Establishment of the improved colonization of Escherichia coli laboratory strain in the intestine mediated by single gene deletion.

In light of the emerging importance of the gut microbiome in human health, there is a need to improve the colonization efficiency of therapeutic bacteria called probiotics. Despite their recognized potential, artificially administered bacteria exhibit poor colonization in the intestine, limiting their therapeutic efficacy. Addressing this challenge requires innovative strategies; however, reported examples are limited. In nature, including in the intestinal tract, bacteria live via biofilm formation. Recently, it has been reported that RNase I, a member of the RNase T2 family conserved among almost all species, including bacteria, inhibits biofilm formation in Escherichia coli. In this study, we focus on these results and investigate the relationship between high biofilm formation and intestinal attachment using a non-settling E. coli laboratory strain as a probiotic model. The intestinal colonization abilities were evaluated through a microfluidic device mimicking the intestinal tract and through oral administration to mice. The in vitro and in vivo experiments showed that the E. coli strain lacking RNase I exhibited remarkable stability in intestinal colonization. We investigated the observation of colonization using fluorescence in situ hybridization, and inoculated E. coli cells were aggregated with the gut microbiome in the cecum and colon. This study proposes a technique to improve the intestinal colonization of bacteria by simply manipulating a single gene disruption, and it is expected to contribute to future research on the colonization of useful bacteria.

Targeted insertion of conditional expression cassettes into the mouse genome using the modified i-PITT.

BACKGROUND: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. RESULTS: To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. CONCLUSIONS: Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.

Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novel genome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.

The Philippines stingless bee propolis promotes hair growth through activation of Wnt/ß-catenin signaling pathway.

Although hair loss is not a horrible disease, it sometimes reduces the patients' quality of life (QOL) and increases their mental stress. Currently, there is no effective treatment for hair loss. It is known that honeybee propolis has various biological activities, including stimulating the proliferation of hair matrix keratinocytes. However, little is known with the hair promoting activity of stingless bee propolis. Hence, this study investigates the hair growth-promoting activity of Philippines stingless bee propolis extract and the underlying a molecular mechanism of promoting hair growth. For the evaluation of hair growth stimulating activity, 99.5% ethanolic extract of Philippines stingless bee propolis is examined using the simple shaving model in C57BL/6N mice. Melaninization of dorsal skin and histological analysis of hair follicles (HFs) revealed that propolis promotes hair growth by stimulating HFs development. The expression of mRNA (Wnt3a, Ctnnb1/ß-catenin, Lef1, and Bmp2) and protein (WNT3A and ß-catenin) of selected Wnt/ß-catenin associated genes explains Philippines stingless bee propolis promoting HFs development by activating Wnt/ß-catenin signaling pathway. These results suggest that the treatment of propolis strongly promotes hair growth by stimulating the development of HFs via activation of Wnt/ß-catenin signaling pathway. This further indicates the potential of Philippines stingless bee propolis as a novel promising agricultural product for hair growth.

Heat Shock Transcription Factor 2 Is Significantly Involved in Neurodegenerative Diseases, Inflammatory Bowel Disease, Cancer, Male Infertility, and Fetal Alcohol Spectrum Disorder: The Novel Mechanisms of Several Severe Diseases.

HSF (heat shock transcription factor or heat shock factor) was discovered as a transcription factor indispensable for heat shock response. Although four classical HSFs were discovered in mammals and two major HSFs, HSF1 and HSF2, were cloned in the same year of 1991, only HSF1 was intensively studied because HSF1 can give rise to heat shock response through the induction of various HSPs' expression. On the other hand, HSF2 was not well studied for some time, which was probably due to an underestimate of HSF2 itself. Since the beginning of the 21st century, HSF2 research has progressed and many biologically significant functions of HSF2 have been revealed. For example, the roles of HSF2 in nervous system protection, inflammation, maintenance of mitosis and meiosis, and cancer cell survival and death have been gradually unveiled. However, we feel that the fact HSF2 has a relationship with various factors is not yet widely recognized; therefore, the biological significance of HSF2 has been underestimated. We strongly hope to widely communicate the significance of HSF2 to researchers and readers in broad research fields through this review. In addition, we also hope that many readers will have great interest in the molecular mechanism in which HSF2 acts as an active transcription factor and gene bookmarking mechanism of HSF2 during cell cycle progression, as is summarized in this review.

