GGT1 is a SNP eQTL gene involved in STAT3 activation and associated with the development of Post-ERCP pancreatitis.

Post-ERCP pancreatitis (PEP) is an acute pancreatitis caused by endoscopic-retrograde-cholangiopancreatography (ERCP). About 10% of patients develop PEP after ERCP. Here we show that gamma-glutamyltransferase 1 (GGT1)-SNP rs5751901 is an eQTL in pancreatic cells associated with PEP and a positive regulator of the IL-6 amplifier. More PEP patients had the GGT1 SNP rs5751901 risk allele (C) than that of non-PEP patients at Hokkaido University Hospital. Additionally, GGT1 expression and IL-6 amplifier activation were increased in PEP pancreas samples with the risk allele. A mechanistic analysis showed that IL-6-mediated STAT3 nuclear translocation and STAT3 phosphorylation were suppressed in GGT1-deficient cells. Furthermore, GGT1 directly associated with gp130, the signal-transducer of IL-6. Importantly, GGT1-deficiency suppressed inflammation development in a STAT3/NF-κB-dependent disease model. Thus, the risk allele of GGT1-SNP rs5751901 is involved in the pathogenesis of PEP via IL-6 amplifier activation. Therefore, the GGT1-STAT3 axis in pancreas may be a prognosis marker and therapeutic target for PEP.

DDX6 is involved in the pathogenesis of inflammatory diseases via NF-κB activation.

The IL-6 amplifier was originally discovered as a mechanism for the enhanced activation of NF-κB in non-immune cells. In the IL-6 amplifier, IL-6-STAT3 and NF-κB stimulation is followed by an excessive production of IL-6, chemokines, and growth factors to develop chronic inflammation preceding the development of inflammatory diseases. Previously, using a shRNA-mediated genome-wide screening, we found that DEAD-Box Helicase 6 (DDX6) is a candidate positive regulator of the amplifier. Here, we investigate whether DDX6 is involved in the pathogenesis of inflammatory diseases via the IL-6 amplifier. We found that DDX6-silencing in non-immune cells suppressed the NF-κB pathway and inhibited activation of the IL-6 amplifier, while the forced expression of DDX6 enhanced NF-κB promoter activity independent of the RNA helicase activity of DDX6. The imiquimod-mediated dermatitis model was suppressed by the siRNA-mediated gene downregulation of DDX6. Furthermore, silencing DDX6 significantly reduced the TNF-α-induced phosphorylation of p65/RelA and IκBα, nuclear localization of p65, and the protein levels of IκBα. Mechanistically, DDX6 is strongly associated with p65 and IκBα, but not TRADD, RIP, or TRAF2, suggesting a novel function of DDX6 as an adaptor protein in the NF-κB pathway. Thus, our findings demonstrate a possible role of DDX6 beyond RNA metabolism and suggest DDX6 is a therapeutic target for inflammatory diseases.

GM-CSF Promotes the Survival of Peripheral-Derived Myeloid Cells in the Central Nervous System for Pain-Induced Relapse of Neuroinflammation.

We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common ß chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.

Computer model of IL-6-dependent rheumatoid arthritis in F759 mice.

The interleukin-6 (IL-6) amplifier, which describes the simultaneous activation of signal transducer and activator of transcription 3 (STAT3) and NF-κb nuclear factor kappa B (NF-κB), in synovial fibroblasts causes the infiltration of immune cells into the joints of F759 mice. The result is a disease that resembles human rheumatoid arthritis. However, the kinetics and regulatory mechanisms of how augmented transcriptional activation by STAT3 and NF-κB leads to F759 arthritis is unknown. We here show that the STAT3-NF-κB complex is present in the cytoplasm and nucleus and accumulates around NF-κB binding sites of the IL-6 promoter region and established a computer model that shows IL-6 and IL-17 (interleukin 17) signaling promotes the formation of the STAT3-NF-κB complex followed by its binding on promoter regions of NF-κB target genes to accelerate inflammatory responses, including the production of IL-6, epiregulin, and C-C motif chemokine ligand 2 (CCL2), phenotypes consistent with in vitro experiments. The binding also promoted cell growth in the synovium and the recruitment of T helper 17 (Th17) cells and macrophages in the joints. Anti-IL-6 blocking antibody treatment inhibited inflammatory responses even at the late phase, but anti-IL-17 and anti-TNFα antibodies did not. However, anti-IL-17 antibody at the early phase showed inhibitory effects, suggesting that the IL-6 amplifier is dependent on IL-6 and IL-17 stimulation at the early phase, but only on IL-6 at the late phase. These findings demonstrate the molecular mechanism of F759 arthritis can be recapitulated in silico and identify a possible therapeutic strategy for IL-6 amplifier-dependent chronic inflammatory diseases.

