Stimulation of the farnesoid X receptor promotes M2 macrophage polarization.

FXR is a key molecule that modulates anti-inflammatory activity in the intestinal-liver axis. Although FXR has pleiotropic functions including regulation of liver inflammation and activation of macrophages, it remains unclear whether it is involved in macrophage polarization. In this paper we demonstrated that stimulation of macrophages derived from the bone marrow using an FXR agonist activated polarization toward M2 but not M1 macrophages. The treatment of mice with chitin skewed macrophage polarization towards M2 macrophages, while co-treatment with an FXR agonist further promoted the polarization toward M2 macrophages in vivo. This skewed polarization towards M2 macrophages by an FXR agonist was accompanied by increased expression of signaling molecules related to the retinoic acid receptor. Inhibition of the retinoic acid receptor suppressed FXR agonist-mediated M2 macrophage polarization, indicating that this polarization was, at least, partly dependent on the retinoic acid receptor pathway. These data demonstrate that FXR has a role in polarization toward M2 macrophages and suggest a possible therapeutic potential of FXR agonists in M2 macrophage-related conditions.

Blockade of the CXCR3/CXCL10 axis ameliorates inflammation caused by immunoproteasome dysfunction.

Immunoproteasomes regulate the degradation of ubiquitin-coupled proteins and generate peptides that are preferentially presented by MHC class I. Mutations in immunoproteasome subunits lead to immunoproteasome dysfunction, which causes proteasome-associated autoinflammatory syndromes (PRAAS) characterized by nodular erythema and partial lipodystrophy. It remains unclear, however, how immunoproteasome dysfunction leads to inflammatory symptoms. Here, we established mice harboring a mutation in Psmb8 (Psmb8-KI mice) and addressed this question. Psmb8-KI mice showed higher susceptibility to imiquimod-induced skin inflammation (IMS). Blockade of IL-6 or TNF-α partially suppressed IMS in both control and Psmb8-KI mice, but there was still more residual inflammation in the Psmb8-KI mice than in the control mice. DNA microarray analysis showed that treatment of J774 cells with proteasome inhibitors increased the expression of the Cxcl9 and Cxcl10 genes. Deficiency in Cxcr3, the gene encoding the receptor of CXCL9 and CXCL10, in control mice did not change IMS susceptibility, while deficiency in Cxcr3 in Psmb8-KI mice ameliorated IMS. Taken together, these findings demonstrate that this mutation in Psmb8 leads to hyperactivation of the CXCR3 pathway, which is responsible for the increased susceptibility of Psmb8-KI mice to IMS. These data suggest the CXCR3/CXCL10 axis as a new molecular target for treating PRAAS.

Necroptosis protects against exacerbation of acute pancreatitis.

The sensing of various extrinsic stimuli triggers the receptor-interacting protein kinase-3 (RIPK3)-mediated signaling pathway, which leads to mixed-lineage kinase-like (MLKL) phosphorylation followed by necroptosis. Although necroptosis is a form of cell death and is involved in inflammatory conditions, the roles of necroptosis in acute pancreatitis (AP) remain unclear. In the current study, we administered caerulein to Ripk3- or Mlkl-deficient mice (Ripk3-/- or Mlkl-/- mice, respectively) and assessed the roles of necroptosis in AP. We found that Ripk3-/- mice had significantly more severe pancreatic edema and inflammation associated with macrophage and neutrophil infiltration than control mice. Consistently, Mlkl-/- mice were more susceptible to caerulein-induced AP, which occurred in a time- and dose-dependent manner, than control mice. Mlkl-/- mice exhibit weight loss, edematous pancreatitis, necrotizing pancreatitis, and acinar cell dedifferentiation in response to tissue damage. Genetic deletion of Mlkl resulted in downregulation of the antiapoptotic genes Bclxl and Cflar in association with increases in the numbers of apoptotic cells, as detected by TUNEL assay. These findings suggest that RIPK3 and MLKL-mediated necroptosis exerts protective effects in AP and caution against the use of necroptosis inhibitors for AP treatment.

Quantitative evaluation of SARS-CoV-2 inactivation using a deep ultraviolet light-emitting diode.

Inactivation technology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is certainly a critical measure to mitigate the spread of coronavirus disease 2019 (COVID-19). A deep ultraviolet light-emitting diode (DUV-LED) would be a promising candidate to inactivate SARS-CoV-2, based on the well-known antiviral effects of DUV on microorganisms and viruses. However, due to variations in the inactivation effects across different viruses, quantitative evaluations of the inactivation profile of SARS-CoV-2 by DUV-LED irradiation need to be performed. In the present study, we quantify the irradiation dose of DUV-LED necessary to inactivate SARS-CoV-2. For this purpose, we determined the culture media suitable for the irradiation of SARS-CoV-2 and optimized the irradiation apparatus using commercially available DUV-LEDs that operate at a center wavelength of 265, 280, or 300 nm. Under these conditions, we successfully analyzed the relationship between SARS-CoV-2 infectivity and the irradiation dose of the DUV-LEDs at each wavelength without irrelevant biological effects. In conclusion, total doses of 1.8 mJ/cm2 for 265 nm, 3.0 mJ/cm2 for 280 nm, and 23 mJ/cm2 for 300 nm are required to inactivate 99.9% of SARS-CoV-2. Our results provide quantitative antiviral effects of DUV irradiation on SARS-CoV-2, serving as basic knowledge of inactivation technologies against SARS-CoV-2.