Effector memory CD4+T cells in mesenteric lymph nodes mediate bone loss in food-allergic enteropathy model mice, creating IL-4 dominance.

Intestinal inflammation can be accompanied by osteoporosis, but their relationship, mediated by immune responses, remains unclear. Here, we investigated a non-IgE-mediated food-allergic enteropathy model of ovalbumin (OVA) 23-3 mice expressing OVA-specific T-cell-receptor transgenes. Mesenteric lymph nodes (MLNs) and their pathogenic CD4+T cells were important to enteropathy occurrence and exacerbation when the mice were fed an egg-white (EW) diet. EW-fed OVA23-3 mice also developed bone loss and increased CD44hiCD62LloCD4+T cells in the MLNs and bone marrow (BM); these changes were attenuated by MLN, but not spleen, resection. We fed an EW diet to F1 cross offspring from OVA23-3 mice and a mouse line expressing the photoconvertible protein KikGR to track MLN CD4+T cells. Photoconverted MLN CD44hiCD62LloCD4+T cells migrated predominantly to the BM; pit formation assay proved their ability to promote bone damage via osteoclasts. Significantly greater expression of IL-4 mRNA in MLN CD44hiCD62LloCD4+T cells and bone was observed in EW-fed OVA23-3 mice. Anti-IL-4 monoclonal antibody injection canceled bone loss in the primary inflammation phase in EW-fed mice, but less so in the chronic phase. This novel report shows the specific inflammatory relationship, via Th2-dominant-OVA-specific T cells and IL-4 production, between MLNs and bone, a distant organ, in food-allergic enteropathy.

Effects of orally administered Euglena gracilis and its reserve polysaccharide, paramylon, on gastric dysplasia in A4gnt knockout mice.

Euglena gracilis is widely utilized as food or supplement to promote human and animal health, as it contains rich nutrients. In this study, we administered spray-dried powder of E. gracilis and paramylon, ß-glucan stored in E. gracilis cells, to A4gnt knockout (KO) mice. A4gnt KO mice are a mutant mouse model that spontaneously develops gastric cancer through hyperplasia-dysplasia-adenocarcinoma sequence in the antrum of the stomach, and we observed the effects of E. gracilis and paramylon on the early involvements of A4gnt KO mice. Male and female 10-week-old A4gnt KO mice and their age-matched wildtype C57BL/6J mice were orally administered with 50 mg of E. gracilis or paramylon suspended in saline or saline as a control. After 3-week administration, animals were euthanatized and the stomach was examined histopathologically and immunohistochemically. Gene expression patterns of the stomach, which have been reported to be altered with A4gnt KO, and IgA concentration in small intestine were also analyzed with real-time PCR and ELISA, respectively. Administration of Euglena significantly reduced the number of stimulated CD3-positive T-lymphocytes in pyloric mucosa of A4gnt KO mice and tend to reduce polymorphonuclear leukocytes infiltration. Euglena administration further downregulated the expression of Il11 and Cxcl1 of A4gnt KO mice. Euglena administration also affected IgA concentration in small intestinal contents of A4gnt KO mice. Paramylon administration reduced the number of CD3-positive lymphocytes in pyloric mucosa of A4gnt KO mice, and downregulated the expressions of Il11 and Ccl2 of A4gnt KO mice. Although we found no significant effects on gross and microscopic signs of gastric dysplasia and cell proliferation, the present study suggests that the administration of Euglena and paramylon may ameliorate the early involvements of A4gnt mice through the effects on inflammatory reactions in the gastric mucosa. The cancer-preventing effects should be studied with long-term experiments until actual gastric cancer formation.

Insights on the Functional Role of Beta-Glucans in Fungal Immunity Using Receptor-Deficient Mouse Models.

