Absence of nicotinic acetylcholine receptor α7 subunit amplifies inflammation and accelerates onset of fibrosis: an inflammatory kidney model.
ABSTRACT: Inflammation is regulated by endogenous mechanisms, including anti-inflammatory cytokines, adenosine, and the nicotinic acetylcholine receptor α7 subunit (α7nAChR). We investigated the role of α7nAChR in protection against the progression of tissue injury in a model of severe, macrophage-mediated, cytokine-dependent anti-glomerular basement membrane (GBM) glomerulonephritis (GN), in α7nAChR-deficient (α7(-/-)) mice . At d 7 after the injection of anti-GBM antibody, kidneys from α7(-/-) mice displayed severe glomeruli (P < 0.0001) and tubulointerstitial lesions (P < 0.001) compared to kidneys from WT mice. An important finding was the presence of severe glomerulosclerosis in α7(-/-) mice in this early phase of the disease. Kidneys of α7(-/-) mice showed greater accumulation of inflammatory cells and higher expression of chemokines and cytokines than did those of WT mice. In addition, in α7(-/-) fibrotic kidneys, the expression of fibrin, collagen, TGF-β, and tissue inhibitor of metalloproteinase (TIMP)-2 increased, and the expression of TIMP3 declined. The increase in counterregulatory responses to inflammation in α7(-/-) nephritic kidneys did not compensate for the lack of α7nAChR. These findings indicate that α7nAChR plays a key role in regulating the inflammatory response in anti-GBM GN and that disruption of the endogenous protective α7nAChR amplifies inflammation to accelerate kidney damage and fibrosis.
Project description:Gastrointestinal prokinetic agents function as serotonin-4 receptor (5-HT4R) agonists to activate myenteric plexus neurons to release acetylcholine (ACh), which then induce anti-inflammatory action. Details of this pathway, however, remain unknown. The aim of this study is to clarify the anti-inflammatory mechanism underlying the 5-HT4R agonist, mosapride citrate (MOS)-induced anti-inflammatory action on postoperative ileus (POI). POI models were generated from wild-type C57BL6/J (WT), 5-HT4R knock-out (S4R KO), α7 nicotinic AChR KO (α7 R KO), and M2 muscarinic ACh receptor KO (M2R KO) mice. MOS attenuated leukocyte infiltration in WT. MOS-induced anti-inflammatory action was completely abolished in both S4R KO and S4R KO mice upon wild-type bone marrow transplantation. MOS-induced anti-inflammatory action against macrophage infiltration, but not neutrophil infiltration, was attenuated in α7 R KO mice. Selective α7nAChR agonists (PNU-282987 and AR-R17779) also inhibited only macrophage infiltration in POI. MOS-mediated inhibition of neutrophil infiltration was diminished by atropine, M2AChR antagonist, methoctramine, and in M2R KO mice. Stimulation with 5-HT4R inhibits leukocyte infiltration in POI, possibly through myenteric plexus activation. Released ACh inhibited macrophage and neutrophil infiltration likely by activation of α7nAChR on macrophages and M2AChR. Thus, macrophage and neutrophil recruitment into inflamed sites is regulated by different types of AChR in the small intestine.
Project description:p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic GN (GN) and which of its four isoforms is preferentially involved in kidney inflammation is not definitely known. We thus examined expression and activation of p38MAPK isoforms during anti-glomerular basement membrane (GBM) nephritis. Therefore, p38? conditional knockout mice (MxCre-p38?(?/?)) were used to examine the role of p38? in anti-GBM induced nephritis. Both wild type and MxCre-p38?(?/?) mice developed acute renal failure over time. Histological examinations revealed a reduced monocyte influx and less tubular damage in MxCre-p38?(?/?) mice, whereas glomerular crescent formation and renal fibrosis was similar. Likewise, the levels of pro- and anti-inflammatory cytokines such as TNF, IL-1 and IL-10 were similar, but IL-8 was even up-regulated in MxCre-p38?(?/?) mice. In contrast, we could detect strong down-regulation of chemotactic cytokines such as CCL-2, -5 and -7, in the kidneys of MxCre-p38?(?/?) mice. In conclusion, p38? is the primary p38MAPK isoform expressed in anti-GBM nephritis and selectively affects inflammatory cell influx and tubular damage. Full protection from nephritis is however not achieved as renal failure and structural damage still occurs.
