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1.
PLoS One ; 13(4): e0195657, 2018.
Article in English | MEDLINE | ID: mdl-29641559

ABSTRACT

Studies disrupting the chemokine pathway CX3CL1 (fractalkine)/ CX3CR1 have shown decreased atherosclerosis in animal models but the techniques used to interrupt the pathway have not been easily translatable into human trials. DNA vaccination potentially overcomes the translational difficulties. We evaluated the effect of a DNA vaccine, targeted to CX3CR1, on atherosclerosis in a murine model and examined possible mechanisms of action. DNA vaccination against CX3CR1, enhanced by dendritic cell targeting using DEC-205 single chain variable region fragment (scFv), was performed in 8 week old ApoE-/- mice, fed a normal chow diet. High levels of anti-CX3CR1 antibodies were induced in vaccinated mice. There were no apparent adverse reactions to the vaccine. Arterial vessels of 34 week old mice were examined histologically for atherosclerotic plaque size, macrophage infiltration, smooth muscle cell infiltration and lipid deposition. Vaccinated mice had significantly reduced atherosclerotic plaque in the brachiocephalic artery. There was less macrophage infiltration but no significant change to the macrophage phenotype in the plaques. There was less lipid deposition in the lesions, but there was no effect on smooth muscle cell migration. Targeted DNA vaccination to CX3CR1 was well tolerated, induced a strong immune response and resulted in attenuated atherosclerotic lesions with reduced macrophage infiltration. DNA vaccination against chemokine pathways potentially offers a potential therapeutic option for the treatment of atherosclerosis.


Subject(s)
Antigens, CD/immunology , Atherosclerosis/immunology , Atherosclerosis/prevention & control , CX3C Chemokine Receptor 1/immunology , Dendritic Cells/immunology , Lectins, C-Type/immunology , Minor Histocompatibility Antigens/immunology , Receptors, Cell Surface/immunology , Vaccines, DNA/immunology , Animals , Atherosclerosis/blood , Atherosclerosis/pathology , Cholesterol/blood , Cytokines/blood , Lipid Metabolism/immunology , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Myocytes, Smooth Muscle/pathology , Vaccination
2.
Nephrol Dial Transplant ; 32(5): 781-791, 2017 May 01.
Article in English | MEDLINE | ID: mdl-27566305

ABSTRACT

BACKGROUND: Endothelial cells are known to contribute to kidney fibrosis via endothelial-mesenchymal transition (EndoMT). Matrix metalloproteinase 9 (MMP-9) is known to be profibrotic. However, whether MMP-9 contributes to kidney fibrosis via EndoMT is unknown. METHODS: Primary mouse renal peritubular endothelial cells (MRPECs) were isolated and treated by recombinant human transforming growth factor beta 1 (rhTGF-ß1) with or without MMP-9 inhibitor or by recombinant human MMP-9 (rhMMP-9) alone. Kidney fibrosis was induced by unilateral ureteral obstruction (UUO) in MMP-9 knockout (KO) and wide-type (WT) control mice. The effects of MMP-9 on EndoMT of MRPECs and kidney fibrosis were examined. RESULTS: We showed that MRPECs underwent EndoMT after rhTGF-ß1 treatment or in UUO kidney as evidenced by decreased expression of endothelial markers, vascular endothelial cadherin (VE-cadherin) and CD31, and increased levels of mesenchymal markers, α-smooth muscle actin (α-SMA) and vimentin. The expression of fibrosis markers was also up-regulated significantly after rhTGF-ß1 treatment in MRPECs. The EndoMT and fibrosis markers were significantly less in rhTGF-ß1-treated MMP-9 KO MRPECs, whereas MMP-9 alone was sufficient to induce EndoMT in MRPECs. UUO kidney of MMP-9 KO mice showed significantly less interstitial fibrosis and EndoMT in MRPECs. Notch signaling shown by Notch intracellular domain (NICD) was increased, while Notch-1 was decreased in rhTGF-ß1-treated MRPECs of MMP-9 WT but not MMP-9 KO mice. Inhibition of MMP-9 or Notch signaling prevented rhTGF-ß1- or rhMMP-9-induced α-SMA and NICD upregulation in MRPECs. UUO kidney of MMP-9 KO mice had less staining of Notch signaling transcription factor Hey-1 in VE-cadherin-positive MRPECs than WT controls. CONCLUSIONS: Our results demonstrate that MMP-9-dependent Notch signaling plays an important role in kidney fibrosis through EndoMT of MRPECs.


