macrophage phenotype following spinal cord contusion injury.

Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we use mammalian-compatible engineered chondroitinase ABC (ChABC) delivered via lentiviral vector (LV-ChABC) to explore the consequences of large-scale CSPG digestion for spinal cord repair. We demonstrate significantly reduced secondary injury pathology in adult rats following spinal contusion injury and LV-ChABC treatment, with reduced cavitation and enhanced preservation of spinal neurons and axons at 12 weeks postinjury, compared with control (LV-GFP)-treated animals. To understand these neuroprotective effects, we investigated early inflammatory changes following LV-ChABC treatment. Increased expression of the phagocytic macrophage marker CD68 at 3 d postinjury was followed by increased CD206 expression at 2 weeks, indicating that large-scale CSPG digestion can alter macrophage phenotype to favor alternatively activated M2 macrophages. Accordingly, ChABC treatment in vitro induced a significant increase in CD206 expression in unpolarized monocytes stimulated with conditioned medium from spinal-injured tissue explants. LV-ChABC also promoted the remodelling of specific CSPGs as well as enhanced vascularity, which was closely associated with CD206-positive macrophages. Neuroprotective effects of LV-ChABC corresponded with improved sensorimotor function, evident as early as 1 week postinjury, a time point when increased neuronal survival correlated with reduced apoptosis. Improved function was maintained into chronic injury stages, where improved axonal conduction and increased serotonergic innervation were also observed. Thus, we demonstrate that ChABC gene therapy can modulate secondary injury processes, with neuroprotective effects that lead to long-term improved functional outcome and reveal novel mechanistic evidence that modulation of macrophage phenotype may underlie these effects.

Gene network and proteomic analyses of cardiac responses to pathological and physiological stress.

BACKGROUND: The molecular mechanisms underlying similarities and differences between physiological and pathological left ventricular hypertrophy (LVH) are of intense interest. Most previous work involved targeted analysis of individual signaling pathways or screening of transcriptomic profiles. We developed a network biology approach using genomic and proteomic data to study the molecular patterns that distinguish pathological and physiological LVH. METHODS AND RESULTS: A network-based analysis using graph theory methods was undertaken on 127 genome-wide expression arrays of in vivo murine LVH. This revealed phenotype-specific pathological and physiological gene coexpression networks. Despite >1650 common genes in the 2 networks, network structure is significantly different. This is largely because of rewiring of genes that are differentially coexpressed in the 2 networks; this novel concept of differential wiring was further validated experimentally. Functional analysis of the rewired network revealed several distinct cellular pathways and gene sets. Deeper exploration was undertaken by targeted proteomic analysis of mitochondrial, myofilament, and extracellular subproteomes in pathological LVH. A notable finding was that mRNA-protein correlation was greater at the cellular pathway level than for individual loci. CONCLUSIONS: This first combined gene network and proteomic analysis of LVH reveals novel insights into the integrated pathomechanisms that distinguish pathological versus physiological phenotypes. In particular, we identify differential gene wiring as a major distinguishing feature of these phenotypes. This approach provides a platform for the investigation of potentially novel pathways in LVH and offers a freely accessible protocol (http://sites.google.com/site/cardionetworks) for similar analyses in other cardiovascular diseases.