Project description:Oxidative stress is implicated in tissue inflammation, and plays an important role in the pathogenesis of immune-mediated nephritis. Using the anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN) mouse model, we found that increased expression of glutathione S-transferase Mu 2 (GSTM2) was related to reduced renal damage caused by anti-GBM antibodies. Furthermore, mesenchymal stem cell (MSC)-based therapy has shed light on the treatment of immune-mediated kidney diseases. The aim of this study was to investigate if MSCs could be utilized as vehicles to deliver the GSTM2 gene product into the kidney and to evaluate its potential therapeutic effect on anti-GBM-GN.The human GSTM2 gene (hGSTM2) was transduced into mouse bone marrow-derived MSCs via a lentivirus vector to create a stable cell line (hGSTM2-MSC). The cultured hGSTM2-MSCs were treated with 0.5 mM H2O2, and apoptotic cells were measured by terminal dUTP nick-end labeling (TUNEL) assay. The 129/svj mice, which were challenged with anti-GBM antibodies, were injected with 10? hGSTM2-MSCs via the tail vein. Expression of hGSTM2 and inflammatory cytokines in the kidney was assayed by quantitative PCR and western blotting. Renal function of mice was evaluated by monitoring proteinuria and levels of blood urea nitrogen (BUN), and renal pathological changes were analyzed by histochemistry. Immunohistochemical analysis was performed to measure inflammatory cell infiltration and renal cell apoptosis.MSCs transduced with hGSTM2 exhibited similar growth and differentiation properties to MSCs. hGSTM2-MSCs persistently expressed hGSTM2 and resisted H2O2-induced apoptosis. Upon injection into 129/svj mice, hGSTM2-MSCs migrated to the kidney and expressed hGSTM2. The anti-GBM-GN mice treated with hGSTM2-MSCs exhibited reduced proteinuria and BUN (58% and 59% reduction, respectively) and ameliorated renal pathological damage, compared with control mice. Mice injected with hGSTM2-MSCs showed alleviated renal inflammatory cell infiltration and reduced expression of chemokine (C-C motif) ligand 2 (CCL2), interleukin (IL)-1? and IL-6 (53%, 46% and 52% reduction, respectively), compared with controls. Moreover, hGSTM2-MSCs increased expression of renal superoxide dismutase and catalase, which may associate with detoxifying reactive oxygen species to prevent oxidative renal damage.Our data suggest that the enhanced protective effect of GSTM2-transduced MSCs against anti-GBM-GN might be associated with inhibition of oxidative stress-induced renal cell apoptosis and inflammation, through over-expression of hGSTM2 in mouse kidneys.
Project description:Invariant natural killer T (iNKT) cells represent a particular subset of T lymphocytes capable of producing several cytokines, which exert regulatory or effector functions, following stimulation of the T cell receptor. In this study, we investigated the influence of iNKT cells on the development of experimental anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). After injection of anti-GBM serum, the number of kidney iNKT cells rapidly increased. iNKT cell-deficient mice (Jalpha18-/-) injected with anti-GBM serum demonstrated worse renal function, increased proteinuria, and greater glomerular and tubular injury compared with similarly treated wild-type mice. We did not detect significant differences in Th1/Th2 polarization in renal tissue that might have explained the severity of disease in Jalpha18-/- mice. Interestingly, expression of both TGF-beta and TGF-beta-induced (TGFBI) mRNA was higher in wild-type kidneys compared with Jalpha18-/- kidneys, suggesting a possible protective role for TGF-beta in anti-GBM GN. Administration of an anti-TGF-beta neutralizing antibody significantly enhanced the severity of disease in wild-type, but not Jalpha18-/-, mice. In conclusion, in experimental anti-GBM GN, iNKT cells attenuate disease severity and TGF-beta has a renoprotective role.