Subject(s)
Endothelium/pathology , Fibrosis/pathology , Kidney Diseases/pathology , Matrix Metalloproteinase 9/metabolism , Mesoderm/pathology , Receptors, Notch/metabolism , Animals , Endothelium/metabolism , Fibrosis/metabolism , Humans , Kidney Diseases/metabolism , Male , Mesoderm/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , Signal Transduction , Transforming Growth Factor beta1/metabolism , Ureteral Obstruction/metabolism , Ureteral Obstruction/pathology
3.
Int J Clin Exp Pathol ; 7(1): 38-47, 2014.
Article in English | MEDLINE | ID: mdl-24427324

ABSTRACT

Regulatory T cells (Treg) are important for maintaining immune homeostasis. Adoptive transfer of Tregs is protective in renal disease models in both immunocompetent and immunodeficient mice. However the involvement of TCR recognition of renal antigens remains to be clarified. To address this question, we made use of Tregs from the DO11.10 mouse (a TCR transgenic (Tg) mouse), that recognise the non-murine antigen Ovalbumin (OVA) and therefore are not activated by renal antigens. DO11.10 Tregs were assessed functionally in vitro and demonstrated equivalent suppression to WT BALB/c Tregs. Adriamycin Nephropathy (AN) was induced in mice which had been transfused with CD4+CD25+Tregs isolated from DO11.10 or BALB/c mice. To eliminate the memory/activation state as a cause of differences in activity, the protective capacity of DO11.10 Tregs pre-activated with OVA in vivo was assessed. Transfer of WT BALB/c Tregs significantly attenuated the development of AN with less glomerulosclerosis, tubular atrophy and macrophage infiltration as compared to AN mice without Treg transfer. However, mice receiving either naïve or pre-activated DO11.10 Tregs were not protected from AN. The lack of protection by DO11.10 Tregs was not due to failure to traffic to the affected kidney. These results suggest that antigen recognition in the kidney is important for Treg protection against injury.


Subject(s)
Lymphocyte Activation/immunology , Nephritis/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Antigens/immunology , Disease Models, Animal , Flow Cytometry , Immunohistochemistry , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Ovalbumin/immunology , Real-Time Polymerase Chain Reaction
4.
World J Nephrol ; 2(3): 84-9, 2013 Aug 06.
Article in English | MEDLINE | ID: mdl-24255890

ABSTRACT

Matrix metalloproteinases (MMPs) are members of the neutral proteinase family. They were previously thought to be anti-fibrotic because of their ability to degrade and remodel of extracellular matrix. However, recent studies have shown that MMPs are implicated in initiation and progression of kidney fibrosis through tubular cell epithelial-mesenchymal transition (EMT) as well as activation of resident fibroblasts, endothelial-mesenchymal transition (EndoMT) and pericyte-myofibroblast transdifferentiation. Interstitial macrophage infiltration has also been shown to correlate with the severity of kidney fibrosis in various chronic kidney diseases. MMPs secreted by macrophages, especially MMP-9, has been shown by us to be profibrotic by induction of tubular cells EMT. EMT is mainly induced by transforming growth factor-ß (TGF-ß). However, MMP-9 was found by us and others to be up-regulated by TGF-ß1 in kidney tubular epithelial cells and secreted by activated macrophages, resulting in EMT and ultimately kidney fibrosis. Therefore, MMP-9 may serve as a potential therapeutic target to prevent kidney fibrosis in chronic kidney disease. This review, by a particular focus on EMT, seeks to provide a comprehensive understanding of MMPs, especially MMP-9, in kidney fibrosis.

5.
BMC Immunol ; 14: 6, 2013 Feb 05.
Article in English | MEDLINE | ID: mdl-23384230

ABSTRACT

BACKGROUND: Macrophages have heterogeneous phenotypes and complex functions within both innate and adaptive immune responses. To date, most experimental studies have been performed on macrophages derived from bone marrow, spleen and peritoneum. However, differences among macrophages from these particular sources remain unclear. In this study, the features of murine macrophages from bone marrow, spleen and peritoneum were compared. RESULTS: We found that peritoneal macrophages (PMs) appear to be more mature than bone marrow derived macrophages (BMs) and splenic macrophages (SPMs) based on their morphology and surface molecular characteristics. BMs showed the strongest capacity for both proliferation and phagocytosis among the three populations of macrophage. Under resting conditions, SPMs maintained high levels of pro-inflammatory cytokines expression (IL-6, IL-12 and TNF-α), whereas BMs produced high levels of suppressive cytokines (IL-10 and TGF-ß). However, SPMs activated with LPS not only maintained higher levels of (IL-6, IL-12 and TNF-α) than BMs or PMs, but also maintained higher levels of IL-10 and TGF-ß. CONCLUSIONS: Our results show that BMs, SPMs and PMs are distinct populations with different biological functions, providing clues to guide their further experimental or therapeutic use.


Subject(s)
Bone Marrow Cells/cytology , Macrophages, Peritoneal/cytology , Macrophages/cytology , Spleen/cytology , Animals , Antigen Presentation/immunology , CD4-Positive T-Lymphocytes/immunology , Cell Membrane/metabolism , Cell Proliferation , Cell Shape , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Dextrans/metabolism , Flow Cytometry , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/metabolism , Gene Expression Regulation , Lymphocyte Activation/immunology , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Phagocytosis/immunology , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism
6.
Lab Invest ; 93(4): 434-49, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23358111

ABSTRACT

A pro-fibrotic role of matrix metalloproteinase-9 (MMP-9) in tubular cell epithelial-mesenchymal transition (EMT) is well established in renal fibrosis; however studies from our group and others have demonstrated some previously unrecognized complexity of MMP-9 that has been overlooked in renal fibrosis. Therefore, the aim of this study was to determine the expression pattern, origin and the exact mechanism underlying the contribution of MMP-9 to unilateral ureteral obstruction (UUO), a well-established model of renal fibrosis via MMP-9 inhibition. Renal MMP-9 expression in BALB/c mice with UUO was examined on day 1, 3, 5, 7, 9, 11 and 14. To inhibit MMP-9 activity, MMP-2/9 inhibitor or MMP-9-neutralizing antibody was administered daily for 4 consecutive days from day 0-3, 6-9 or 10-13 and tissues harvested at day 14. In UUO, there was a bi-phasic early- and late-stage upregulation of MMP-9 activity. Interestingly, tubular epithelial cells (TECs) were the predominant source of MMP-9 during early stage, whereas TECs, macrophages and myofibroblasts produced MMP-9 during late-stage UUO. Early- and late-stage inhibition of MMP-9 in UUO mice significantly reduced tubular cell EMT and renal fibrosis. Moreover, MMP-9 inhibition caused a significant reduction in MMP-9-cleaved osteopontin and macrophage infiltration in UUO kidney. Our in vitro study showed MMP-9-cleaved osteopontin enhanced macrophage transwell migration and MMP-9 of both primary TEC and macrophage induced tubular cell EMT. In summary, our result suggests that MMP-9 of both TEC and macrophage origin may directly or indirectly contribute to the pathogenesis of renal fibrosis via osteopontin cleavage, which, in turn further recruit macrophage and induce tubular cell EMT. Our study also highlights the time dependency of its expression and the potential of stage-specific inhibition strategy against renal fibrosis.


Subject(s)
Kidney Diseases/immunology , Kidney/pathology , Matrix Metalloproteinase 9/metabolism , Osteopontin/metabolism , Ureteral Obstruction/metabolism , Animals , Cell Movement , Cells, Cultured , Epithelial Cells/physiology , Epithelial-Mesenchymal Transition , Fibrosis , Kidney/metabolism , Kidney Diseases/metabolism , Kidney Diseases/pathology , Macrophages/physiology , Mice , Mice, Inbred BALB C , Snail Family Transcription Factors , Transcription Factors/metabolism , beta Catenin/metabolism
7.
J Cell Sci ; 126(Pt 1): 67-76, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23203799

ABSTRACT

Transforming growth factor ß1 (TGF-ß1) is known to be both anti-inflammatory and profibrotic. Cross-talk between TGF-ß/Smad and Wnt/ß-catenin pathways in epithelial-mesenchymal transition (EMT) suggests a specific role for ß-catenin in profibrotic effects of TGF-ß1. However, no such mechanistic role has been demonstrated for ß-catenin in the anti-inflammatory effects of TGF-ß1. In the present study, we explored the role of ß-catenin in the profibrotic and anti-inflammatory effects of TGF-ß1 by using a cytosolic, but not membrane, ß-catenin knockdown chimera (F-TrCP-Ecad) and the ß-catenin/CBP inhibitor ICG-001. TGF-ß1 induced nuclear Smad3/ß-catenin complex, but not ß-catenin/LEF-1 complex or TOP-flash activity, during EMT of C1.1 (renal tubular epithelial) cells. F-TrCP-Ecad selectively degraded TGF-ß1-induced cytoplasmic ß-catenin and blocked EMT of C1.1 cells. Both F-TrCP-Ecad and ICG-001 blocked TGF-ß1-induced Smad3/ß-catenin and Smad reporter activity in C1.1 cells, suggesting that TGF-ß1-induced EMT depends on ß-catenin binding to Smad3, but not LEF-1 downstream of Smad3, through canonical Wnt. In contrast, in J774 macrophages, the ß-catenin level was low and was not changed by interferon-γ (IFN-γ) or lipopolysaccharide (LPS) with or without TGF-ß1. TGF-ß1 inhibition of LPS-induced TNF-α and IFN-γ-stimulated inducible NO synthase (iNOS) expression was not affected by F-TrCP-Ecad, ICG-001 or by overexpression of wild-type ß-catenin in J774 cells. Inhibition of ß-catenin by either F-TrCP-Ecad or ICG-001 abolished LiCl-induced TOP-flash, but not TGF-ß1-induced Smad reporter, activity in J774 cells. These results demonstrate for the first time that ß-catenin is required as a co-factor of Smad in TGF-ß1-induced EMT of C1.1 epithelial cells, but not in TGF-ß1 inhibition of macrophage activation. Targeting ß-catenin may dissociate the TGF-ß1 profibrotic and anti-inflammatory effects.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Lymphoid Enhancer-Binding Factor 1/metabolism , Smad3 Protein/metabolism , Transforming Growth Factor beta1/pharmacology , beta Catenin/metabolism , Animals , Blotting, Western , Cell Line , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Immunoprecipitation , Lymphoid Enhancer-Binding Factor 1/genetics , Mice , Microscopy, Fluorescence , Protein Binding/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Smad3 Protein/genetics , beta Catenin/genetics
8.
Kidney Int ; 83(2): 223-32, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23223173

ABSTRACT

The CD40-CD154 costimulatory pathway has been shown to be critical for both T- and B-cell activation in autoimmune disease. Here, we assessed the effects of blocking this pathway using CD40 DNA vaccine enhanced by dendritic cell targeting on the development of active Heymann nephritis, a rat model of human membranous nephropathy. DNA vaccination delivers plasmid DNA encoding the target antigen, either alone or in combination with enhancing elements, to induce both humoral and cellular immune responses. To determine whether CD40 DNA vaccine targeting the encoded CD40 directly to dendritic cells would improve the efficacy of the vaccination against self-protein CD40, we utilized a plasmid encoding a single-chain Fv antibody specific for the dendritic cell-restricted antigen-uptake receptor DEC205 (scDEC), the target gene CD40, and the adjuvant tetanus sequence p30. This vaccine plasmid was compared to a control plasmid without scDEC. Rats vaccinated with scDEC-CD40 had significantly less proteinuria and renal injury than did rats receiving scControl-CD40 and were protected from developing Heymann nephritis. Thus, CD40 DNA vaccination targeted to dendritic cells limits the development of Heymann nephritis.


Subject(s)
CD40 Antigens/genetics , Dendritic Cells/immunology , Glomerulonephritis, Membranous/prevention & control , Vaccines, DNA/immunology , Animals , Autoantibodies/blood , CD40 Antigens/immunology , CD40 Antigens/physiology , CD40 Ligand/physiology , CTLA-4 Antigen/genetics , Immunoglobulin G/metabolism , Lymphocyte Activation , Male , Mice , Rats , Rats, Inbred Lew , Single-Chain Antibodies/genetics , Vaccination
9.
Int J Clin Exp Med ; 5(1): 24-33, 2012.
Article in English | MEDLINE | ID: mdl-22328945

ABSTRACT

Monocytes utilise a variety of chemokines to traffic to atherosclerotic plaques. CX3C chemokine ligand 1 (CX3CL1 & Fractalkine) and its receptor CX3CR1 and monocyte chemoattractant protein 1 (CCL2) have been identified as chemokines/receptors that have an important role in the migration and recruitment of monocytes during the pathogenesis of several inflammatory diseases including atherosclerosis. DNA vectors containing single chain variable region fragment (scFv) for DC-targeted receptor DEC205 were cloned with mouse CX3CR1 and CCL2 genes respectively, and vaccinated into C57/BL6 mice weekly for 3 weeks. Induced anti-CX3CR1 and anti-CCL2 in vaccinated mice was examined by ELISA and Western Blot analysis, while the cellular response was examined by ELISPOT. The inhibition of chemotaxis of J774 macrophages to Py-4-1 endothelial cells was examined by in vitro transwell migration assay using serum collected from vaccinated mice. All vaccinated mice generated anti-CX3CR1 and anti-CCL2 Ab and cellular response by 8 weeks after DNA vaccination. Macrophage migration towards TNF-α activated endothelial cells was significantly inhibited by serum containing both anti-CX3CR1 or anti-CCL2 Ab from vaccinated mice. These results demonstrate that DC-targeting of DNA vaccines to self-antigens generates functional immune responses which can inhibit specific key chemotactic targets. This suggests a potential therapeutic role for chemokine/receptor DNA vaccination in atherosclerosis, where chemotaxis has a pivotal role in the inflammatory process.

10.
Kidney Int ; 81(9): 892-902, 2012 May.
Article in English | MEDLINE | ID: mdl-22318423

ABSTRACT

Plasmacytoid dendritic cells play important roles in inducing immune tolerance, preventing allograft rejection, and regulating immune responses in both autoimmune disease and graft-versus-host disease. In order to evaluate a possible protective effect of plasmacytoid dendritic cells against renal inflammation and injury, we purified these cells from mouse spleens and adoptively transferred lipopolysaccharide (LPS)-treated cells, modified ex vivo, into mice with adriamycin nephropathy. These LPS-treated cells localized to the kidney cortex and the lymph nodes draining the kidney, and protected the kidney from injury during adriamycin nephropathy. Glomerulosclerosis, tubular atrophy, interstitial expansion, proteinuria, and creatinine clearance were significantly reduced in mice with adriamycin nephropathy subsequently treated with LPS-activated plasmacytoid dendritic cells as compared to the kidney injury in mice given naive plasmacytoid dendritic cells. In addition, LPS-pretreated cells, but not naive plasmacytoid dendritic cells, convert CD4+CD25- T cells into Foxp3+ regulatory T cells and suppress the proinflammatory cytokine production of endogenous renal macrophages. This may explain their ability to protect against renal injury in adriamycin nephropathy.


Subject(s)
Adoptive Transfer , Dendritic Cells/drug effects , Dendritic Cells/transplantation , Kidney Diseases/prevention & control , Kidney/immunology , Lipopolysaccharides/pharmacology , Animals , Atrophy , Biomarkers/blood , CD4-Positive T-Lymphocytes/immunology , Cells, Cultured , Chronic Disease , Coculture Techniques , Creatinine/blood , Cytokines/metabolism , Dendritic Cells/immunology , Disease Models, Animal , Doxorubicin , Forkhead Transcription Factors/metabolism , Glomerulonephritis/immunology , Glomerulonephritis/prevention & control , Inflammation Mediators/metabolism , Interleukin-2 Receptor alpha Subunit/metabolism , Kidney/metabolism , Kidney/pathology , Kidney/physiopathology , Kidney Diseases/blood , Kidney Diseases/chemically induced , Kidney Diseases/immunology , Kidney Diseases/pathology , Kidney Diseases/physiopathology , Lymph Nodes/immunology , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Nephritis, Interstitial/immunology , Nephritis, Interstitial/prevention & control , Phenotype , Proteinuria/immunology , Proteinuria/prevention & control , T-Lymphocytes, Regulatory/immunology , Toll-Like Receptor 4/metabolism
11.
J Biomed Biotechnol ; 2011: 567305, 2011.
Article in English | MEDLINE | ID: mdl-22007144

ABSTRACT

E-Cadherin/ß-catenin complex plays an important role in maintaining epithelial integrity and disrupting this complex affect not only the adhesive repertoire of a cell, but also the Wnt-signaling pathway. Aberrant expression of the complex is associated with a wide variety of human malignancies and disorders of fibrosis resulting from epithelial-mesenchymal transition. These associations provide insights into the complexity that is likely responsible for the fibrosis/tumor suppressive action of E-cadherin/ß-catenin.


Subject(s)
Cadherins/metabolism , Epithelial-Mesenchymal Transition/physiology , Epithelium/metabolism , Wnt Signaling Pathway/physiology , beta Catenin/metabolism , Cadherins/genetics , Epithelial Cells/metabolism , Epithelial Cells/pathology , Epithelial-Mesenchymal Transition/genetics , Epithelium/pathology , Fibrosis , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , beta Catenin/genetics
12.
J Am Soc Nephrol ; 22(7): 1229-39, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21719780

ABSTRACT

The kidney contains receptors for the cytokine IL-25, but the effects of IL-25 in CKD are unknown. Here, we induced adriamycin nephropathy in both BALB/c mice and severe combined immunodeficient (SCID) mice, and we injected IL-25 for 7 consecutive days starting at day 5 after adriamycin administration. BALB/c mice treated with IL-25 had less glomerulosclerosis, tubular atrophy, interstitial expansion, and proteinuria than control mice at day 28. IL-25 increased the levels of IL-4 and IL-13 in serum, kidney, renal draining lymph nodes, and CD4+ lymphocytes. IL-25 also directly suppressed effector macrophages in vitro and in vivo and induced alternatively activated (M2) macrophages in vivo. However, in SCID mice and in BALB/c mice treated with IL-4/13-neutralizing antibody, IL-25 failed to protect against renal injury and did not induce M2. In conclusion, IL-25 protects against renal injury in adriamycin nephropathy in mice by, at least in part, inducing Th2 immune responses.


Subject(s)
Glomerulosclerosis, Focal Segmental/metabolism , Interleukins/metabolism , Macrophage Activation/drug effects , Proteinuria/metabolism , Th2 Cells/drug effects , Acute Kidney Injury/chemically induced , Acute Kidney Injury/prevention & control , Animals , Antibiotics, Antineoplastic , Doxorubicin , Glomerulosclerosis, Focal Segmental/chemically induced , Glomerulosclerosis, Focal Segmental/drug therapy , Glomerulosclerosis, Focal Segmental/immunology , Interleukin-13/metabolism , Interleukin-4/metabolism , Interleukins/pharmacology , Interleukins/therapeutic use , Male , Mice , Mice, Inbred BALB C , Mice, SCID , Proteinuria/chemically induced , Proteinuria/drug therapy , Proteinuria/immunology
13.
J Am Soc Nephrol ; 21(6): 933-42, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20299353

ABSTRACT

IL-10/TGF-beta-modified macrophages, a subset of activated macrophages, produce anti-inflammatory cytokines, suggesting that they may protect against inflammation-mediated injury. Here, macrophages modified ex vivo by IL-10/TGF-beta (IL-10/TGF-beta Mu2) significantly attenuated renal inflammation, structural injury, and functional decline in murine adriamycin nephrosis (AN). These cells deactivated effector macrophages and inhibited CD4+ T cell proliferation. IL-10/TGF-beta Mu2 expressed high levels of the regulatory co-stimulatory molecule B7-H4, induced regulatory T cells from CD4+CD25- T cells in vitro, and increased the number of regulatory T cells in lymph nodes draining the kidneys in AN. The phenotype of IL-10/TGF-beta Mu2 did not switch to that of effector macrophages in the inflamed kidney, and these cells did not promote fibrosis. Taken together, these data demonstrate that IL-10/TGF-beta-modified macrophages effectively protect against renal injury in AN and may become part of a therapeutic strategy for chronic inflammatory disease.


Subject(s)
Interleukin-10/pharmacology , Macrophages/drug effects , Macrophages/pathology , Nephrosis/prevention & control , Nephrosis/physiopathology , T-Lymphocytes, Regulatory/pathology , Transforming Growth Factor beta/pharmacology , Animals , B7-1 Antigen/metabolism , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathology , Cell Proliferation/drug effects , Cells, Cultured , Disease Models, Animal , Doxorubicin/adverse effects , Kidney/drug effects , Kidney/metabolism , Kidney/pathology , Male , Mice , Mice, Inbred BALB C , Nephrosis/chemically induced , Nitric Oxide Synthase Type II/metabolism , Phenotype , V-Set Domain-Containing T-Cell Activation Inhibitor 1
14.
Am J Pathol ; 176(3): 1256-70, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20075196

ABSTRACT

As a rich source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages are well-placed to play an important role in renal fibrosis. However, the exact underlying mechanisms and the extent of macrophage involvement are unclear. Tubular cell epithelial-mesenchymal transition (EMT) is an important contributor to renal fibrosis and MMPs to induction of tubular cell EMT. The aim of this study was to investigate the contribution of macrophages and MMPs to induction of tubular cell EMT. The murine C1.1 tubular epithelial cell line and primary tubular epithelial cells were cultured in activated macrophage-conditioned medium (AMCM) derived from lipopolysaccharide-activated J774 macrophages. MMP-9, but not MMP-2 activity was detected in AMCM. AMCM-induced tubular cell EMT in C1.1 cells was inhibited by broad-spectrum MMP inhibitor (GM6001), MMP-2/9 inhibitor, and in AMCM after MMP-9 removal by monoclonal Ab against MMP-9. AMCM-induced EMT in primary tubular epithelial cells was inhibited by MMP-2/9 inhibitor. MMP-9 induced tubular cell EMT in both C1.1 cells and primary tubular epithelial cells. Furthermore, MMP-9 induced tubular cell EMT in C1.1 cells to an extent similar to transforming growth factor-beta. Transforming growth factor-beta-induced tubular cell EMT in C1.1 cells was inhibited by MMP-2/9 inhibitor. Our in vitro study provides evidence that MMPs, specifically MMP-9, secreted by effector macrophages can induce tubular cell EMT and thereby contribute to renal fibrosis.