Understanding the host anti-fungal immunity induced by beta-glucan has been one of the most challenging conundrums in the field of biomedical research. During the last couple of decades, insights on the role of beta-glucan in fungal disease progression, susceptibility, and resistance have been greatly augmented through the utility of various beta-glucan cognate receptor-deficient mouse models. Analysis of dectin-1 knockout mice has clarified the downstream signaling pathways and adaptive effector responses triggered by beta-glucan in anti-fungal immunity. On the other hand, assessment of CR3-deficient mice has elucidated the compelling action of beta-glucans in neutrophil-mediated fungal clearance, and the investigation of EphA2-deficient mice has highlighted its novel involvement in host sensing and defense to oral mucosal fungal infection. Based on these accounts, this review focuses on the recent discoveries made by these gene-targeted mice in beta-glucan research with particular emphasis on the multifaceted aspects of fungal immunity.

A Vaspin-HSPA1L complex protects proximal tubular cells from organelle stress in diabetic kidney disease.

Proximal tubular cells (PTCs) are crucial for maintaining renal homeostasis, and tubular injuries contribute to progression of diabetic kidney disease (DKD). However, the roles of visceral adipose tissue-derived serine protease inhibitor (vaspin) in the development of DKD is not known. We found vaspin maintains PTCs through ameliorating ER stress, autophagy impairment, and lysosome dysfunction in DKD. Vaspin-/- obese mice showed enlarged and leaky lysosomes in PTCs associated with increased apoptosis, and these abnormalities were also observed in the patients with DKD. During internalization into PTCs, vaspin formed a complex with heat shock protein family A (Hsp70) member 1 like (HSPA1L) as well as 78 kDa glucose-regulated protein (GRP78). Both vaspin-partners bind to clathrin heavy chain and involve in the endocytosis. Notably, albumin-overload enhanced extracellular release of HSPA1L and overexpression of HSPA1L dissolved organelle stresses, especially autophagy impairment. Thus, vapsin/HSPA1L-mediated pathways play critical roles in maintaining organellar function of PTCs in DKD.

Tumor-suppressing potential of stingless bee propolis in in vitro and in vivo models of differentiated-type gastric adenocarcinoma.

The protective property of propolis across a wide spectrum of diseases has long been realized, yet the anti-tumor efficacy of this bioactive substance from Philippine stingless bees has remained poorly understood. Here, we showed the tumor-suppressing potential of crude ethanolic extract of Philippine stingless bee propolis (EEP) in in vitro models of gastric cancer highlighting the first indication of remarkable subtype specificity towards differentiated-type human gastric cancer cell lines but not the diffuse-type. Mechanistically, this involved the profound modulation of several cell cycle related gene transcripts, which correlated with the prominent cell cycle arrest at the G0/G1 phase. To reinforce our data, a unique differentiated-type gastric cancer model, A4gnt KO mice, together with age-matched 60 week-old C57BL/6 J mice were randomly assigned to treatment groups receiving distilled water or EEP for 30 consecutive days. EEP treatment induced significant regression of gross and histological lesions of gastric pyloric tumors that consistently corresponded with specific transcriptional regulation of cell cycle components. Also, the considerable p21 protein expression coupled with a marked reduction in rapidly dividing BrdU-labeled S-phase cells unequivocally supported our observation. Altogether, these findings support the role of Philippine stingless bee propolis as a promising adjunct treatment option in differentiated-type gastric cancer.

Analysis of A4gnt Knockout Mice Reveals an Essential Role for Gastric Sulfomucins in Preventing Gastritis Cystica Profunda.

Gastric adenocarcinoma cells secrete sulfomucins, but their role in gastric tumorigenesis remains unclear. To address that question, we generated A4gnt/Chst4 double-knockout (DKO) mice by crossing A4gnt knockout (KO) mice, which spontaneously develop gastric adenocarcinoma, with Chst4 KO mice, which are deficient in the sulfotransferase GlcNAc6ST-2. A4gnt/Chst4 DKO mice lack gastric sulfomucins but developed gastric adenocarcinoma. Unexpectedly, severe gastric erosion occurred in A4gnt/Chst4 DKO mice at as early as 3 weeks of age, and with aging these lesions were accompanied by gastritis cystica profunda (GCP). Cxcl1, Cxcl5, Ccl2, and Cxcr2 transcripts in gastric mucosa of 5-week-old A4gnt/Chst4 DKO mice exhibiting both hyperplasia and severe erosion were significantly upregulated relative to age-matched A4gnt KO mice, which showed hyperplasia alone. However, upregulation of these genes disappeared in 50-week-old A4gnt/Chst4 DKO mice exhibiting high-grade dysplasia/adenocarcinoma and GCP. Moreover, Cxcl1 and Cxcr2 were downregulated in A4gnt/Chst4 DKO mice relative to age-matched A4gnt KO mice exhibiting adenocarcinoma alone. These combined results indicate that the presence of sulfomucins prevents severe gastric erosion followed by GCP in A4gnt KO mice by transiently regulating a set of inflammation-related genes, Cxcl1, Cxcl5, Ccl2, and Cxcr2 at 5 weeks of age, although sulfomucins were not directly associated with gastric cancer development.

