Group2 innate lymphoid cells ameliorate renal fibrosis and dysfunction associated with adenine-induced CKD.

Renal fibrosis is a common pathway of chronic kidney disease (CKD) progression involving primary kidney injury and kidney diseases. Group 2 innate lymphoid cells (ILC2s) mediate type 2 immune responses irrespective of antigen presentation and play a reno-protective role in kidney injury and disease. In the present study, we observed a decrease in kidney-resident ILC2s in CKD and found that enrichment of ILC2s in the kidney ameliorates renal fibrosis. In CKD kidney, ILC2s preferentially produced IL-13 over IL-5 in response to IL-33 stimulation, regardless of ST2L expression. Moreover, GATA3 expression was decreased in ILC2s, and T-bet+ ILC1s and RORγt+ ILC3s were increased in CKD kidney. Adoptive transfer of kidney ILC2s into adenine-induced CKD model mouse improved renal function and fibrosis. Renal fibroblasts cultured with IL33-activated kidney ILC2s suppressed myofibroblast trans-differentiation through Acta2 and Fn-1 regulation. These results suggest that kidney ILC2s prevent CKD progression via improvement of renal fibrosis. Our findings also suggest that ILC2s may contribute to the development of new therapeutic agents and strategies for tissue fibroses.

In vitro investigation of the effects of Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 exopolysaccharides on tight junction damage caused by influenza virus infection.

It is a problem that influenza virus infection increases susceptibility to secondary bacterial infection in lungs leading to lethal pneumonia. We previously reported that exopolysaccharides (EPS) derived from Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1) could prevent against influenza virus infection followed by secondary bacterial infection in vitro. Therefore, the present study assessed whether EPS derived OLL1073R-1 protects the alveolar epithelial barrier disfunction caused by influenza virus infection. After A549 cells treated with EPS or without EPS were infected influenza virus A/Puerto Rico/8/34 (IFV) for 12 h, the levels of tight junction genes expression and inflammatory genes expression were measured by reverse transcription polymerase chain reaction. As results, EPS treatment could protect against low-titer IFV infection, but not high-titer IFV infection, followed by suppression of the increased expression of inflammatory cytokine gene levels and recovery of the decrease in the expression level of ZO-1 gene that was caused by low-titer IFV infection, leading to an improvement trend in the barrier function. Our findings showed that EPS derived from OLL1073R-1 could inhibit low-titer IFV infection leading to maintenance of the epithelial barrier function through the suppression of inflammatory cytokine genes expression.

IL-33 attenuates renal fibrosis via group2 innate lymphoid cells.

Renal fibrosis is a common pathway in the progression of various kidney diseases and injuries. Unilateral ureteral obstruction (UUO) induces renal fibrosis, and immune responses profoundly affect its pathogenesis. Group2 innate lymphoid cells (ILC2s) are strongly activated by interleukin (IL) -33, which is a member of IL-1 family and recognize as alarmin. ILC2s quickly produce large amounts of type 2 cytokines including IL-5 and IL-13, which are involved in inflammation, tissue homeostasis, and wound healing. However, the relationship between renal fibrosis and ILC2s has been unclear. In the present study, we investigated the roles of the ILC2/L-33 axis in renal fibrosis using a UUO model. We found that kidney ILC2s decreased in UUO-affected kidneys compared with their counterpart kidneys despite IL-33 upregulation. There was no effect of reactive oxygen species or TGF-ß from reduced ILC2 caused by UUO. Pretreatment with IL-33 before UUO induced ILC2s and Tregs in kidneys and alleviated renal fibrosis. Furthermore, this protective effect was maintained even when CD4+T cells was depleted. These findings demonstrated that ILC2s play a predominant role in the suppressive function of renal fibrosis mediated by pretreatment with IL-33. In contrast, post-treatment with IL-33 after UUO increased ILC2s in kidneys but had no therapeutic effect on renal fibrosis. Our findings suggest that ILC2s have potential roles in the prevention of renal fibrosis and can serve as a therapeutic and diagnostic target.

Nrf2 Suppresses Allergic Lung Inflammation by Attenuating the Type 2 Innate Lymphoid Cell Response.