Subject(s)
Epithelial Cells/enzymology , Epithelial Cells/pathology , Epithelium/enzymology , Kidney Tubules/pathology , Macrophages/enzymology , Matrix Metalloproteinase 9/metabolism , Mesoderm/enzymology , Actins/metabolism , Animals , Cell Line , Cells, Cultured , Culture Media, Conditioned/pharmacology , Dipeptides/pharmacology , Epithelial Cells/drug effects , Epithelium/pathology , Fibrosis/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Lipopolysaccharides/pharmacology , Macrophage Activation/drug effects , Macrophages/drug effects , Macrophages/pathology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase Inhibitors , Mesoderm/drug effects , Mesoderm/pathology , Mice , Mice, Inbred BALB C , Phenotype , Protein Transport/drug effects , Recombinant Proteins/pharmacology , Ureteral Obstruction/enzymology , Ureteral Obstruction/pathology
15.
Am J Pathol ; 175(2): 580-91, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19590041

ABSTRACT

Epithelial-mesenchymal transition (EMT) plays an important role in organ fibrosis, including that of the kidney. Loss of E-cadherin expression is a hallmark of EMT; however, whether the loss of E-cadherin is a consequence or a cause of EMT remains unknown, especially in the renal system. In this study, we show that transforming growth factor (TGF)-beta1-induced EMT in renal tubular epithelial cells is dependent on proteolysis. Matrix metalloproteinase-mediated E-cadherin disruption led directly to tubular epithelial cell EMT via Slug. TGF-beta1 induced the proteolytic shedding of E-cadherin, which caused the nuclear translocation of beta-catenin, the transcriptional induction of Slug, and the repression of E-cadherin transcription in tubular epithelial cells. These findings reveal a direct role for E-cadherin and for matrix metalloproteinases in causing EMT downstream of TGF-beta1 in fibrotic disease. Specific inhibition rather than activation of matrix metalloproteinases may offer a novel approach for treatment of fibrotic disease.


Subject(s)
Cadherins/metabolism , Cell Dedifferentiation , Epithelium/pathology , Kidney Tubules/pathology , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 9/metabolism , Mesoderm/pathology , Active Transport, Cell Nucleus , Animals , Cadherins/genetics , Cell Line , Cell Nucleus/metabolism , Fibrosis , Rats , Snail Family Transcription Factors , Transcription Factors/metabolism , Transcription, Genetic , Transforming Growth Factor beta1/pharmacology , Up-Regulation , beta Catenin/metabolism
16.
Am J Pathol ; 172(6): 1491-9, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18467704

ABSTRACT

Macrophages are important mediators of injury in most types of human kidney diseases; however, the pathogenic importance of both macrophage number and activation status is unknown. To examine this question, severe-combined immunodeficient mice with adriamycin nephrosis, an experimental model of human focal segmental glomerulosclerosis, were treated intravenously with either resting (1 x 10(6) to 5 x 10(6)) or activated (1 x 10(3) to 1 x 10(6)) macrophages on day 6 postadriamycin administration, and the effects on kidney injury were examined. On day 28, renal injury was worse in the group that received activated macrophages at doses as low as 1 x 10(4) macrophages per mouse compared with control adriamycin nephrotic mice. However, treatment with resting macrophages at doses as high as 5 x 10(6) macrophages per mouse had no significant effect on either renal histology or function. The transferred activated macrophages homed to inflamed kidneys during the middle-to-late stages of the disease, but such homing was not observed for resting macrophages. This study of in vivo cell adoptive transfer supports the importance of macrophage activation status over macrophage number in causing renal injury. These data suggest that therapeutic strategies for treating progressive kidney diseases should target activated macrophages.


Subject(s)
Kidney/pathology , Macrophages/immunology , Nephrosis/immunology , Adoptive Transfer , Animals , Cells, Cultured , Disease Models, Animal , Doxorubicin , Glomerulosclerosis, Focal Segmental/immunology , Glomerulosclerosis, Focal Segmental/pathology , Kidney/immunology , Macrophage Activation , Macrophages/transplantation , Male , Mice , Mice, Inbred BALB C , Mice, SCID , Nephrosis/chemically induced , Nephrosis/pathology
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