Calcineurin inhibitor inhibits tolerance induction by suppressing terminal exhaustion of donor T cells after allo-HCT.

Calcineurin inhibitor-based graft-versus-host disease (GVHD) prophylaxis is standard in allogeneic hematopoietic stem cell transplantation (HCT) but fails to induce long-term tolerance without chronic GVHD (cGVHD) in a considerable number of patients. In this study, we addressed this long-standing question in mouse models of HCT. After HCT, alloreactive donor T cells rapidly differentiated into PD-1+ TIGIT+ terminally exhausted T cells (terminal Tex). GVHD prophylaxis with cyclosporine (CSP) suppressed donor T-cell expression of TOX, a master regulator to promote differentiation of transitory exhausted T cells (transitory Tex), expressing both inhibitory receptors and effector molecules, into terminal Tex, and inhibited tolerance induction. Adoptive transfer of transitory Tex, but not terminal Tex, into secondary recipients developed cGVHD. Transitory Tex maintained alloreactivity and thus PD-1 blockade restored graft-versus-leukemia (GVL) activity of transitory Tex and not terminal Tex. In conclusion, CSP inhibits tolerance induction by suppressing the terminal exhaustion of donor T cells, while maintaining GVL effects to suppress leukemia relapse.

In situ Microinflammation Detection Using Gold Nanoclusters and a Tissue-clearing Method.

Microinflammation enhances the permeability of specific blood vessel sites through an elevation of local inflammatory mediators, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. By a two-dimensional immunohistochemistry analysis of tissue sections from mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), we previously showed that pathogenic immune cells, including CD4+ T cells, specifically accumulate and cause microinflammation at the dorsal vessels of the fifth lumbar cord (L5), resulting in the onset of disease. However, usual pathological analyses by using immunohistochemistry on sections are not effective at identifying the microinflammation sites in organs. Here, we developed a new three-dimensional visualization method of microinflammation using luminescent gold nanoclusters (AuNCs) and the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method. Our protocol is based on the detection of leaked AuNCs from the blood vessels due to an enhanced vascular permeability caused by the microinflammation. When we injected ultrasmall coordinated Au13 nanoclusters intravenously (i.v.) to EAE mice, and then subjected the spinal cords to tissue clearing, we detected Au signals leaked from the blood vessels at L5 by light sheet microscopy, which enabled the visualization of complex tissue structures at the whole organ level, consistent with our previous report that microinflammation occurs specifically at this site. Our method will be useful to specify and track the stepwise development of microinflammation in whole organs that is triggered by the recruitment of pathogenic immune cells at specific blood vessels in various inflammatory diseases.

Combination therapy with bevacizumab and a CCR2 inhibitor for human ovarian cancer: An in vivo validation study.

BACKGROUND: Anti-angiogenic therapy with bevacizumab (BEV), an anti-VEGF antibody, plays a critical role in the treatment of ovarian cancer. However, despite an encouraging initial response, most tumors become resistant to BEV over time, and a new strategy that enables sustainable treatment using BEV is therefore needed. METHODS: To overcome the resistance to BEV in patients with ovarian cancer, we performed a validation study of combination therapy with BEV (10 mg/kg) and the CCR2 inhibitor BMS CCR2 22 (20 mg/kg) (BEV/CCR2i) using 3 consecutive patient-derived xenografts (PDXs) of immunodeficient mice. RESULTS: BEV/CCR2i demonstrated a significant effect of growth suppression in the BEV-resistant serous PDX and BEV-sensitive serous PDX compared with BEV (30.4% after the second cycle and 15.5% after the first cycle, respectively), and treatment cessation did not attenuate this effect. Tissue clearing and immunohistochemistry with an anti-α-SMA antibody suggested that BEV/CCR2i suppressed angiogenesis from the host mice more than BEV. In addition, human CD31 immunohistochemistry revealed that BEV/CCR2i decreased microvessels originating from the patients to a significantly greater degree than BEV. Regarding the BEV-resistant clear cell PDX, the effect of BEV/CCR2i was unclear during the first five cycles, but the following two cycles of increased-dose BEV/CCR2i (CCR2i 40 mg/kg) significantly suppressed tumor growth compared with BEV (28.3%) by inhibiting the CCR2B-MAPK pathway. CONCLUSIONS: BEV/CCR2i showed a sustained anticancer immunity-independent effect in human ovarian cancer that was more significant in serous carcinoma than in clear cell carcinoma.