The Keap1-Nrf2 system plays a pivotal role in the oxidative stress response by inducing a number of cytoprotective genes. Under stress, damaged epithelial cells release cytokines that activate type 2 innate lymphoid cells (ILC2s), which mediate the allergic immune response. In this article, we investigated the role of the Keap1-Nrf2 pathway in ILC2 homeostasis and allergic inflammation using Nrf2 knockout mice. ILC2s from Nrf2-deficient mice showed a transient, upregulated IL-33 response and underwent hyperproliferation in response to a combined stimulation of IL-33 with IL-2, IL-7, or TSLP. This enhanced proliferation was correlated with an increased activation of downstream signals, including JAK1, Akt, and Erk1/2. In contrast, activating Nrf2 with a chemical inducer (CDDO-Im) decreased the viability of the wild-type but not of the Nrf2-deficient ILC2s. This effect on viability resembled that exerted by the corticosteroid dexamethasone; however, unlike the latter, the Nrf2-dependent cell death was mediated by neither caspase 3-dependent apoptosis nor necroptosis. Using a mouse intratracheal IL-33 administration allergy model, we found that the activation of Nrf2 by CDDO-Im in vivo decreased the number of pulmonary ILC2s and eosinophils. These findings indicated that Nrf2 is an important regulator of the allergic response by determining the survival and death of ILC2s, and these findings suggest that Nrf2 activation is a potential therapeutic strategy for steroid-resistant allergy alleviation.

Leucine-Rich Repeat Kinase 2 Controls Inflammatory Cytokines Production through NF-κB Phosphorylation and Antigen Presentation in Bone Marrow-Derived Dendritic Cells.

Leucine-rich repeat kinase 2 (LRRK2) is the causal molecule of familial Parkinson's disease. Although the characteristics of LRRK2 have gradually been revealed, its true physiological functions remain unknown. LRRK2 is highly expressed in immune cells such as B2 cells and macrophages, suggesting that it plays important roles in the immune system. In the present study, we investigate the roles of LRRK2 in the immune functions of dendritic cells (DCs). Bone marrow-derived DCs from both C57BL/6 wild-type (WT) and LRRK2 knockout (KO) mice were induced by culture with granulocyte/macrophage-colony stimulating factor (GM/CSF) in vitro. We observed the differentiation of DCs, the phosphorylation of the transcriptional factors NF-κB, Erk1/2, and p-38 after lipopolysaccharide (LPS) stimulation and antigen-presenting ability by flow cytometry. We also analyzed the production of inflammatory cytokines by ELISA. During the observation period, there was no difference in DC differentiation between WT and LRRK2-KO mice. After LPS stimulation, phosphorylation of NF-κB was significantly increased in DCs from the KO mice. Large amounts of inflammatory cytokines were produced by DCs from KO mice after both stimulation with LPS and infection with Leishmania. CD4+ T-cells isolated from antigen-immunized mice proliferated to a significantly greater degree upon coculture with antigen-stimulated DCs from KO mice than upon coculture with DCs from WT mice. These results suggest that LRRK2 may play important roles in signal transduction and antigen presentation by DCs.

HIF-PHD inhibitor regulates the function of group2 innate lymphoid cells and polarization of M2 macrophages.

Hypoxia-inducible factor-prolyl hydroxylase (HIF-PHD) inhibitors are therapeutic agents for renal anemia that work through HIF2-mediated upregulation of erythropoietin (EPO) and have also been reported to suppress renal fibrosis. Group 2 innate lymphoid cells (ILC2s) have been proven to be involved in the pathogenesis of fibrosis in various organs, including the kidney. However, the relationship between the HIF pathway, renal fibrosis, and kidney ILC2s remains unclear. In the present study, we found that HIF activation by HIF-PHD inhibitors suppressed type 2 cytokine production from kidney ILC2s. The enhanced HIF pathway downregulated the IL-33 receptor ST2L on ILC2s, and phosphorylation of downstream p38 MAPK was attenuated. M2 macrophages that promote renal fibrosis were polarized by ILC2 supernatants, but reduced cytokine production from ILC2s treated with HIF-PHD inhibitors suppressed this polarization. Our findings suggest that HIF-PHD inhibitors are potential therapeutic agents for renal fibrosis that are mediated by the alteration of ILC2 function.

Exploring the significance of interleukin-33/ST2 axis in minimal change disease.

Minimal change disease (MCD), a common cause of idiopathic nephrotic syndrome, has been postulated to exhibit an association with allergic conditions. Recent studies revealed the crucial role of interleukin (IL)-33 in type 2 innate immunity. We hypothesized that development of MCD involves an IL-33-related immune response. We examined 49 patients with biopsy-proven MCD, 6 healthy volunteers, and 29 patients in remission. In addition to clinical features, serum and urinary levels of IL-33 and soluble suppression of tumorigenicity 2 protein (sST2), a secreted form of the receptor of IL-33, were analyzed. Although IL-33 was barely detectable in either MCD or control samples, sST2 levels at diagnosis were elevated in MCD patients. Serum sST2 levels of MCD patients were correlated with serum total protein level (r = - 0.36, p = 0.010) and serum creatinine level (r = 0.34, p = 0.016). Furthermore, the elevated sST2 levels were observed to decrease following remission. Immunofluorescence revealed IL-33 expression in the podocytes among MCD patients, with a significant increase compared with controls. In vitro, mouse podocyte cells incubated with serum from a MCD patient at disease onset showed increased IL-33 secretion. These results suggest an IL-33-related immune response plays a role in MCD.

Effects of NKT Cells on Metabolic Disorders Caused by High-Fat Diet Using CD1d-Knockout Mice.

Purpose: The purpose of this study was to investigate whether NKT cells play an important role in preventing or exacerbating diseases caused by high-fat diet (HFD) using CD1d-knockout (KO) mice which lack NKT cells. Methods: Five-week-old male Balb/c (wild-type; WT) or CD1dKO mice were fed with control-diet (CTD) or HFD for 16 weeks. Results: The present study revealed four main findings. First, CD1dKO mice were susceptible to obesity caused by HFD in comparison to WT mice. Second, clinical conditions of fatty liver caused by HFD were comparable between CD1dKO mice and WT mice. Third, HFD-fed WT mice showed high levels of serum biochemical markers, involved in lipid metabolisms, in comparison to WT mice fed a CTD. Notably, the serum concentrations of ALT, T-CHO, TG and HDL-C in CD1dKO mice fed a HFD were almost comparable to those of CD1dKO mice fed a CTD. Fourth, the expression of peroxisome proliferator-activated receptor (PPAR) γ, low-density lipoprotein receptor (LDLR), CD36 of epididymal adipose tissue enhanced and proprotein convertase subtilisin/kexin type (PCSK) 9 in serum decreased. Conclusion: NKT cells were responsible for protection against HFD-induced obesity. However, CD1dKO mice were resistant to serum biochemical marker abnormalities after HFD feeding. One possible explanation is that the epididymal adipose tissue of CD1dKO mice could take up greater amounts of excess lipids in serum in comparison to WT mice.

The essential role of intestinal microbiota in cytomegalovirus reactivation.