Suppression of IL-17F, but not of IL-17A, provides protection against colitis by inducing Treg cells through modification of the intestinal microbiota.

The cytokines IL-17A and IL-17F have 50% amino-acid identity and bind the same receptor; however, their functional differences have remained obscure. Here we found that Il17f-/- mice resisted chemically induced colitis, but Il17a-/- mice did not, and that Il17f-/- CD45RBhiCD4+ T cells induced milder colitis in lymphocyte-deficient Rag2-/- mice, accompanied by an increase in intestinal regulatory T cells (Treg cells). Clostridium cluster XIVa in colonic microbiota capable of inducing Treg cells was increased in both Il17f-/- mice and mice given transfer Il17f-/- T cells, due to decreased expression of a group of antimicrobial proteins. There was substantial production of IL-17F, but not of IL-17A, not only by naive T cells but also by various colon-resident cells under physiological conditions. Furthermore, antibody to IL-17F suppressed the development of colitis, but antibody to IL-17A did not. These observations suggest that IL-17F is an effective target for the treatment of colitis.

Orally administered brown seaweed-derived ß-glucan effectively restrained development of gastric dysplasia in A4gnt KO mice that spontaneously develop gastric adenocarcinoma.

ß-Glucan refers to a heterogeneous group of chemically defined storage polysaccharides containing ß-(1,3)-d-linked glucose polymers with branches connected by either ß-(1,4) or ß-(1,6) glycosidic linkage. To date, an extensive amount of scientific evidence supports their multifunctional biological activities, but their potential involvement in the progression of premalignant lesions remains to be clarified. A4gnt KO mice that lack α1,4-N-acetylglucosamine-capped O-glycans in gastric gland mucin are a unique animal model for gastric cancer because the mutant mice spontaneously develop gastric cancer through hyperplasia-dysplasia-adenocarcinoma sequence. In particular, A4gnt KO mice show gastric dysplasia during 10-20 weeks of age. Here we investigated the putative gastro-protective activity of brown seaweed-derived ß-glucan (Laminaran) against development of gastric dysplasia, precancerous lesion for gastric cancer in A4gnt KO mice. The mutant mice at 12 weeks of age were randomly assigned into three treatment groups namely, wildtype control + distilled water (normal control), A4gnt KO mice + distilled water (untreated control), and A4gnt KO mice + 100 mg/kg Laminaran. After 3 weeks, the stomach was removed and examined for morphology and gene expression patterns. In contrast to the untreated control group, administration of Laminaran substantially attenuated gastric dysplasia development and counterbalanced the increased induction in cell proliferation and angiogenesis. Furthermore, Laminaran treatment effectively overcame the A4gnt KO-induced alteration in the gene expression profile of selected cytokines as revealed by real-time PCR analysis. Collectively, our present findings indicate that ß-glucan can potentially restrain the development of gastric dysplasia to mediate their tissue-preserving activity.

Establishment and analysis of a novel mouse line carrying a conditional knockin allele of a cancer-specific FBXW7 mutation.