Dupuytren's contracture-associated SNPs increase SFRP4 expression in non-immune cells including fibroblasts to enhance inflammation development.

Dupuytren's contracture (DC) is an inflammatory fibrosis characterized by fibroproliferative disorders of the palmar aponeurosis, for which there is no effective treatment. Although several genome-wide association studies have identified risk alleles associated with DC, the functional linkage between these alleles and the pathogenesis remains elusive. We here focused on two single nucleotide polymorphisms (SNPs) associated with DC, rs16879765 and rs17171229, in secreted frizzled related protein 4 (SFRP4). We investigated the association of SRFP4 with the IL-6 amplifier, which amplifies the production of IL-6, growth factors and chemokines in non-immune cells and aggravates inflammatory diseases via NF-κB enhancement. Knockdown of SFRP4 suppressed activation of the IL-6 amplifier in vitro and in vivo, whereas the overexpression of SFRP4 induced the activation of NF-κB-mediated transcription activity. Mechanistically, SFRP4 induced NF-κB activation by directly binding to molecules of the ubiquitination SFC complex, such as IkBα and ßTrCP, followed by IkBα degradation. Furthermore, SFRP4 expression was significantly increased in fibroblasts derived from DC patients bearing the risk alleles. Consistently, fibroblasts with the risk alleles enhanced activation of the IL-6 amplifier. These findings indicate that the IL-6 amplifier is involved in the pathogenesis of DC, particularly in patients harboring the SFRP4 risk alleles. Therefore, SFRP4 is a potential therapeutic target for various inflammatory diseases and disorders, including DC.

A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.

Heterogenous expression of endoglin marks advanced renal cancer with distinct tumor microenvironment fitness.

Intratumoral heterogeneity, including in clear cell renal cell carcinoma, is a potential cause of drug resistance and metastatic cancer progression. We specified the heterogeneous population marked by endoglin (also known as CD105) in a preclinical model of clear cell renal cell carcinoma progression. Highly malignant derivatives of human clear cell renal cell carcinoma OS-RC-2 cells were established as OS5Ks by serial orthotopic inoculation in our previous study. Expression of both ENG (encoding endoglin) mRNA and protein were heterogeneously upregulated in OS5Ks, and the endoglin-positive (ENG+ ) population exhibited growth dependency on endoglin in anchorage-independent cultures. Despite the function of endoglin as a type III receptor, transforming growth factor ß and bone morphogenetic protein-9 signaling were unlikely to contribute to the proliferative phenotype. Although endoglin has been proposed as a marker for renal cancer-initiating cells, the OS5K-3 ENG+ population did not enrich other reported cancer-initiating cell markers or differentiate into the ENG- population. Mouse tumor inoculation models revealed that the tumor-forming capabilities of OS5K-3 ENG+ and ENG- cells in vivo were highly dependent on the microenvironment, with the renal microenvironment most preferable to ENG+ cells. In conclusion, the renal microenvironment, rather than the hypothesized ENG+ cell-centered hierarchy, maintains cellular heterogeneity in clear cell renal cell carcinoma. Therefore, the effect of the microenvironment should be considered when evaluating the proliferative capability of renal cancer cells in the experimental settings.

Visualization of the cancer cell cycle by tissue-clearing technology using the Fucci reporter system.

Tissue-clearing technology is an emerging imaging technique currently utilized not only in neuroscience research but also in cancer research. In our previous reports, tissue-clearing methods were used for the detection of metastatic tumors. Here, we showed that the cell cycles of primary and metastatic tumors were visualized by tissue-clearing methods using a reporter system. First, we established cancer cell lines stably expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) reporter with widely used cancer cell lines A549 and 4T1. Fluorescence patterns of the Fucci reporter were investigated in various tumor inoculation models in mice. Interestingly, fluorescence patterns of the Fucci reporter of tumor colonies were different between various organs, and even among colonies in the same organs. The effects of antitumor drugs were also evaluated using these Fucci reporter cells. Of the three antitumor drugs studied, 5-fluorouracil treatment on 4T1-Fucci cells resulted in characteristic fluorescent patterns by the induction of G2 /M arrest both in vitro and in vivo. Thus, the combination of a tissue-clearing method with the Fucci reporter is useful for analyzing the mechanisms of cancer metastasis and drug resistance.