Project description:Sinomenine has long been used for the treatment of rheumatoid arthritis in China. However, its anti-inflammatory mechanism is still debatable because the in vitro minimal effective concentration (≥250 μM) is hardly reached in either synovium or serum after oral administration at a therapeutic dose. Recent findings suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might mediate the inhibitory effect of sinomenine on macrophage activation, which attracts us to explore the anti-arthritis mechanism of sinomenine by taking neuroendocrine-inflammation axis into consideration. Here, we showed that orally administered sinomenine ameliorated the systemic inflammation of collagen-induced arthritis (CIA) rats, which was significantly diminished by either vagotomy or the antagonists of nicotinic acetylcholine receptors (especially the antagonist of α7nAChR), but not by the antagonists of muscarinic receptor. Sinomenine might bind to α7nAChR through interacting with the residues Tyr184 and Tyr191 in the pocket. In addition, the generation of vasoactive intestinal polypeptide (VIP) from the gut of CIA rats and cultured neuron-like cells was selectively enhanced by sinomenine through the activation of α7nAChR-PI3K/Akt/mTOR pathway. The elevated levels of VIP in the serum and small intestine of rats were negatively correlated with the scores of joint destruction. The crucial role of VIP in the anti-arthritic effect of sinomenine was confirmed by using VIP hybrid, a non-specific antagonist of VIP receptor. Taken together, intestine-sourced VIP mediates the anti-arthritic effect of sinomenine, which is generated by the activation of α7nAChR.
Project description:A subset of genes in the human genome are uniquely human and not found in other species. One example is CHRFAM7A, a dominant-negative inhibitor of the antiinflammatory α7 nicotinic acetylcholine receptor (α7nAChR/CHRNA7) that is also a neurotransmitter receptor linked to cognitive function, mental health, and neurodegenerative disease. Here we show that CHRFAM7A blocks ligand binding to both mouse and human α7nAChR, and hypothesized that CHRFAM7A-transgenic mice would allow us to study its biological significance in a tractable animal model of human inflammatory disease, namely SIRS, the systemic inflammatory response syndrome that accompanies severe injury and sepsis. We found that CHRFAM7A increased the hematopoietic stem cell (HSC) reservoir in bone marrow and biased HSC differentiation to the monocyte lineage in vitro. We also observed that while the HSC reservoir was depleted in SIRS, HSCs were spared in CHRFAM7A-transgenic mice and that these mice also had increased immune cell mobilization, myeloid cell differentiation, and a shift to inflammatory monocytes from granulocytes in their inflamed lungs. Together, the findings point to a pathophysiological inflammatory consequence to the emergence of CHRFAM7A in the human genome. To this end, it is interesting to speculate that human genes like CHRFAM7A can account for discrepancies between the effectiveness of drugs like α7nAChR agonists in animal models and human clinical trials for inflammatory and neurodegenerative disease. The findings also support the hypothesis that uniquely human genes may be contributing to underrecognized human-specific differences in resiliency/susceptibility to complications of injury, infection, and inflammation, not to mention the onset of neurodegenerative disease.
Project description:In macrophages the α7 nicotinic acetylcholine receptor (α7nAChR) modulates production of inflammatory cytokines, cholesterol accumulation and lipoprotein uptake. Recently, our laboratory showed that selective stimulation of the α7nAChR protects macrophages from apoptosis, an effect that is absent in α7nAChR-deficient macrophages. All these observations are suggestive of a potential role of macrophage α7nAChR in atherosclerosis. Mouse models of the disease with bone marrow deletion of α7nAChR represent an attractive approach to address the in vivo relevance of these in vitro findings. However, recent studies that focused on the impact of hematopoietic deficiency of α7nAChR on early atherosclerotic lesions of low density lipoprotein receptor knockout (LDLRKO) mice, yielded controversial results. The question also remained whether macrophage α7nAChR modulates the characteristics of advanced lesions. Here we used LDLR knockout mice transplanted with bone marrow from wild-type or α7nAChR knockout animals to revisit the effect of hematopoietic deficiency of α7nAChR on early lesions and to examine, for the first time, its impact on advanced plaques. Aortic sinus atherosclerotic lesions were analyzed following 8 and 14 weeks on a high fat diet. Early lesions in mice with α7nAChR deficient bone marrow were not different from those in control animals. However, advanced lesions of mice with bone marrow deletion of α7nAChR exhibited reduction in size, macrophage content and cell proliferation. These studies are the first in examining the impact of hematopoietic deficiency of α7nAChR on the characteristics of advanced atherosclerotic lesions in a mouse model of the disease and provide novel evidence underscoring a potential pro-atherogenic role of macrophage α7nAChR.