F-box and WD40 domain protein 7 (FBXW7) is a component of the SKP1-CUL1-F-box protein (SCF) complex that mediates the ubiquitination of diverse oncogenic target proteins. The exploration of FBXW7 mutations in human primary cancer has revealed three mutation hotspots at conserved arginine residues (Arg465, Arg479, and Arg505) in the WD40 domain, which are critical for substrate recognition. To study the function of human FBXW7 R465C , the most frequent mutation in human malignancies, we generated a novel conditional knockin mouse line of murine Fbxw7 R468C corresponding to human FBXW7 R465C . Systemic heterozygous knockin of the Fbxw7 R468C mutation resulted in perinatal lethality due to defects in lung development, and occasionally caused an eyes-open at birth phenotype and cleft palate. Furthermore, mice carrying liver-specific heterozygous and homozygous Fbxw7 R468C alleles cooperated with an oncogenic Kras mutation to exhibit bile duct hyperplasia within 8 months of birth and cholangiocarcinoma-like lesions within 8 weeks of birth, respectively. In addition, the substrates affected by the mutant Fbxw7 differed between the embryos, embryonic fibroblasts, and adult liver. This novel conditional knockin Fbxw7 R468C line should be useful to gain a more profound understanding of carcinogenesis associated with mutation of FBXW7.

The essential role of phospho-T38 CPI-17 in the maintenance of physiological blood pressure using genetically modified mice.

PKC-potentiated phosphorylation-dependent inhibitory protein of protein phosphatase 1 (CPI-17), an endogenous myosin phosphatase inhibitory protein, is considered a key molecule for Ca2+ sensitization of the contractile apparatus. Here, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 to generate CPI-17-deficient [knockout (KO)] and threonine 38 (T38)-phospho-resistant mice [threonine mutant into alanine (TA)], and then effects of CPI-17 on vascular contractility in vitro and mean blood pressure (MBP) in vivo were investigated. In isolated thoracic aorta, phorbol 12, 13-dibutyrate induced a sustained contraction of wild-type (WT) mice, whereas no contraction showed from TA or KO mice. A high concentration of KCl solution-induced contraction was not different between transgenic and WT mice. In contrast, phenylephrine (PE)-induced contractions in both mutant strains were significantly smaller than those of WT mice in association with a low level of myosin phosphorylation, suggesting that at least part of PE-induced contraction is regulated by phosphorylation of CPI-17 at T38. Finally, the physiologic role of CPI-17 in the regulation of blood pressure was investigated using radio telemetry. MBP was decreased significantly in both transgenic mice, even with a compensatory increase in heart rate. In summary, we generated KO and constitutively phospho-resistant mouse models of CPI-17 for the first time. p-CPI-17 at T38, possibly by PKC, could be important to maintain vascular contractility and blood pressure in vivo. -Yang, Q., Fujii, W., Kaji, N., Kakuta, S., Kada, K., Kuwahara, M., Tsubone, H., Ozaki, H., Hori, M. The essential role of phospho-T38 CPI-17 in the maintenance of physiological blood pressure using genetically modified mice.

Herpes simplex virus-1 evasion of CD8+ T cell accumulation contributes to viral encephalitis.

Herpes simplex virus-1 (HSV-1) is the most common cause of sporadic viral encephalitis, which can be lethal or result in severe neurological defects even with antiviral therapy. While HSV-1 causes encephalitis in spite of HSV-1-specific humoral and cellular immunity, the mechanism by which HSV-1 evades the immune system in the central nervous system (CNS) remains unknown. Here we describe a strategy by which HSV-1 avoids immune targeting in the CNS. The HSV-1 UL13 kinase promotes evasion of HSV-1-specific CD8+ T cell accumulation in infection sites by downregulating expression of the CD8+ T cell attractant chemokine CXCL9 in the CNS of infected mice, leading to increased HSV-1 mortality due to encephalitis. Direct injection of CXCL9 into the CNS infection site enhanced HSV-1-specific CD8+ T cell accumulation, leading to marked improvements in the survival of infected mice. This previously uncharacterized strategy for HSV-1 evasion of CD8+ T cell accumulation in the CNS has important implications for understanding the pathogenesis and clinical treatment of HSV-1 encephalitis.

Adenocarcinoma of Barrett's esophagus in a dog.