Human cytomegalovirus (HCMV) is a member of Herpesviridae. It has been reported that HCMV is reactivated in the breast milk of HCMV-seropositive lactating women. As we have reported various aspects of the roles of indigenous microbiota, its role in the murine CMV (MCMV) reactivation was examined in this study. MCMV was latently infected in the salivary gland, mammary tissues, and colon in the pregnant mice. When the salivary gland, mammary tissues, and colon were removed 5 days after delivery, MCMV reactivation of latent infection in each organ was confirmed by the detection of MCMV IE1 mRNA using reverse transcription-quantitative PCR. MCMV reactivation was observed in 100% of the mice during pregnancy. Next, for the elimination of intestinal microbiota, the pregnant mice were treated with low-dose or high-dose non-absorbable antibiotics. Although the numbers of aerobe/anaerobe in cecal content in low-dose antibiotic-treated mice were comparable to those in untreated controls, high-dose antibiotic treatment decreased the number of aerobe/anaerobe microbes from ca.9.0 Log10 to ca.3.0 Log10 (cfu/g). However, it could not be confirmed in 16S rRNA analysis that specific bacterial phylum or genus was eliminated by this high-dose treatment. Interestingly, MCMV reactivation was also observed in 100% of low-dose antibiotic-treated mice, whereas, in high-dose antibiotic-treated mice, MCMV reactivation was not observed in the salivary gland or colon. MCMV IE1 mRNA was detected only in 33% of the mammary tissues of those high-dose-treated mice. These results suggest that the indigenous microbiota played a crucial role in the reactivation of latent infection. IMPORTANCE Human cytomegalovirus (HCMV) infection via breast milk is a serious problem for very preterm infants such as developing a sepsis-like syndrome, cholestasis, or bronchopulmonary dysplasia, among others. It has been reported that HCMV is reactivated in the breast milk of HCMV-seropositive lactating women. In this study, the roles of indigenous microbiota in the murine CMV (MCMV) reactivation were examined using a mouse model. In MCMV latently infected mice, MCMV reactivation was observed in 100% of the mice during pregnancy. For the elimination of intestinal microbiota, MCMV-latent mice were treated with non-absorbable antibiotics. After delivery, MCMV reactivation was not observed in antibiotic-treated mice. This result suggested that the indigenous microbiota played a crucial role in the reactivation of latent infection.

Low-magnification narrow-band imaging for small gastric neoplasm detection on screening endoscopy.

Background and Aims: Microsurface patterns of the gastric mucosa can be observed using magnifying narrow-band imaging (M-NBI). However, the efficacy of M-NBI at low-magnification (LM-NBI) screening for detecting small gastric neoplasms is unclear. Methods: This prospective study was conducted at a single institution. LM-NBI, defined as minimal magnification that could reveal the microsurface pattern of the gastric mucosa, was performed after routine white-light imaging (WLI) observation of the stomach. Depending on the phase in which the neoplastic lesions were initially found, they were divided into the WLI group and the LM-NBI group, and the characteristics of these neoplastic lesions were investigated accordingly. Results: Sixty-five epithelial lesions (adenomas or noninvasive carcinomas) of 20 mm or less in diameter were identified in this study. Sixteen lesions were detected only with LM-NBI. Smaller lesions were detected using LM-NBI (P = .01). WLI took about 160 to 260 seconds, while LM-NBI required about 70 to 80 seconds. All lesions in the LM-NBI group had a background of map-like redness (n = 5) or atrophic/metaplastic mucosa (n = 11). Conclusions: LM-NBI was able to detect lesions overlooked by WLI, especially those in areas of map-like redness or atrophic/metaplastic mucosa of the stomach. Approximately one-quarter of newly diagnosed neoplasms were retrieved on routine examination during an extra 1.5 minutes.

The Roles of Kidney-Resident ILC2 in Renal Inflammation and Fibrosis.

Innate lymphoid cells (ILCs) are a recently discovered lymphocyte population with high cytokine productive capacity. Type-2 ILCs (ILC2s) are the most studied, and they exert a rapid type-2 immune response to eliminate helminth infections. Massive and sustainable ILC2 activation induces allergic tissue inflammation, so it is important to maintain correct ILC2 activity for immune homeostasis. The ILC2-activating cytokine IL-33 is released from epithelial cells upon tissue damage, and it is upregulated in various kidney disease mouse models and in kidney disease patients. Various kidney diseases eventually lead to renal fibrosis, which is a common pathway leading to end-stage renal disease and is a chronic kidney disease symptom. The progression of renal fibrosis is affected by the innate immune system, including renal-resident ILC2s; however, the roles of ILC2s in renal fibrosis are not well understood. In this review, we summarize renal ILC2 function and characterization in various kidney diseases and highlight the known and potential contributions of ILC2s to kidney fibrosis.

Efficient immunogenic peptide antigen delivery to dendritic cells using an ESCRT-mediated extracellular vesicle formation method.

In the activation of cell-mediated adaptive immune responses that play major roles in the elimination of virus-infected or tumor cells, it is important that dendritic cells present antigen peptides on major histocompatibility complex (MHC) class I molecules and activate pathogen-specific cytotoxic T lymphocytes (CTL). As exogenous peptide antigens are generally presented on MHC class II but not class I, the development of a method for exogenous antigen delivery that facilitates MHC class I presentation is necessary for a potentially effective vaccine that is expected to provoke cell-mediated adaptive immune responses. Here, we developed extracellular vesicles that incorporate antigenic proteins by utilizing endosomal sorting complexes required for transport (ESCRT)-mediated vesicle formation pathway. Furthermore, we proved that these vesicles could deliver their contents to the cytoplasm of dendritic cells and activate antigen-specific CTLs. These technologies could be applied to the development of novel CTL-inducing peptide vaccines.

Systemic chemotherapy of TS-1 and cisplatin for gastric signet-ring cell carcinoma presenting as cardiac tamponade.

A 59-year-old man complaining of dyspnea, anterior chest oppression, and hypotension was diagnosed to have cardiac tamponade due to massive pericardial effusion. A cytological analysis of the pericardial effusion disclosed adenocarcinoma. An endoscopic study revealed gastric cancer in the lesser curvature wall of the middle body of the stomach, and signet-ring cell carcinoma was confirmed histologically. The gastric cancer was complicated by malignant pericardial effusion, and metastasis to the mediastinal lymph nodes. The patient was treated with pericardiocentesis followed by systemic chemotherapy consisting of TS-1 and cisplatin (CDDP). After 5 months, pericardial effusion disappeared and the primary gastric tumor decreased in size. Our experience suggests that the systemic chemotherapy of TS-1 and CDDP may be effective for controlling advanced gastric signet-cell carcinoma accompanied by malignant pericardial effusion.

Leucine-rich repeat kinase 2 is a regulator of B cell function, affecting homeostasis, BCR signaling, IgA production, and TI antigen responses.

LRRK2 is the causal molecule of autosomal dominant familial Parkinson's disease. B2 cells express a much higher LRRK2 mRNA level than B1 cells. To reveal the function of LRRK2 in B cells, we analyzed B cell functions in LRRK2-knockout (LRRK2(-/-)) mice. LRRK2(-/-) mice had significantly higher counts of peritoneal B1 cells than wild-type mice. After BCR stimulation, phosphor-Erk1/2 of splenic B2 cells was enhanced to a higher degree in LRRK2(-/-) mice. LRRK2(-/-) mice had a significantly higher serum IgA level, and TNP-Ficoll immunization increased the titer of serum anti-TNP IgM antibody. LRRK2 may play important roles in B cells.

13C-Urea breath test, using a new compact nondispersive isotope-selective infrared spectrophotometer: comparison with mass spectrometry.

BACKGROUND: The (13)C-urea breath test ((13)C-UBT) is the most commonly used noninvasive method of detecting Helicobacter pylori infection. The isotope ratio mass spectrometer (IRMS) is the most commonly used device for this test, but the UBiT-IR300 infrared spectrophotometer, which, by comparison, is a more compact, less expensive, and easier to use analytical device, has now become widely used in the clinical setting in Japan. The objective of this study was to examine the diagnostic performance of the (13)C-UBT, using the UBiT-IR300. METHODS: A multicenter open-label study was performed, in which the (13)C-UBT was conducted using 100 mg of (13)C-urea. Analysis of (13)CO(2) in the expired breath was performed by infrared spectroscopy and mass spectrometry, and assessment of H. pylori infection was performed by culture, histological examination, and rapid urease test. RESULTS: In 255 cases of H. pylori infection diagnosed by biopsy methods, the (13)C-UBTs, performed with two different (13)C-ruea formulations, and using infrared spectroscopy for evaluation, showed a sensitivity of 97.7%, specificity of 98.0%, and accuracy of 97.8% (total number of evaluable cases, n = 505). The rate of agreement in the assessment of H. pylori infection between infrared spectroscopy and mass spectrometry was 100% ( n = 505). The regression equation for infrared spectroscopy to mass spectrometry was y = 0.9822x - 0.0809 ( n = 2542), with a correlation coefficient of r = 0.99989 ( P = 0.0001). CONCLUSIONS: Diagnosis of H. pylori infection can be performed using infrared spectroscopy as well as mass spectrometry.

Comparison between a new 13C-urea breath test, using a film-coated tablet, and the conventional 13C-urea breath test for the detection of Helicobacter pylori infection.

BACKGROUND: In Japan, urea breath-testing includes mouth rinsing with water immediately after the ingestion of (13)C-urea solution, to prevent false-positive results that are caused by oral bacteria with urease activity. Our objective was to evaluate the diagnostic performance of a urea breath test using a film-coated (13)C-urea tablet and omitting mouth rinsing. METHODS: The study was a multicenter trial comparing the solution- and tablet-based urea breath tests (UBTs). Helicobacter pylori status was determined by histology, culture, and rapid urease testing. RESULTS: Of the 255 subjects who completed the study, evaluation of the tablet-based UBT was possible in 254, and comparison of the tablet-based UBT and the solution-based UBT was possible in 250 patients. When the assessment achieved by a combination of biopsy-based methods was used as a reference standard, the sensitivity, specificity, and accuracy of the tablet-based method were determined to be 97.7%, 98.4%, and 98.0%, respectively. When the results of the solution-based UBT were used as a reference standard, the sensitivity, specificity, and accuracy of the tablet-based UBT were determined to be 96.9%, 97.6%, and 97.2%, respectively. CONCLUSIONS: The (13)C-urea tablet-based method proved to be a simple and accurate test for the diagnosis of H. Pylori infection. Mouth rinsing was not required.

Allo-antigen stimulated CD8+ T-cells suppress NF-κB and Ets-1 DNA binding activity, and inhibit phosphorylated NF-κB p65 nuclear localization in CD4+ T-cells.

CD8+ T-cells of asymptomatic HIV-1 carriers (AC) suppress human immunodeficiency virus type 1 (HIV-1) replication in a class I major histocompatibility complex (MHC-I)-restricted and -unrestricted manner. In order to investigate the mechanism of MHC-I-unrestricted CD8+ T-cell-mediated HIV-1 suppression, we previously established allo-antigen stimulated CD8+T-cells from HIV-1-uninfected donors. These allo-antigen stimulated CD8+ T-cells suppressed HIV-1 replication in acutely infected autologous CD4+ T-cells when directly co-cultured. To elucidate the mechanism of HIV-1 replication suppression, we analyzed DNA-binding activity and phosphorylation of transcriptional factors associated with HIV-1 replication by electrophoresis mobility shift assay and Western blotting. When CD4+ T-cells were cultured with allo-antigen stimulated CD8+ T-cells, the reduction of NF-κB and Ets-1 DNA-binding activity was observed. Nuclear localization of NF-κB p65 and Ets-1 was suppressed in CD4+ T-cells. Although NF-κB p65 and Ets-1 are known to be regulated by protein kinase A (PKA), no difference was observed in the expression and phosphorylation of the PKA catalytic subunit in CD4+ T-cells cultured with PHA-treated CD8+ T-cells or allo-antigen stimulated CD8+ T-cells. Cyclic AMP is also known to enter through gap junctions, but the suppression of HIV-1 replication mediated by allo-antigen stimulated CD8+ T-cells was not affected by the gap junction inhibitor. The nuclear transport of phosphorylated NF-κB p65 (Ser276) was inhibited only in CD4+ T-cells cultured with allo-antigen stimulated CD8+ T-cells. Our results indicate that allo-antigen stimulated CD8+ T-cells suppress the transcriptional activity of NF-κB p65 or Ets-1 in an antigen-nonspecific manner, and inhibit the nuclear transport of phosphorylated NF-κB p65 (Ser276).

LRRK2 is expressed in B-2 but not in B-1 B cells, and downregulated by cellular activation.

LRRK2, the causal molecule of familial Parkinson's disease, is expressed strongly by one of the B cell subsets, B-2 cells, but not by the other subset, B-1 cells, in the mouse peritoneal cavity, spleen, and peripheral blood. Bone marrow pre-B cells or T cells exhibited little LRRK2 expression. LRRK2 expression was dramatically downregulated upon activation of B-2 cells with various types of stimulation. These results suggest that LRRK2, whose true function has not yet been clarified, may play some important role(s) in the development and function of B cells, particularly the maintenance of B-2 cells in a resting status.