Whole-organ analysis of TGF-ß-mediated remodelling of the tumour microenvironment by tissue clearing.

Tissue clearing is one of the most powerful strategies for a comprehensive analysis of disease progression. Here, we established an integrated pipeline that combines tissue clearing, 3D imaging, and machine learning and applied to a mouse tumour model of experimental lung metastasis using human lung adenocarcinoma A549 cells. This pipeline provided the spatial information of the tumour microenvironment. We further explored the role of transforming growth factor-ß (TGF-ß) in cancer metastasis. TGF-ß-stimulated cancer cells enhanced metastatic colonization of unstimulated-cancer cells in vivo when both cells were mixed. RNA-sequencing analysis showed that expression of the genes related to coagulation and inflammation were up-regulated in TGF-ß-stimulated cancer cells. Further, whole-organ analysis revealed accumulation of platelets or macrophages with TGF-ß-stimulated cancer cells, suggesting that TGF-ß might promote remodelling of the tumour microenvironment, enhancing the colonization of cancer cells. Hence, our integrated pipeline for 3D profiling will help the understanding of the tumour microenvironment.

Protocol for Imaging and Analysis of Mouse Tumor Models with CUBIC Tissue Clearing.

Tissue-clearing technologies have developed rapidly in the past decade, especially for use in neuroscience research. We previously reported that CUBIC, which is one tissue-clearing method, is useful for applications in cancer research. CUBIC cancer analysis can be used to detect cancer metastasis with single-cell resolution at whole mouse body/organ level. This approach can also analyze the tumor characteristics with high-quality 3D images. Here, we describe a detailed CUBIC cancer protocol from tissue clearing, capturing 3D images and post-immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Kubota et al. (2017).

Whole-Body Profiling of Cancer Metastasis with Single-Cell Resolution.

Stochastic and proliferative events initiated from a single cell can disrupt homeostatic balance and lead to fatal disease processes such as cancer metastasis. To overcome metastasis, it is necessary to detect and quantify sparsely distributed metastatic cells throughout the body at early stages. Here, we demonstrate that clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC)-based cancer (CUBIC-cancer) analysis with a refractive index (RI)-optimized protocol enables comprehensive cancer cell profiling of the whole body and organs. We applied CUBIC-cancer analysis to 13 mouse models using nine cancer cell lines and spatiotemporal quantification of metastatic cancer progression at single-cell resolution. CUBIC-cancer analysis suggests that the epithelial-mesenchymal transition promotes not only extravasation but also cell survival at metastatic sites. CUBIC-cancer analysis is also applicable to pharmacotherapeutic profiling of anti-tumor drugs. CUBIC-cancer analysis is compatible with in vivo bioluminescence imaging and 2D histology. We suggest that a scalable analytical pipeline with these three modalities may contribute to addressing currently incurable metastatic diseases.

Chemical Principles in Tissue Clearing and Staining Protocols for Whole-Body Cell Profiling.

Mammalian bodies have more than a billion cells per cubic centimeter, which makes whole-body cell (WBC) profiling of an organism one of the ultimate challenges in biology and medicine. Recent advances in tissue-clearing technology have enabled rapid and comprehensive cellular analyses in whole organs and in the whole body by a combination of state-of-the-art technologies of optical imaging and image informatics. In this review, we focus mainly on the chemical principles in currently available techniques for tissue clearing and staining to facilitate our understanding of their underlying mechanisms. Tissue clearing is usually conducted by the following steps: (a) fixation, (b) permeabilization, (c) decolorizing, and (d) refractive index (RI) matching. To phenotype individual cells after tissue clearing, it is important to visualize genetically encoded fluorescent reporters and/or to stain tissues with fluorescent dyes, fluorescent labeled antibodies, or nucleic acid probes. Although some technical challenges remain, the chemical principles in tissue clearing and staining for WBC profiling will enable various applications, such as identifying cellular circuits across multiple organs and measuring their dynamics in stochastic and proliferative cellular processes, for example, autoimmune and malignant neoplastic diseases.