An endoscopic examination revealed a mass in the distal esophagus of a 9-year-old intact male bulldog. Histopathologically, the mass was composed of cuboidal to columnar neoplastic epithelial cells and extended from the squamous epithelium of the esophageal mucosa, indicating that the tumor was derived from Barrett's esophagus. Moreover, highly atypical foci that exhibited a cribriform pattern and high mitotic indices were also observed. The epithelial cells on the surface of the lesion often produced mucus that was positive for Alcian blue and immunohistochemically positive for MUC5AC. The neoplastic epithelial cells were diffusely positive for cytokeratin 7 and p53, and occasionally positive for cytokeratin 20. Based on these findings, the tumor was diagnosed as an adenocarcinoma. This report describes the clinical and pathological features of a spontaneous case of adenocarcinoma of Barrett's esophagus in a dog.

Involvement of interleukin-1 type 1 receptors in lipopolysaccharide-induced sickness responses.

Sickness responses to lipopolysaccharide (LPS) were examined in mice with deletion of the interleukin (IL)-1 type 1 receptor (IL-1R1). IL-1R1 knockout (KO) mice displayed intact anorexia and HPA-axis activation to intraperitoneally injected LPS (anorexia: 10 or 120µg/kg; HPA-axis: 120µg/kg), but showed attenuated but not extinguished fever (120µg/kg). Brain PGE2 synthesis was attenuated, but Cox-2 induction remained intact. Neither the tumor necrosis factor-α (TNFα) inhibitor etanercept nor the IL-6 receptor antibody tocilizumab abolished the LPS induced fever in IL-1R1 KO mice. Deletion of IL-1R1 specifically in brain endothelial cells attenuated the LPS induced fever, but only during the late, 3rd phase of fever, whereas deletion of IL-1R1 on neural cells or on peripheral nerves had little or no effect on the febrile response. We conclude that while IL-1 signaling is not critical for LPS induced anorexia or stress hormone release, IL-1R1, expressed on brain endothelial cells, contributes to the febrile response to LPS. However, also in the absence of IL-1R1, LPS evokes a febrile response, although this is attenuated. This remaining fever seems not to be mediated by IL-6 receptors or TNFα, but by some yet unidentified pyrogenic factor.

Pre-Transplantation Blockade of TNF-α-Mediated Oxygen Species Accumulation Protects Hematopoietic Stem Cells.

Hematopoietic stem cell (HSC) transplantation (HSCT) for malignancy requires toxic pre-conditioning to maximize anti-tumor effects and donor-HSC engraftment. While this induces bone marrow (BM)-localized inflammation, how this BM environmental change affects transplanted HSCs in vivo remains largely unknown. We here report that, depending on interval between irradiation and HSCT, residence within lethally irradiated recipient BM compromises donor-HSC reconstitution ability. Both in vivo and in vitro we demonstrate that, among inflammatory cytokines, TNF-α plays a role in HSC damage: TNF-α stimulation leads to accumulation of reactive oxygen species (ROS) in highly purified hematopoietic stem/progenitor cells (HSCs/HSPCs). Transplantation of flow-cytometry-sorted murine HSCs reveals damaging effects of accumulated ROS on HSCs. Short-term incubation either with an specific inhibitor of tumor necrosis factor receptor 1 signaling or an antioxidant N-acetyl-L-cysteine (NAC) prevents TNF-α-mediated ROS accumulation in HSCs. Importantly, pre-transplantation exposure to NAC successfully demonstrats protective effects in inflammatory BM on graft-HSCs, exhibiting better reconstitution capability than that of nonprotected control grafts. We thus suggest that in vivo protection of graft-HSCs from BM inflammation is a feasible and attractive approach, which may lead to improved hematopoietic reconstitution kinetics in transplantation with myeloablative conditioning that inevitably causes inflammation in recipient BM. Stem Cells 2017;35:989-1002.

Visualization of Probiotic-Mediated Ca2+ Signaling in Intestinal Epithelial Cells In Vivo.

Probiotics, such as lactic acid bacteria (LAB) and Bacillus subtilis var. natto, have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs), because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca2+ biosensor Yellow Cameleon (YC3.60) transgenic mouse line and established 5D (x, y, z, time, and Ca2+) intravital imaging systems of lymphoid tissues including those in Peyer's patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed B. subtilis natto, a probiotic TTCC012 strain, is shown to directly induce Ca2+ signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the Lactococcus lactis strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca2+ signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria.