Implication of the IL-10-Expression Signature in the Pathogenicity of Leptospira-Infected Macrophages.

Leptospirosis, an emerging infectious disease caused by pathogenic Leptospira spp., occurs in ecoregions with heavy rainfall and has public health implications. Macrophages are the major anti-Leptospira phagocytes that infiltrate the kidneys during renal leptospirosis, which is caused by leptospires residing in the renal tubules. The pathogenicity of Leptospira spp. in immune effector cells such as macrophages is not well understood. To evaluate this pathogenesis, we characterized and compared the transcriptome-wide alterations in macrophages infected with pathogenic and nonpathogenic Leptospira spp. Using transcriptome data and quantitative reverse transcription PCR analysis, at 2 h postinfection, the hypoxia-inducible factor-1α-dependent glycolysis pathway was implicated in pathogenic Leptospira-infected macrophages but not in nonpathogenic leptospiral infections. Immune-related biological processes were mostly activated in pathogenic Leptospira-infected macrophages, and flow cytometry investigations revealed that classically activated macrophages represent the predominant polarization status. At 24 h after infection, biological pathways associated with interleukin-10, IL-10, signaling the induction of macrophage tolerance, as well as higher levels of IL-10 mRNA and protein expression, were observed in nonpathogenic Leptospira-infected macrophages compared to in pathogenic leptospiral infection. Following leptospiral infection of macrophages, strong IL-10-expressing transcriptome signatures were observed following nonpathogenic leptospiral infection. The transcriptional programs generated in Leptospira-infected macrophages revealed an inflammatory milieu following the production of a critical anti-inflammatory cytokine, IL-10, which is implicated in controlling the pathogenicity of activated macrophages. These findings imply that IL-10-mediated anti-inflammatory responses and tolerance in activated macrophages induced by nonpathogenic Leptospira spp. infection reduce inflammation and tissue damage, thus providing a potential therapeutic target for leptospirosis. IMPORTANCE Activation of macrophages by Leptospira spp. infection is thought to be involved in the pathogenesis of leptospirosis. To evaluate the innate macrophage responses to Leptospira spp., specifically pathogenic versus nonpathogenic Leptospira spp., we characterized the entire transcriptome-wide alterations in infected macrophages. We showed that hypoxia-inducible factor-1α and immune-related pathways are activated in pathogenic leptospiral-infected macrophages. We confirmed the significantly high levels of IL-10-expressing signatures and tolerance in activated macrophages caused by nonpathogenic Leptospira infection. Furthermore, nonpathogenic leptospiral infections attenuated macrophage activation responses. These findings suggest a potential therapeutic strategy for the immune microenvironment caused by macrophage activation driven by IL-10 overexpression, which may contribute to regulating inflammation in leptospirosis.

Injection of hybrid 3D spheroids composed of podocytes, mesenchymal stem cells, and vascular endothelial cells into the renal cortex improves kidney function and replenishes glomerular podocytes.

Podocytes are highly differentiated epithelial cells that are crucial for maintaining the glomerular filtration barrier in the kidney. Podocyte injury followed by depletion is the major cause of pathological progression of kidney diseases. Although cell therapy has been considered a promising alternative approach to kidney transplantation for the treatment of kidney injury, the resultant therapeutic efficacy in terms of improved renal function is limited, possibly owing to significant loss of engrafted cells. Herein, hybrid three-dimensional (3D) cell spheroids composed of podocytes, mesenchymal stem cells, and vascular endothelial cells were designed to mimic the glomerular microenvironment and as a cell delivery vehicle to replenish the podocyte population by cell transplantation. After creating a native glomerulus-like condition, the expression of multiple genes encoding growth factors and basement membrane factors that are strongly associated with podocyte maturation and functionality was significantly enhanced. Our in vivo results demonstrated that intrarenal transplantation of podocytes in the form of hybrid 3D cell spheroids improved engraftment efficiency and replenished glomerular podocytes. Moreover, the proteinuria of the experimental mice with hypertensive nephropathy was effectively reduced. These data clearly demonstrated the potential of hybrid 3D cell spheroids for repairing injured kidneys.

