Early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination

Mesenchymal stem/stromal cells (MSCs) can be isolated from most adult tissues and hold considerable promise for tissue regenerative therapies. Some of the potential advantages that MSCs have over other adult stem cell types include: (1) their relative ease of isolation, culture and expansion; (2) their immunomodulatory properties; (3) they can provide trophic support to injured tissues; (4) they can be transduced by retroviral vectors at a high efficiency; (5) they have an ability to home to sites of inflammation and injury. Collectively these characteristics suggest that MSCs are attractive vehicles for cell and gene therapy applications. In the current study, we investigated whether transplantation of human adipose-derived MSCs (Ad-MSCs) engineered to overexpress the anti-inflammatory cytokine interleukin (IL)-4 was efficacious in experimental autoimmune encephalomyelitis (EAE). Ad-MSCs transduced with a bicistronic lentiviral vector encoding mouse IL-4 and enhanced green fluorescent protein (Ad-IL4-MSCs) stably expressed, relatively high levels of both transgenes. Importantly the phenotypic and functional attributes of Ad-IL4-MSCs, such as the expression of homing molecules and differentiation capacity, was not altered by the transduction process. Notably, the early administration of Ad-IL4-MSCs in mice with EAE at the time of T-cell priming attenuated clinical disease. This protective effect was associated with a reduction in peripheral MOG-specific T-cell responses and a shift from a pro- to an anti-inflammatory cytokine response. These data suggest that the delivery of Ad-MSCs genetically engineered to express anti-inflammatory cytokines may provide a rational approach to promote immunomodulation and tissue protection in a number of inflammatory and degenerative diseases including multiple sclerosis.     

Early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination

Mesenchymal stem/stromal cells (MSCs) can be isolated from most adult tissues and hold considerable promise for tissue regenerative therapies. Some of the potential advantages that MSCs have over other adult stem cell types include: (1) their relative ease of isolation, culture and expansion; (2) their immunomodulatory properties; (3) they can provide trophic support to injured tissues; (4) they can be transduced by retroviral vectors at a high efficiency; (5) they have an ability to home to sites of inflammation and injury. Collectively these characteristics suggest that MSCs are attractive vehicles for cell and gene therapy applications. In the current study, we investigated whether transplantation of human adipose-derived MSCs (Ad-MSCs) engineered to overexpress the anti-inflammatory cytokine interleukin (IL)-4 was efficacious in experimental autoimmune encephalomyelitis (EAE). Ad-MSCs transduced with a bicistronic lentiviral vector encoding mouse IL-4 and enhanced green fluorescent protein (Ad-IL4-MSCs) stably expressed, relatively high levels of both transgenes. Importantly the phenotypic and functional attributes of Ad-IL4-MSCs, such as the expression of homing molecules and differentiation capacity, was not altered by the transduction process. Notably, the early administration of Ad-IL4-MSCs in mice with EAE at the time of T-cell priming attenuated clinical disease. This protective effect was associated with a reduction in peripheral MOG-specific T-cell responses and a shift from a pro- to an anti-inflammatory cytokine response. These data suggest that the delivery of Ad-MSCs genetically engineered to express anti-inflammatory cytokines may provide a rational approach to promote immunomodulation and tissue protection in a number of inflammatory and degenerative diseases including multiple sclerosis.     

Renal Dnase1 Enzyme Activity and Protein Expression Is Selectively Shut Down in Murine and Human Membranoproliferative Lupus Nephritis

Background

Deposition of chromatin-IgG complexes within glomerular membranes is a key event in the pathogenesis of lupus nephritis. We recently reported an acquired loss of renal Dnase1 expression linked to transformation from mild to severe membranoproliferative lupus nephritis in (NZBxNZW)F1 mice. As this may represent a basic mechanism in the progression of lupus nephritis, several aspects of Dnase1 expression in lupus nephritis were analyzed.

Methodology/Principal Findings

Total nuclease activity and Dnase1 expression and activity was evaluated using in situ and in vitro analyses of kidneys and sera from (NZBxNZW)F1 mice of different ages, and from age-matched healthy controls. Immunofluorescence staining for Dnase1 was performed on kidney biopsies from (NZBxNZW)F1 mice as well as from human SLE patients and controls. Reduced serum Dnase1 activity was observed in both mesangial and end-stage lupus nephritis. A selective reduction in renal Dnase1 activity was seen in mice with massive deposition of chromatin-containing immune complexes in glomerular capillary walls. Mice with mild mesangial nephritis showed normal renal Dnase1 activity. Similar differences were seen when comparing human kidneys with severe and mild lupus nephritis. Dnase1 was diffusely expressed within the kidney in normal and mildly affected kidneys, whereas upon progression towards end-stage renal disease, Dnase1 was down-regulated in all renal compartments. This demonstrates that the changes associated with development of severe nephritis in the murine model are also relevant to human lupus nephritis.

Conclusions/Significance

Reduction in renal Dnase1 expression and activity is limited to mice and SLE patients with signs of membranoproliferative nephritis, and may be a critical event in the development of severe forms of lupus nephritis. Reduced Dnase1 activity reflects loss in the expression of the protein and not inhibition of enzyme activity.     

Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis

In an effort to identify potential biomarkers in lupus nephritis, urine from mice with spontaneous lupus nephritis was screened for the presence of VCAM-1, P-selectin, TNFR-1, and CXCL16, four molecules that had previously been shown to be elevated in experimental immune nephritis, particularly at the peak of disease. Interestingly, all four molecules were elevated approximately 2- to 4-fold in the urine of several strains of mice with spontaneous lupus nephritis, including the MRL/lpr, NZM2410, and B6.Sle1.lpr strains, correlating well with proteinuria. VCAM-1, P-selectin, TNFR-1, and CXCL16 were enriched in the urine compared with the serum particularly in active disease, and were shown to be expressed within the diseased kidneys. Finally, all four molecules were also elevated in the urine of patients with lupus nephritis, correlating well with urine protein levels and systemic lupus erythematosus disease activity index scores. In particular, urinary VCAM-1 and CXCL16 showed superior specificity and sensitivity in distinguishing subjects with active renal disease from the other systemic lupus erythematosus patients. These studies uncover VCAM-1, P-selectin, TNFR-1, and CXCL16 as a quartet of molecules that may have potential diagnostic significance in lupus nephritis. Longitudinal studies are warranted to establish the clinical use of these potential biomarkers.     

Restoration of SMN expression in mesenchymal stem cells derived from gene-targeted patient-specific iPSCs

Spinal muscular atrophy (SMA) is primarily a neurodegenerative disease caused by the homozygous deletion of the survival motor neuron 1 (SMN1) gene, thereby reducing SMN protein expression. Mesenchymal stem cells (MSCs) have been implicated in the treatment of SMA. In the present study, we overexpressed exogenous SMN1 at the ribosomal DNA (rDNA) locus of induced pluripotent stem cells (iPSCs) generated from a SMA patient using an rDNA-targeting vector. The gene-targeted patient iPSCs differentiated into MSCs (SMN1-MSCs). A 2.1-fold higher expression level of SMN protein was detected in SMN1-MSCs than that detected in MSCs derived from patient iPSCs, and the results of the immunofluorescence analysis showed no difference in the quantity of SMN nuclear structures (gems) between SMN1-MSCs and MSCs derived from normal human iPSCs (h-MSCs). These findings provide a novel strategy for obtaining gene-targeted MSCs for potential clinical applications in autologous cell-based therapy.     

Amelioration of lupus nephritis by serum amyloid P component gene therapy with distinct mechanisms varied from different stage of the disease

Background

Our previous study revealed that administration of syngeneic female BALB/c mice with excessive self activated lymphocyte-derived DNA (ALD-DNA) could induce systemic lupus erythematosus (SLE) disease, indicating that overload of self-DNA might exceed normal clearance ability and comprise the major source of autoantigens in lupus mice. Serum amyloid P component (SAP), an acute-phase serum protein with binding reactivity to DNA in mice, was proved to promote the clearance of free DNA and prevent mice against self-antigen induced autoimmune response. It is reasonable to hypothesize that SAP treatment might contribute to alleviation of SLE disease, whereas its role in ALD-DNA-induced lupus nephritis is not fully understood.

Methodology/Principal Findings

The ratios of SAP to DNA significantly decreased and were negatively correlated with the titers of anti-dsDNA antibodies in ALD-DNA-induced lupus mice, indicating SAP was relatively insufficient in lupus mice. Herein a pcDNA3-SAP plasmid (pSAP) was genetically constructed and intramuscularly injected into BALB/c mice. It was found that SAP protein purified from the serum of pSAP-treated mice bound efficiently to ALD-DNA and inhibited ALD-DNA-mediated innate immune response in vitro. Treatment of ALD-DNA-induced lupus mice with pSAP in the early stage of SLE disease with the onset of proteinuria reversed lupus nephritis via decreasing anti-dsDNA autoantibody production and immune complex (IC) deposition. Further administration of pSAP in the late stage of SLE disease that had established lupus nephritis alleviated proteinuria and ameliorated lupus nephritis. This therapeutic effect of SAP was not only attributable to the decreased levels of anti-dsDNA autoantibodies, but also associated with the decreased infiltration of lymphocytes and the reduced production of inflammatory markers.

Conclusion/Significance

These results suggest that SAP administration could effectively alleviated lupus nephritis via modulating anti-dsDNA antibody production and the inflammation followed IC deposition, and SAP-based intervening strategy may provide new approaches for treating SLE disease.     

Aberrant macrophages mediate defective kidney repair that triggers nephritis in lupus-susceptible mice

CSF-1, required for macrophage (M?) survival, proliferation, and activation, is upregulated in the tubular epithelial cells (TECs) during kidney inflammation. CSF-1 mediates M?-dependent destruction in lupus-susceptible mice with nephritis and, paradoxically, M?-dependent renal repair in lupus-resistant mice after transient ischemia/reperfusion injury (I/R). We now report that I/R leads to defective renal repair, nonresolving inflammation, and, in turn, early-onset lupus nephritis in preclinical MRL/MpJ-Faslpr/Fas(lpr) mice (MRL-Fas(lpr) mice). Moreover, defective renal repair is not unique to MRL-Fas(lpr) mice, as flawed healing is a feature of other lupus-susceptible mice (Sle 123) and MRL mice without the Fas(lpr) mutation. Increasing CSF-1 hastens renal healing after I/R in lupus-resistant mice but hinders healing, exacerbates nonresolving inflammation, and triggers more severe early-onset lupus nephritis in MRL-Fas(lpr) mice. Probing further, the time-related balance of M1 "destroyer" M? shifts toward the M2 "healer" phenotype in lupus-resistant mice after I/R, but M1 M? continue to dominate in MRL-Fas(lpr) mice. Moreover, hypoxic TECs release mediators, including CSF-1, that are responsible for stimulating the expansion of M1 M? inherently poised to destroy the kidney in MRL-Fas(lpr) mice. In conclusion, I/R induces CSF-1 in injured TECs that expands aberrant M? (M1 phenotype), mediating defective renal repair and nonresolving inflammation, and thereby hastens the onset of lupus nephritis.     

Over-expression of HO-1 on mesenchymal stem cells promotes angiogenesis and improves myocardial function in infarcted myocardium

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with diverse cytoprotective effects, and reported to have an important role in angiogenesis recently. Here we investigated whether HO-1 transduced by mesenchymal stem cells (MSCs) can induce angiogenic effects in infarcted myocardium. HO-1 was transfected into cultured MSCs using an adenoviral vector. 1 × 10⁶ Ad-HO-1-transfected MSCs (HO-1-MSCs) or Ad-Null-transfected MSCs (Null-MSCs) or PBS was respectively injected into rat hearts intramyocardially at 1 h post-myocardial infarction. The results showed that HO-1-MSCs were able to induce stable expression of HO-1 in vitro and in vivo. The capillary density and expression of angiogenic growth factors, VEGF and FGF2 were significantly enhanced in HO-1-MSCs-treated hearts compared with Null-MSCs-treated and PBS-treated hearts. However, the angiogenic effects of HO-1 were abolished by treating the animals with HO inhibitor, zinc protoporphyrin. The myocardial apoptosis was marked reduced with significantly reduced fibrotic area in HO-1-MSCs-treated hearts; Furthermore, the cardiac function and remodeling were also significantly improved in HO-1-MSCs-treated hearts. Our current findings support the premise that HO-1 transduced by MSCs can induce angiogenic effects and improve heart function after acute myocardial infarction.     

The granulocyte colony stimulating factor pathway regulates autoantibody production in a murine induced model of systemic lupus erythematosus

Introduction

An NZB-derived genetic locus (Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF.

Methods

Leukocytes from B6.Sle2c2 and B6 congenic mice, which carry a different allele of the G-CSF receptor, were compared for their responses to G-CSF. Autoantibody production was induced with the chronic graft-versus-host-disease (cGVHD) model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta^®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus.

Results

B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4⁺ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice.

Conclusion

The different in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus.     

CXCR4 Receptor Overexpression in Mesenchymal Stem Cells Facilitates Treatment of Acute Lung Injury in Rats

Novel therapeutic regimens for tissue renewal incorporate mesenchymal stem cells (MSCs) as they differentiate into a variety of cell types and are a stem cell type that is easy to harvest and to expand in vitro. However, surface chemokine receptors, such as CXCR4, which are involved in the mobilization of MSCs, are expressed only on the surface of a small proportion of MSCs, and the lack of CXCR4 expression may underlie the low efficiency of homing of MSCs toward tissue damage, which results in a poor curative effect. Here, a rat CXCR4 expressing lentiviral vector was constructed and introduced into MSCs freshly prepared from rat bone marrow. The influence of CXCR4 expression on migration, proliferation, differentiation, and paracrine effects of MSCs was examined in vitro. The in vivo properties of CXCR4-MSCs were also investigated in a model of acute lung injury in rats induced by lipopolysaccharide. Expression of CXCR4 in MSCs significantly enhanced the chemotactic and paracrine characteristics of the cells in vitro but did not affect self-renewal or differentiation into alveolar and vascular endothelial cells. In vivo, CXCR4 improved MSC homing and colonization of damaged lung tissue, and furthermore, the transplanted CXCR4-MSCs suppressed the development of acute lung injury in part by modulating levels of inflammatory molecules and the neutrophil count. These results indicated that efficient mobilization of MSCs to sites of tissue injury may be due to CXCR4, and therefore, increased expression of CXCR4 may improve their therapeutic potential in the treatment of diseases where tissue damage develops.     

Murine lupus susceptibility locus Sle1c2 mediates CD4+ T cell activation and maps to estrogen-related receptor γ

Sle1c is a sublocus of the NZM2410-derived Sle1 major lupus susceptibility locus. We have shown previously that Sle1c contributes to lupus pathogenesis by conferring increased CD4(+) T cell activation and increased susceptibility to chronic graft-versus-host disease (cGVHD), which mapped to the centromeric portion of the locus. In this study, we have refined the centromeric sublocus to a 675-kb interval, termed Sle1c2. Mice from recombinant congenic strains expressing Sle1c2 exhibited increased CD4(+) T cell intrinsic activation and cGVHD susceptibility, similar to mice with the parental Sle1c. In addition, B6.Sle1c2 mice displayed a robust expansion of IFN-γ-expressing T cells. NZB complementation studies showed that Sle1c2 expression exacerbated B cell activation, autoantibody production, and renal pathology, verifying that Sle1c2 contributes to lupus pathogenesis. The Sle1c2 interval contains two genes, only one of which, Esrrg, is expressed in T cells. B6.Sle1c2 CD4(+) T cells expressed less Esrrg than B6 CD4(+) T cells, and Esrrg expression was correlated negatively with CD4(+) T cell activation. Esrrg encodes an orphan nuclear receptor that regulates oxidative metabolism and mitochondrial functions. In accordance with reduced Esrrg expression, B6.Sle1c2 CD4(+) T cells present reduced mitochondrial mass and altered mitochondrial functions as well as altered metabolic pathway utilization when compared with B6 CD4(+) T cells. Taken together, we propose Esrrg as a novel lupus susceptibility gene regulating CD4(+) T cell function through their mitochondrial metabolism.     

Lupus Nephritis: Enigmas,Conflicting Models and an Emerging Concept

Autoantibodies to components of chromatin, which include double-stranded DNA (dsDNA), histones and nucleosomes, are central in the pathogenesis of lupus nephritis. How anti-chromatin autoantibodies exert their nephritogenic activity, however, is controversial. One model assumes that autoantibodies initiate inflammation when they cross-react with intrinsic glomerular structures such as components of membranes, matrices or exposed nonchromatin ligands released from cells. Another model suggests glomerular deposition of autoantibodies in complex with chromatin, thereby inducing classic immune complex–mediated tissue damage. Recent data suggest acquired error of renal chromatin degradation due to the loss of renal DNaseI enzyme activity is an important contributing factor to the development of lupus nephritis in lupus-prone (NZBxNZW)F1 mice and in patients with lupus nephritis. Down-regulation of DNaseI expression results in reduced chromatin fragmentation and in deposition of extracellular chromatin–IgG complexes in glomerular basement membranes in individuals who produce IgG anti-chromatin autoantibodies. The main focus of the present review is to discuss whether exposed chromatin fragments in glomeruli are targeted by potentially nephritogenic anti-dsDNA autoantibodies or if the nephritogenic activity of these autoantibodies is explained by cross-reaction with intrinsic glomerular constituents or if both models coexist in diseased kidneys. In addition, the role of silencing of the renal DNaseI gene and the biological consequences of reduced chromatin fragmentation in nephritic kidneys are discussed.     

Epigallocatechin-3-gallate prevents lupus nephritis development in mice via enhancing the Nrf2 antioxidant pathway and inhibiting NLRP3 inflammasome activation

Patients with lupus nephritis show an impaired oxidative status and increased levels of interleukin (IL)-1β and IL-18, which are closely linked to inflammation and correlated with disease activity. Although epigallocatechin-3-gallate (EGCG), the major bioactive polyphenol present in green tea with antioxidant and free radical scavenging activities, has been reported to have anti-inflammatory effects by inhibiting nuclear factor-kappa B (NF-κB)-mediated inflammatory responses in vivo, its effectiveness for the treatment of lupus nephritis is still unknown. In the present study, 12-week-old New Zealand black/white (NZB/W) F1 lupus-prone mice were treated daily with EGCG by gavage until sacrificed at 34 weeks old for clinical, pathological, and mechanistic evaluation. We found that the administration (1) prevented proteinuria, renal function impairment, and severe renal lesions; (2) increased renal nuclear factor E2-related factor 2 (Nrf2) and glutathione peroxidase activity; (3) reduced renal oxidative stress, NF-κB activation, and NLRP3 mRNA/protein expression and protein levels of mature caspase-1, IL-1β, and IL-18; and (4) enhanced splenic regulatory T (Treg) cell activity. Our data clearly demonstrate that EGCG has prophylactic effects on lupus nephritis in these mice that are highly associated with its effects of enhancing the Nrf2 antioxidant signaling pathway, decreasing renal NLRP3 inflammasome activation, and increasing systemic Treg cell activity.     

A therapeutic role for mesenchymal stem cells in acute lung injury independent of hypoxia-induced mitogenic factor

Bone marrow mesenchymal stem cells (BM-MSCs) have therapeutic potential in acute lung injury (ALI). Hypoxia-induced mitogenic factor (HIMF) is a lung-specific growth factor that participates in a variety of lung diseases. In this study, we evaluated the therapeutic role of BM-MSC transplantation in lipopolysaccharide (LPS)- induced ALI and assessed the importance of HIMF in MSC transplantation. MSCs were isolated and identified, and untransduced MSCs, MSCs transduced with null vector or MSCs transduced with a vector encoding HIMF were transplanted into mice with LPS-induced ALI. Histopathological changes, cytokine expression and indices of lung inflammation and lung injury were assessed in the various experimental groups. Lentiviral transduction did not influence the biological features of MSCs. In addition, transplantation of BM-MSCs alone had significant therapeutic effects on LPS-induced ALI, although BM-MSCs expressing HIMF failed to improve the histopathological changes observed with lung injury. Unexpectedly, tumour necrosis factor α levels in lung tissues, lung oedema and leucocyte infiltration into lungs were even higher after the transplantation of MSCs expressing HIMF, followed by a significant increase in lung hydroxyproline content and α-smooth muscle actin expression on day 14, as compared to treatment with untransduced MSCs. BM-MSC transplantation improved LPS-induced lung injury independent of HIMF.     

Negative role of colony-stimulating factor-1 in macrophage, T cell, and B cell mediated autoimmune disease in MRL-Fas(lpr) mice

Inflammation in the kidney and other tissues (lung, and salivary and lacrimal glands) is characteristic of MRL-Fas(lpr) mice with features of lupus. Macrophages (Mphi) are prominent in these tissues. Given that 1) Mphi survival, recruitment, proliferation, and activation during inflammation is dependent on CSF-1, 2) Mphi mediate renal resident cell apoptosis, and 3) CSF-1 is up-regulated in MRL-Fas(lpr) mice before, and during nephritis, we hypothesized that CSF-1-deficient MRL-Fas(lpr) mice would be protected from Mphi-mediated nephritis, and the systemic illness. To test this hypothesis, we compared CSF-1-deficient MRL-Fas(lpr) with wild-type strains. Renal pathology is suppressed and function improved in CSF-1-deficient MRL-Fas(lpr) mice. There are far fewer intrarenal Mphi and T cells in CSF-1-deficient MRL-Fas(lpr) vs wild-type kidneys. This leukocytic reduction results from suppressed infiltration, and intrarenal proliferation, but not enhanced apoptosis. The CSF-1-deficient MRL-Fas(lpr) kidneys remain preserved as indicated by greatly reduced indices of injury (nephritogenic cytokines, tubular apoptosis, and proliferation). The renal protective mechanism in CSF-1-deficient mice is not limited to reduced intrarenal leukocytes; circulating Igs and autoantibodies, and renal Ig deposits are decreased. This may result from enhanced B cell apoptosis and fewer B cells in CSF-1-deficient MRL-Fas(lpr) mice. Furthermore, the systemic illness including, skin, lung, and lacrimal and salivary glands pathology, lymphadenopathy, and splenomegaly are dramatically suppressed in CSF-1-deficient MRL-Fas(lpr) as compared with wild-type mice. These results indicate that CSF-1 is an attractive therapeutic target to combat Mphi-, T cell-, and B cell-mediated autoimmune lupus.     

Pre-B cell leukemia homeobox 1 is associated with lupus susceptibility in mice and humans

Sle1a.1 is part of the Sle1 susceptibility locus, which has the strongest association with lupus nephritis in the NZM2410 mouse model. In this study, we show that Sle1a.1 results in the production of activated and autoreactive CD4(+) T cells. Additionally, Sle1a.1 expression reduces the peripheral regulatory T cell pool, as well as induces a defective response of CD4(+) T cells to the retinoic acid expansion of TGF-β-induced regulatory T cells. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d overexpression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells and to decrease their apoptotic response to retinoic acid. PBX1-d is expressed more frequently in the CD4(+) T cells from lupus patients than from healthy controls, and its presence correlates with an increased central memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance.     

HO-1 modified mesenchymal stem cells modulate MMPs/TIMPs system and adverse remodeling in infarcted myocardium

The present study was to determine the effects of the heme oxygenase-1 (HO-1) modified mesenchymal stem cells (MSCs) transplantation into acute MI hearts on normalizing the ratio of MMPs/TIMPs and remodeling in infarcted myocardium. HO-1 was transfected into cultured MSCs using an adenoviral vector. 1 × 10⁶ Ad-HO-1-transfected MSCs (HO-1-MSCs) or Ad-Null-transfected MSCs (Null-MSCs) or PBS was respectively injected into rat hearts 1 h intramyocardially after myocardial infarction. The cardiac performance was significantly improved and left ventricular dilatation was significantly attenuated in HO-1-MSCs transplanted hearts. Moreover, a significant increase in microvessel density was observed in HO-1-MSCs transplanted hearts. TIMP2,3 expression in HO-1-MSCs transplanted hearts was significantly increased, and MMP2,9 expression in HO-1-MSCs transplanted hearts was significantly lower than Null-MSCs transplanted and PBS-treated hearts. TIMP1 expression did not vary significantly. Null-MSCs transplantation did not decrease the expression of MMP2,9 significantly compared with PBS-treated hearts. The ratio of TIMP2 to MMP2, and TIMP3 to MMP9 in cell-grafted hearts was increased significantly. HO-1-MSCs transplantation normalize the ratio of MMPs/TIMPs, contributing to the reversion of myocardial extracellular remodeling.     

Genetic dissection of the murine lupus susceptibility locus Sle2: contributions to increased peritoneal B-1a cells and lupus nephritis map to different loci

Lupus pathogenesis in the NZM2410 mouse model results from the expression of multiple interacting susceptibility loci. Sle2 on chromosome 4 was significantly linked to glomerulonephritis in a linkage analysis of a NZM2410 x B6 cross. Yet, Sle2 expression alone on a C57BL/6 background did not result in any clinical manifestation, but in an abnormal B cell development, including the accumulation of B-1a cells in the peritoneal cavity and spleen. Analysis of B6.Sle2 congenic recombinants showed that at least three independent loci, New Zealand White-derived Sle2a and Sle2b, and New Zealand Black-derived Sle2c, contribute to an elevated number of B-1a cells, with Sle2c contribution being the strongest of the three. To determine the contribution of these three Sle2 loci to lupus pathogenesis, we used a mapping by genetic interaction strategy, in which we bred them to B6.Sle1.Sle3 mice. We then compared the phenotypes of these triple congenic mice with that of previously characterized B6.Sle1.Sle2.Sle3, which express the entire Sle2 interval in combination with Sle1 and Sle3. Sle2a and Sle2b, but not Sle2c, contributed significantly to lupus pathogenesis in terms of survival rate, lymphocytic expansion, and kidney pathology. These results show that the Sle2 locus contains several loci affecting B cell development, with only the two NZW-derived loci having the least effect of B-1a cell accumulation significantly contributing to lupus pathogenesis.     

Myeloid dendritic cells from B6.NZM Sle1/Sle2/Sle3 lupus-prone mice express an IFN signature that precedes disease onset

Patients with systemic lupus erythematosus show an overexpression of type I IFN-responsive genes that is referred to as "IFN signature." We found that B6.NZMSle1/Sle2/Sle3 (Sle1,2,3) lupus-prone mice also express an IFN signature compared with non-autoimmune C57BL/6 mice. In vitro, myeloid dendritic cells (mDCs) (GM-CSF bone marrow-derived dendritic cells; BMDCs) from Sle1,2,3 mice constitutively overexpressed IFN-responsive genes such as IFN-β, Oas-3, Mx-1, ISG-15, and CXCL10 and members of the IFN signaling pathway STAT1, STAT2, and IRF7. The IFN signature was similar in Sle1,2,3 BMDCs from young, pre-autoimmune mice and from mice with high titers of autoantibodies, suggesting that the IFN signature in mDCs precedes disease onset and is independent from the autoantibodies. Sle1,2,3 BMDCs hyperresponded to stimulation with IFN-α and the TLR7 and TLR9 agonists R848 and CpGs. We propose that this hyperresponse is induced by the IFN signature and only partially contributes to the signature, as oligonucleotides inhibitory for TLR7 and TLR9 only partially suppressed the constitutive IFN signature, and pre-exposure to IFN-α induced the same hyperresponse in wild-type BMDCs as in Sle1,2,3 BMDCs. In vivo, mDCs and to a lesser extent T and B cells from young prediseased Sle1,2,3 mice also expressed the IFN signature, although they lacked the strength that BMDCs showed in vitro. Sle1,2,3 plasmacytoid DCs expressed the IFN signature in vitro but not in vivo, suggesting that mDCs may be more relevant before disease onset. We propose that Sle1,2,3 mice are useful tools to study the role of the IFN signature in lupus pathogenesis.     

Mesenchymal stromal cell injection protects against oxidative stress in Escherichia coli–induced acute lung injury in mice

Background aimsStem cells may be a promising therapy for acute respiratory distress syndrome. Recent in vivo and in vitro studies suggested that the mesenchymal stromal cells (MSCs) have anti-oxidative stress properties. We hypothesized that intravenous injection of bone marrow–derived mesenchymal stem cells (MSCs) could attenuate Escherichia coli–induced acute lung injury (ALI) in mice by controlling the oxidative stress status.MethodsEighty mice were randomly divided into four groups: group 1 (control group) received 25 μL of saline as a vehicle; group 2 contained E coli–induced ALI mice; group 3 included mice that received MSCs before induction of ALI; group 4 included mice that received MSCs after induction of ALI. Lung samples were isolated and assayed for oxidative stress variables and histopathologic analysis. Total anti-oxidant capacity was measured in broncho-alveolar lavage.ResultsPre- and post-injury MSC injection increased survival, reduced pulmonary edema and attenuated lung injuries in ALI mice. Histologically, MSCs exhibited a considerable degree of preservation of the pulmonary alveolar architecture. An increase of anti-oxidant enzyme activities and a decrease of myeloperoxidase activity and malondialdehyde levels in the MSC recipient groups versus the ALI group were found. Furthermore, the total anti-oxidant capacity and reduced glutathione levels were significantly increased in MSCs recipient groups versus the ALI group. Weak +ve inducible nitric oxide synthase immuno-expression in groups that received MSCs was detected. Pre-injury MSC injection showed better effects than did post-injury MSC injection.ConclusionsSystemic bone marrow–derived MSC injection was effective in modulating the oxidative stress status in E coli–induced acute lung injury in mice.