Noncanonical immunomodulatory activity of complement regulator C4BP(ß-) limits the development of lupus nephritis.

Lupus nephritis is a chronic autoimmune-inflammatory condition that can lead to end-stage kidney disease. Presently available immunosuppressive treatments for lupus nephritis are suboptimal and can induce significant side effects. Recently, we characterized a novel immunomodulatory activity of the minor isoform of the classical pathway complement inhibitor, C4BP(ß-). We show here that C4BP(ß-) treatment prevented the development of proteinuria and albuminuria, decreased significantly the formation of anti-dsDNA antibodies and, locally, mitigated renal glomerular IgG and C3 deposition and generation of apoptotic cells. There was a consequent histological improvement and increased survival in lupus-prone mice. The therapeutic efficacy of C4BP(ß-) was analogous to that of the broad-acting immunosuppressant cyclophosphamide. Remarkably, a comparative transcriptional profiling analysis revealed that the kidney gene expression signature resulting from C4BP(ß-) treatment turned out to be 10 times smaller than that induced by cyclophosphamide treatment. C4BP(ß-) immunomodulation induced significant downregulation of transcripts relevant to lupus nephritis indicating immunopathogenic cell infiltration, including activated T cells (Lat), B cells (Cd19, Ms4a1, Tnfrsf13c), inflammatory phagocytes (Irf7) and neutrophils (Prtn3, S100a8, S100a9). Furthermore, cytokine profiling and immunohistochemistry confirmed that C4BP(ß-), through systemic and local CXCL13 downregulation, was able to prevent ectopic lymphoid structures neogenesis in aged mice with lupus nephritis. Thus, due to its anti-inflammatory and immunomodulatory activities and high specificity, C4BP(ß-) could be considered for further clinical development in patients with systemic lupus erythematosus.

CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury.

Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte-endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing. In the warm ischemia model different doses were used, resulting in clear renal function improvement and a structural renoprotective effect. Renal ischemia activated the CD40 gene and protein expression, which was inhibited by intravenous siRNA administration. CD40 gene silencing improved renal inflammatory status, as seen by the reduction of CD68 and CD3 T-cell infiltrates, attenuated pro-inflammatory, and enhanced anti-inflammatory mediators. Furthermore, siRNA administration decreased a spleen pro-inflammatory monocyte subset and reduced TNFα secretion by splenic T cells. In the cold ischemia model with syngeneic and allogeneic renal transplantation, the most effective dose induced similar functional and structural renoprotective effects. Our data show the efficacy of our siRNA in modulating both the local and the systemic inflammatory milieu after an ischemic insult. Thus, CD40 silencing could emerge as a novel therapeutic strategy in solid organ transplantation.

Cold ischaemia, innate immunity and deterioration of the glomerular filtration barrier in antibody-mediated acute rejection.

BACKGROUND: In renal transplantation, cold ischaemia (CI) determines acute rejection through innate immunity among others. Acute rejection episodes are a risk factor for late allograft dysfunction and proteinuria. This implies some alteration of the glomerular filtration barrier (GFB). Besides its effects on acute rejection, we hypothesized that CI might somehow damage the GFB being directly responsible for late proteinuria. METHODS: On rat kidney allografts suffering from antibody-mediated acute rejection with or without CI and compared with syngeneic grafts, we quantified the gene expression of innate and adaptive immune mediators and assessed the capillary glomerular basement membranes (CapBM) by immunostaining collagen-IV (ColIV). ColIV was also assessed in equivalent groups from a previous chronic study followed up for 24 weeks. RESULTS: CI up-regulated enzymes critical in the stabilization of collagen chains, increasing ColIV deposition and thickening the CapBM. CI increased the C4d and IgG deposits within grafts, amplified innate immunity (heat shock protein 70, fibronectin, Toll-like-receptor-4 and MyD88) and synergized with alloreactivity in triggering adaptive response through CD40. CONCLUSIONS: Initial CI increased the ColIV deposition in CapBM, damaging the GFB and being responsible for part of the proteinuria associated with late allograft dysfunction. This deterioration of the GFB is related to the early innate immunity activation and subsequent up-regulation of CD40 in acute rejected grafts. In chronic rejected allografts, thickened CapBM may be a consequence of an unresolved immune-inflammatory response worsened by CI.

In vivo CD40 Silencing by siRNA Infusion in Rodents and Evaluation by Kidney Immunostaining.

The co-stimulatory molecule CD40 and its ligand CD40L play a key role in the regulation of immunological processes and are involved in the pathophysiology of autoimmune and inflammatory diseases. Inhibition of the CD40-CD40L axis is a promising therapy, and a number of strategies and techniques have been designed to hinder its functionality. Our group has broad experience in silencing CD40 using RNAi technology, and here we summarize protocols for the systemic administration of a specific anti-CD40 siRNA in different rodents models, in addition to the subsequent quantification of CD40 expression in murine kidneys by immunostaining. The use of RNAi technology with specific siRNAs to silence genes is becoming an essential method to investigate gene functions and is rapidly emerging as a therapeutic tool. Graphic abstract: CD40 siRNA mechanism.

The gene silencing of IRF5 and BLYSS effectively modulates the outcome of experimental lupus nephritis.

Systemic lupus erythematosus is a highly complex and heterogeneous autoimmune disease mostly mediated by B cells. It is characterized by circulating self-reactive antibodies that deposit and form immune complexes in kidney, leading to irreparable tissue damage and resulting in lupus nephritis. In a New Zealand Black X New Zealand White F1 mouse model, we tested two different small interfering RNA (siRNA) silencing treatments against interferon regulatory factor 5 (IRF5) and B cell-activating factor (BLYSS) expression and their combination in a second set of animals. The administration of these two siRNAs separately prevented the progression of proteinuria and albuminuria at similar levels to that in cyclophosphamide animals. These treatments effectively resulted in a reduction of serum anti-double-stranded DNA (dsDNA) antibodies and histopathological renal score compared with non-treated group. Treated groups showed macrophage, T cell, and B cell infiltrate reduction in renal tissue. Moreover, kidney gene expression analysis revealed that siRNA treatments modulated very few pathways in contrast to cyclophosphamide, despite showing similar therapeutic effects. Additionally, the combined therapy tested in a second set of animals, in which the disease appeared more virulent, exhibited better results than monotherapies in the disease progression, delaying the disease onset and ameliorating the disease outcome. Herein, we provide the potential therapeutic effect of both selective IRF5 and BLYSS silencing as an effective and potential treatment, particularly in early phases of the disease.

Genetic mutations in a Spanish population with chronic pancreatitis.

BACKGROUND/AIMS: Mutations in the PRSS1 and the SPINK1 genes have variably been associated with alcohol-related, idiopathic and hereditary chronic pancreatitis (CP). The aim of this study was to determine for the first time the significance of PRSS1, SPINK1 mutations and genetic variants of AAT in a group of Spanish patients with CP. METHODS: 104 consecutive patients with CP were included, as well as 84 healthy control subjects. The R122H and N29I mutations in the PRSS1 gene, the N34S mutation in the SPINK1 gene and PiS and PiZ mutations in the AAT gene were analyzed by RFLP-PCR methods. RESULTS: No R122H mutation was found in the PRSS1 gene, and N29I mutation was detected in 7.7% of CP patients. A N29I mutation was observed in 3.9% of patients with alcohol-related pancreatitis (ACP). A total of 5.8% of CP patients were identified with the N34S mutation. Genotype MS, SS and MZ were detected in 18.3, 3.8 and 1.3% of CP patients, respectively. CONCLUSION: The percentage of N29I mutations in ACP patients was higher than that reported in other studies, while the percentage of N34S and AAT mutations in ACP and idiopathic CP patients was similar.

The Timing of Immunomodulation Induced by Mesenchymal Stromal Cells Determines the Outcome of the Graft in Experimental Renal Allotransplantation.

The immunomodulatory characteristics of mesenchymal stromal cells (MSCs) may lead to multifaceted strategies in rejection of organ transplantation. This study was designed to investigate, first, the effect of the donor-type MSCs from Wistar rats on the immune system of immunocompetent Lewis rats and, second, the rejection responses in a renal transplantation model of Wistar to Lewis. In the first experimental model, MSCs from the bone marrow induced a systemic immune response in the immunocompetent Lewis rats, characterized by two different phases. In the initial phase (days 1-3 after MSCs infusion), the main findings were a decrease in the percentage of the main peripheral blood (PB) lymphocyte subpopulations [T cells, B cells, and natural killer (NK) cells], an increase in the FOXP3 MFI in Tregs, and an elevated concentration of circulating proinflammatory cytokines (IL-1ß and TNF-α). In the late phase (days 4-6), the percentage of T cells, B cells, and NK cells returned to baseline levels; the concentration of circulating IL-1ß and TNF-α decreased; and the level of anti-inflammatory cytokines (IL-10 and IL-4) increased with respect to the initial phase. In the allogeneic kidney transplantation model, rats were randomized into four groups: nontreated, cyclosporine oral administration, and two groups of rats treated with two different schedules of MSC infusion: 4 days (MSCs-4) and 7 days (MSCs-7) before kidney transplantation and in both a further infusion at the day of transplantation. Both MSC treatments decreased the percentage of T, B, and NK cells in PB. Creatinine levels, survival, and histological parameters were better in MSCs-7 than in MSCs-4. We can conclude that MSCs, by themselves, produce changes in the immune system; they do not need a pathological condition to produce immunomodulatory responses. In the renal allograft model, the optimal time schedule for MSC infusion before grafting was 7 days to prevent acute rejection.

Silencing of CD40 in vivo reduces progression of experimental atherogenesis through an NF-κB/miR-125b axis and reveals new potential mediators in the pathogenesis of atherosclerosis.

BACKGROUND AND AIMS: CD40/CD40L signaling exerts a critical role in the development of atherosclerosis, and microRNAs (miRNAs) are key regulators in vascular inflammation and plaque formation. In this work, we investigated mRNA/miRNA expression during progression of atherosclerotic lesions through CD40 silencing. METHODS: We silenced CD40 with a specific siRNA in ApoE-/- mice and compared expression of mRNA/miRNA in ascending aorta with scrambled treated mice. RESULTS: siRNA-CD40 treated mice significantly reduced the extension and severity of atherosclerotic lesions, as well as the number of F4/80+, galectin-3+ macrophages and NF-κB+ cells in the intima. Genome-wide mRNA/miRNA profiling allowed the identification of transcripts, which were significantly upregulated during atherosclerosis; among them, miR-125b and miR-30a, Xpr1, a regulator of macrophage differentiation, Taf3, a core transcription factor and the NF-κB activator Ikkß, whereas, the NF-κB inhibitor Ikbα was downregulated during disease progression. All those changes were reversed upon CD40 silencing. Interestingly, TAF3, XPR1 and miR-125b were also overexpressed in human atherosclerotic plaques. Murine Taf3 and Xpr1 were detected in the perivascular adipose tissue (PVAT), and Taf3 also in intimal foam cells. Finally, expression of miR-125b was regulated by the CD40-NF-κB signaling axis in RAW264.7 macrophages. CONCLUSIONS: CD40 silencing with a specific siRNA ameliorates progression of experimental atherosclerosis in ApoE-/- mice, and evidences a role for NF-κB, Taf3, Xpr1, and miR-125b in the pathogenesis of atherosclerosis.

Datasets for the validation of the "in vivo" siRNA-silencing of CD40 and for the detection of new markers of atherosclerosis progression in ApoE-deficient mice.

Data presented in this Data in Brief article correspond to the article "in vivo" silencing of CD40 reduces progression of experimental atherogenesis through a NFκB/miR-125b axis and reveals new potential mediators in the pathogenesis of atherosclerosis" (M. Hueso, L. De Ramon, E. Navarro, E. Ripoll, J.M. Cruzado, J.M. Grinyo, J. Torras, 2016) [1]. Here, we describe the validation of the silencing of CD40 expression with a specific siRNA in ApoE-/- mouse aortas, and its systemic effects on splenic lymphocytic subpopulations as well as on the infiltration of aortic intima by F4/80+, galectin-3+ macrophages or by NF-κB+ cells. We also show the output of a Gene Ontology and TLDA analysis which allowed the detection of potential mediators of atherosclerosis progression. We provide the scientific community with a set of genes whose expression is increased during atherosclerosis progression but downregulated upon CD40 silencing.

RNAi-Based Therapy in Experimental Ischemia-Reperfusion Injury. The New Targets.

BACKGROUND: Ischemia reperfusion injury is an important pathophysiological process in many fields such as transplantation, stroke, atherosclerosis, trauma and myocardial infarction. Recent advances in gene silencing may help to reduce ischemic effects, targeting molecules related to this pathological process. METHODS AND RESULTS: Here, we review the different silencing approaches in ischemic injury, highlighting the role of co-stimulatory molecules in renal transplantation. CONCLUSION: Gene silencing appears as a new strategy to prevent the inflammation and injury associated with ischemia.

JAK3-STAT pathway blocking benefits in experimental lupus nephritis.

BACKGROUND: Lupus nephritis (LN) is a complex chronic autoimmune disease of unknown etiology characterized by loss of tolerance against several self-antigens. Cytokines are known to be central players in LN pathogenesis. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is one important pathway that mediates signal transduction of several cytokines. In this study, we examined the pathogenic role of this pathway and how CP-690,550 treatment influences LN outcome. METHODS: Six-month-old NZB/NZWF1 mice were divided into two different treatment groups: (1) control animals given vehicle treatment, cyclophosphamide, and mycophenolate mofetil treatment as positive controls of the therapy and (2) mice treated with CP-690,550, a JAK3 inhibitor. Mice were treated for 12 weeks. We evaluated renal function, anti-double-stranded DNA (anti-dsDNA) antibody, renal histology changes, kidney complement and immunoglobulin G (IgG) deposits, T-cell and macrophage infiltration, kidney inflammatory gene expression, and circulating cytokine changes. RESULTS: CP-690,550 treatment significantly reduced proteinuria and improved renal function and histological lesions of the kidney. Compared with vehicle-treated animals, those undergoing CP-690,550 treatment showed significantly diminished anti-dsDNA antibody and complement component C3 and IgG deposition in glomeruli. We also observed a significant reduction of T-cell and macrophage infiltration. Kidney gene expression revealed a reduction in inflammatory cytokines and complement and related macrophage-attracting genes. Circulating inflammatory cytokines were also reduced with treatment. CONCLUSIONS: On the basis of our results, we conclude that the JAK-STAT pathway is implicated in the progression of renal inflammation in NZB/WF1 mice and that targeting JAK3 with CP-690,550 is effective in slowing down the course of experimental LN. Thus, CP-690,550 could become a new therapeutic tool in LN and other autoimmune diseases.

Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells.

The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV. The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 µm). As a control, non-conditioned culture medium was used (control medium). To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV.

New approaches for the treatment of lupus nephritis in the 21st century: from the laboratory to the clinic.

Systemic lupus erythematosus is a complex autoimmune disorder affecting multiple organ systems. Glomerulonephritis leading to severe proteinuria, chronic renal failure and end-stage renal disease remains one of the most severe complications of systemic lupus erythematosus and is associated with significant morbidity and mortality. Conventional lupus nephritis (LN) treatment based on cyclophosphamide, steroids and, recently, mycophenolatemofetil has improved the outcome of the disease over the last 50 years, although failure to achieve remission or treatment resistance has been reported in 18-57% of patients. Chronic complications such as long-term toxicity dampen their ability to maintain disease remission. There is a need to develop more specific pharmacological agents for patients to provide choices that are equally effective, less toxic and have fewer complications. During the last 10 years, experimental studies based on different pathogenesis pathways of LN have provided an enormous amount of knowledge and have offered the possibility to target the disease with selective approaches. In this article, we summarize the new experimental strategies that have recently been utilized to target LN, focusing on mechanisms of action.

CD40 gene silencing reduces the progression of experimental lupus nephritis modulating local milieu and systemic mechanisms.

Lupus nephritis (LN) is an autoimmune disorder in which co-stimulatory signals have been involved. Here we tested a cholesterol-conjugated-anti-CD40-siRNA in dendritic cells (DC) in vitro and in a model of LPS to check its potency and tissue distribution. Then, we report the effects of Chol-siRNA in an experimental model of mice with established lupus nephritis. Our in vitro studies in DC show a 100% intracellular delivery of Chol-siRNA, with a significant reduction in CD40 after LPS stimuli. In vivo in ICR mice, the CD40-mRNA suppressive effects of our Chol-siRNA on renal tissue were remarkably sustained over a 5 days after a single preliminary dose of Chol-siRNA. The intra-peritoneal administration of Chol-siRNA to NZB/WF1 mice resulted in a reduction of anti-DNA antibody titers, and histopathological renal scores as compared to untreated animals. The higher dose of Chol-siRNA prevented the progression of proteinuria as effectively as cyclophosphamide, whereas the lower dose was as effective as CTLA4. Chol-siRNA markedly reduced insterstitial CD3+ and plasma cell infiltrates as well as glomerular deposits of IgG and C3. Circulating soluble CD40 and activated splenic lymphocyte subsets were also strikingly reduced by Chol-siRNA. Our data show the potency of our compound for the therapeutic use of anti-CD40-siRNA in human LN and other autoimmune disorders.

Mesenchymal stem cell therapy prevents interstitial fibrosis and tubular atrophy in a rat kidney allograft model.

In solid organ transplantation, mesenchymal stem cell (MSC) therapy is strongly emerging among other cell therapies due to the positive results obtained in vitro and in vivo as an immunomodulatory agent and their potential regenerative role. We aimed at testing whether a single dose of MSCs, injected at 11 weeks after kidney transplantation for the prevention of chronic mechanisms, enhanced regeneration and provided protection against the inflammatory and fibrotic processes that finally lead to the characteristic features of chronic allograft nephropathy (CAN). Either bone marrow mononuclear cells (BMCs) injection or no-therapy (NT) were used as control treatments. A rat kidney transplantation model of CAN with 2.5 h of cold ischemia was used, and functional, histological, and molecular parameters were assessed at 12 and 24 weeks after transplantation. MSC and BMC cell therapy preserves renal function at 24 weeks and abrogates proteinuria, which is typical of this model (NT24w: 68.9 ± 26.5 mg/24 h, MSC24w: 16.6 ± 2.3 mg/24 h, BMC24w: 24.1 ± 5.3 mg/24 h, P<0.03). Only MSC-treated animals showed a reduction in interstitial fibrosis and tubular atrophy (NT24w: 2.3 ± 0.29, MSC24w: 0.4 ± 0.2, P<0.03), less T cells (NT: 39.6 ± 9.5, MSC: 8.1 ± 0.9, P<0.03) and macrophages (NT: 20.9 ± 4.7, MSC: 5.9 ± 1.7, P<0.05) infiltrating the parenchyma and lowered expression of inflammatory cytokines while increasing the expression of anti-inflammatory factors. MSCs appear to serve as a protection from injury development rather than regenerate the damaged tissue, as no differences were observed in Ki67 expression, and kidney injury molecule-1, Clusterin, NGAL, and hepatocyte growth factor expression were only up-regulated in nontreated animals. Considering the results, a single delayed MSC injection is effective for the long-term protection of kidney allografts.