Enhanced early immune response of leptospiral outer membrane protein LipL32 stimulated by narrow band mid-infrared exposure.

Previous studies revealed significant impact on cancer cell by mid-infrared (MIR) radiation. However, the effects of narrow band MIR on immune reaction and infectious disease are still unknown. In this study, an enhanced innate immune response was observed through the interaction between Leptospiral outer membrane protein (LipL32) and toll-like receptor 2 (TLR2). Thereafter, human kidney proximal tubular cells (HK-2 cells) initiated a serial reaction of enhanced MCP-1 production. The 6 µm narrow bandwidth light source emitted by waveguide thermal emitter (WTE) was applied to induce carbonyl group (CO bond) stretching vibration during the stage of antigen-receptor complex formation. The amount of MCP-1 gene expression had 2.5 folds increase after narrow band MIR illumination comparing to non-MIR illumination at low dose LipL32 condition. Besides, both ELISA and confocal microscopy results also revealed that the chemokine concentration increased significantly after narrow band MIR illumination either at low or high concentration of LipL32. Furthermore, a specific phenomenon that narrow band MIR can amplify the signal of weak immune response by enhancing sensitivity of the interaction between antigen and receptor was observed. This study exhibits clear evidence that the narrow band MIR exposure can modulate the early immune response of infectious disease and play a potential role to develop host-directed therapy in the future.

Inhibition of spleen tyrosine kinase (syk) suppresses renal fibrosis through anti-inflammatory effects and down regulation of the MAPK-p38 pathway.

Renal fibrosis results from an excessive accumulation of extracellular matrix that occurs in most types of chronic kidney disease. Among the many fibrogenic factors that regulate renal fibrotic processes, transforming growth factor-ß1 (TGF-ß1) and inflammation after injury play critical roles. Spleen tyrosine kinase (Syk) is important for signaling processes implicated in autoimmune, inflammatory, and allergic diseases. We examined the effects of Syk inhibition on renal fibrosis in vivo and on TGF-ß1-induced renal fibroblast activation in vitro. A unilateral ureteral obstruction (UUO) model was induced in male B6 mice. Mice with UUO were administered a Syk inhibitor or saline intraperitoneally 1 day before UUO surgery and daily thereafter. Both kidneys were harvested 7 days after surgery for further analysis. For the in vitro experiments, NRK-49F rat fibroblasts were pre-incubated with a Syk inhibitor before TGF-ß1 stimulation. The inhibitory effects of Syk inhibition on signaling pathways down-stream of TGF-ß1 were analyzed. In the UUO mouse model, administration of a Syk inhibitor attenuated extracellular matrix protein deposition and expression of α-smooth muscle actin, type I collagen, and fibronectin in a dose-dependent manner. In addition, macrophage infiltration in UUO kidney was reduced by Syk inhibition. Pre-incubation of NRK-49F cells with a Syk inhibitor suppressed TGF-ß1-induced myofibroblast activation. Furthermore, inhibitory effects of Syk inhibition on TGF-ß1-mediated myofibroblast activation were associated with down-regulation of MAPK-p38. These results suggest that Syk inhibition reduces tubulointerstitial fibrosis in UUO mice and inhibits TGF-ß1-induced kidney myofibroblast activation. Syk inhibition could have therapeutic potential for the treatment of renal tubulointerstitial fibrosis.

The AMPK agonist AICAR inhibits TGF-ß1 induced activation of kidney myofibroblasts.

Activation of interstitial myofibroblasts and excessive production of extracellular matrix proteins are common pathways that contribute to chronic kidney disease. In a number of tissues, AMP-activated kinase (AMPK) activation has been shown to inhibit fibrosis. Here, we examined the inhibitory effect of the AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), on renal fibrosis in vivo and TGF-ß1-induced renal fibroblasts activation in vitro. A unilateral ureteral obstruction (UUO) model was induced in male BALB/c mice. Mice with UUO were administered AICAR (500 mg/Kg/day) or saline intraperitoneally 1 day before UUO surgery and daily thereafter. Both kidneys were harvested 7 days after surgery for further analysis. For the in vitro studies, NRK-49F rat fibroblasts were pre-incubated with AICAR before TGF-ß1 stimulation. The inhibitory effects of AICAR on signaling pathways down-stream of TGF-ß1 were analyzed. In UUO model mice, administration of AICAR attenuated extracellular matrix protein deposition and the expression of α-smooth muscle actin (α-SMA), type I collagen and fibronectin. Pre-incubation of NRK-49F cells with AICAR inhibited TGF-ß1-induced myofibroblast activation. Silencing of AMPKα1 by siRNA or by blocking AMPK activation with Compound C diminished the inhibitory effect of AICAR. Moreover, the inhibitory effects of AICAR on TGF-ß1-mediated myofibroblast activation were associated with down-regulation of ERK 1/2 and STAT3. Our results suggest that AICAR reduces tubulointerstitial fibrosis in UUO mice and inhibits TGF-ß1-induced kidney myofibroblast activation. AMPK activation by AICAR may have therapeutic potential for the treatment of renal tubulointerstitial fibrosis.

Essential calcium-binding cluster of Leptospira LipL32 protein for inflammatory responses through the Toll-like receptor 2 pathway.

Leptospirosis is the most widespread zoonosis caused by the pathogenic Leptospira worldwide. LipL32, a 32-kDa lipoprotein, is the most abundant protein on the outer membrane of Leptospira and has an atypical poly(Asp) motif ((161)DDDDDGDD(168)). The x-ray crystallographic structure of LipL32 revealed that the calcium-binding cluster of LipL32 includes several essential residues Asp(132), Thr(133), Asp(164), Asp(165), and Tyr(178). The goals of this study were to determine possible roles of the Ca(2+)-binding cluster for the interaction of LipL32 and Toll-like receptor 2 (TLR2) in induced inflammatory responses of human kidney cells. Site-directed mutagenesis was employed to individually mutate Ca(2+)-binding residues of LipL32 to Ala, and their effects subsequently were observed. These mutations abolished primarily the structural integrity of the calcium-binding cluster in LipL32. The binding assay and atomic force microscopy analysis further demonstrated the decreased binding capability of LipL32 mutants to TLR2. Inflammatory responses induced by LipL32 variants, as determined by TLR2 pathway intermediates hCXCL8/IL-8, hCCL2/MCP-1, hMMP7, and hTNF-α, were also lessened. In conclusion, the calcium-binding cluster of LipL32 plays essential roles in presumably sustaining LipL32 conformation for its proper association with TLR2 to elicit inflammatory responses in human renal cells.

High-dose intratympanic gentamicin instillations for treatment of Meniere's disease: long-term results.

CONCLUSIONS: Administration of high-dose gentamicin for intractable Meniere's disease appears to be effective in achieving long-term control of vertigo. However, the safety of this route of administration with respect to the patient's hearing has not yet been sufficiently established. OBJECTIVES: The study aimed to analyze the long-term results of patients receiving high-dose intratympanic gentamicin (ITG) instillation for refractory Meniere's disease. PATIENTS AND METHODS: Fourteen patients with Meniere's disease according to 1995 American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) guidelines who had failed medical (12 subjects) or surgical (2 subjects) treatment were included. Intratympanic injections of 27 mg/ml gentamicin were performed three times daily for 4 days. Vertigo control, the patients' functional level, and their hearing threshold were all analyzed. Criteria described in 1995 by AAO-HNS were used. RESULTS: The overall successful vertigo control rate was 92.9% over the 2-year follow-up and 85.7% at long-term follow-up (average 10 years). Hearing level as pure-tone average was worse in four patients (28.5%) after 2 years follow-up and in six patients (42.8%) after long-term follow-up, respectively. Profound sensorineural hearing loss occurred as a result of gentamicin injection in one patient (7%).