Project description:In this study, we employed high-throughput RNA sequencing (RNA-Seq) to identify the Smad3-dependent lncRNAs related to renal inflammation and fibrosis in Smad3 knockout (KO) mouse models of unilateral ureteral obstructive nephropathy (UUO) and immunologically-induced anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). 12 kidney tissue samples of Smad3 KO/WT mice from normal control, UUO at day 5 or anti-GBM GN at day 10 models (n=2 in each group) for whole transcriptome RNA-sequencing.
Project description:The role of the chemokine, macrophage inflammatory protein-2 (MIP-2), during anti-glomerular basement membrane (GBM) antibody (Ab) glomerulonephritis (GN) was studied. Rat MIP-2 cDNA had been cloned previously. Recombinant rat MIP-2 (rMIP-2) from Escherichia coli exhibited neutrophil chemotactic activity and produced neutrophil influx when injected into the rat bladder wall. By using a riboprobe derived from the cDNA and an anti-rMIP-2 polyclonal Ab, MIP-2 was found to be induced in glomeruli with anti-GBM Ab GN as mRNA by 30 min and protein by 4 h, with both disappearing by 24 h. The expression of MIP-2 correlated with glomerular neutrophil influx. A single dose of the anti-MIP-2 Ab 30 min before anti-GBM Ab was effective in reducing neutrophil influx (40% at 4 h, P < 0.01) and periodic acid-Schiff deposits containing fibrin (54% at 24 h, P < 0.01). The anti-rMIP-2 Ab had no effect on anti-GBM Ab binding (paired-label isotope study). Functional improvement in the glomerular damage was evidenced by a reduction of abnormal proteinuria (P < 0.05). These results suggest that MIP-2 is a major neutrophil chemoattractant contributing to influx of neutrophils in Ab-induced glomerular inflammation in the rat.
Project description:We investigated a glomerulonephritis (GN) model in rats induced by nephrotoxic serum (NTS) which contains antibodies against the glomerular basement membrane (GBM). The anti-GBM GN model in rats is widely used since its biochemical and histopathological characteristics are similar to crescentic nephritis and Goodpasture's disease in humans (Pusey, 2003). Male Wistar Kyoto (WKY) and Sprague-Dawley (SD) rats were dosed once with 1, 2.5 and 5 ml/kg nephrotoxic serum (NTS) or 1.5 and 5 ml/kg NTS, respectively. GN and tubular damage were observed histopathologically in all treated rats after 14 days. To obtain insight into molecular processes during GN pathogenesis, mRNA expression was investigated in WKY and SD kidneys using Affymetrix's GeneChip Rat genome 230_2.0 arrays (GSE64265). The immunopathological processes during GN are still not fully understood and likely involve both innate and adaptive immunity. In the present study, several hundred mRNAs were found deregulated, which functionally were mostly associated with inflammation and regeneration. The ?-chain of the major histocompatibility complex class II RT1.B (Rt1-Bb) and complement component 6 (C6) were identified as two mRNAs differentially expressed between WKY and SD rat strains which could be related to known different susceptibilities to NTS of different rat strains; both were increased in WKY and decreased in SD rats (Pavkovic et al., 2015 ). Increased Rt1-Bb expression in WKY rats could indicate a stronger and more persistent cellular reaction of the adaptive immune system in this strain, in line with findings indicating adaptive immune reactions during GN. The complement cascade is also known to be essential for GN development, especially terminal cascade products like C6.