Distinct Roles of IL-1ß and IL-18 in NLRC4-Induced Autoinflammation.

The NLRC4 inflammasome assembles in response to detection of bacterial invasion, and NLRC4 activation leads to the production of IL-1ß and IL-18 together with pyroptosis-mediated cell death. Missense activating mutations in NLRC4 cause autoinflammatory disorders whose symptoms are distinctly dependent on the site of mutation and other aspects of the genetic background. To determine the involvement of IL-1ß and IL-18 in the inflammation induced by NLRC4 mutation, we depleted IL-1ß, IL-18, or both cytokines in Nlrc4-transgenic mice in which mutant Nlrc4 is expressed under the MHC class II promoter (Nlrc4-H443P-Tg mice). The deletion of the Il1b or Il18 gene in Nlrc4-H443P-Tg mice reduced the neutrophil numbers in the spleen, and mice with deletion of both genes had an equivalent number of neutrophils compared to wild-type mice. Deletion of Il1b ameliorated but did not eliminate bone marrow hyperplasia, while mice deficient in Il18 showed no bone marrow hyperplasia. In contrast, tail bone deformity remained in the presence of Il18 deficiency, but Il1b deficiency completely abolished bone deformity. The decreased bone density in Nlrc4-H443P-Tg mice was counteracted by Il1b but not Il18 deficiency. Our results demonstrate the distinct effects of IL-1ß and IL-18 on NLRC4-induced inflammation among tissues, which suggests that blockers for each cytokine should be utilized depending on the site of inflammation.

Notch Balances Th17 and Induced Regulatory T Cell Functions in Dendritic Cells by Regulating Aldh1a2 Expression.

Dendritic cells (DCs) are important for adaptive immune responses through the activation of T cells. The molecular interplay between DCs and T cells determines the magnitude of T cell responses or outcomes of functional differentiation of T cells. In this study, we demonstrated that DCs in mice that are Rbpj deficient in CD11c+ cells (Rbpj-/- mice) promoted the differentiation of IL-17A-producing Th17 cells. Rbpj-deficient DCs expressed little Aldh1a2 protein that is required for generating retinoic acid. Those DCs exhibited a reduced ability for differentiating regulatory T cells induced by TGF-ß. Rbpj protein directly regulated Aldh1a2 transcription by binding to its promoter region. The overexpression of Aldh1a2 in Rbpj-deficient DCs negated their Th17-promoting ability. Transfer of naive CD4+ T cells into Rag1-deficient Rbpj-/- mice enhanced colitis with increased Th17 and reduced induced regulatory T cells (iTreg) compared with control Rag1-deficient mice. The cotransfer of iTreg and naive CD4+ T cells into Rag1-deficient Rbpj-/- mice improved colitis compared with transfer of naive CD4+ T cell alone. Furthermore, cotransfer of DCs from Rbpj-/- mice that overexpressed Aldh1a2 or Notch-stimulated DCs together with naive CD4+ T cells into Rbpj-/-Rag1-deficient mice led to reduced colitis with increased iTreg numbers. Therefore, our studies identify Notch signaling in DCs as a crucial balancer of Th17/iTreg, which depends on the direct regulation of Aldh1a2 transcription in DCs.

Differentiation of preadipocytes and mature adipocytes requires PSMB8.

The differentiation of adipocytes is tightly regulated by a variety of intrinsic molecules and also by extrinsic molecules produced by adjacent cells. Dysfunction of adipocyte differentiation causes lipodystrophy, which impairs glucose and lipid homeostasis. Although dysfunction of immunoproteasomes causes partial lipodystrophy, the detailed molecular mechanisms remain to be determined. Here, we demonstrate that Psmb8, a catalytic subunit for immunoproteasomes, directly regulates the differentiation of preadipocytes and additionally the differentiation of preadipocytes to mature adipocytes. Psmb8(-/-) mice exhibited slower weight gain than wild-type mice, and this was accompanied by reduced adipose tissue volume and smaller size of mature adipocytes compared with controls. Blockade of Psmb8 activity in 3T3-L1 cells disturbed the differentiation to mature adipocytes. Psmb8(-/-) mice had fewer preadipocyte precursors, fewer preadipocytes and a reduced ability to differentiate preadipocytes toward mature adipocytes. Our data demonstrate that Psmb8-mediated immunoproteasome activity is a direct regulator of the differentiation of preadipocytes and their ultimate maturation.

Dysfunctional immunoproteasomes in autoinflammatory diseases.

Recent progress in DNA sequencing technology has made it possible to identify specific genetic mutations in familial disorders. For example, autoinflammatory syndromes are caused by mutations in gene coding for immunoproteasomes. These diseases include Japanese autoinflammatory syndrome with lipodystrophy, Nakajo-Nishimura syndrome, joint contractures, muscular atrophy, microcytic anemia, panniculitis-associated lipodystrophy syndrome, and chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature syndrome. Causal mutations of these syndromes are present in gene coding for subunits of the immunoproteasome. Importantly, a genetically modified mouse that lacks the catalytic subunit of immunoproteasomes does not always develop an autoinflammatory syndrome. Analysis of causal gene mutations, assessment of patients' phenotypic changes, and appropriate animal models will be indispensable for clarifying the underlying mechanisms responsible for the development of autoinflammatory syndromes and establishing curative approaches.

CD98 Heavy Chain Is a Potent Positive Regulator of CD4+ T Cell Proliferation and Interferon-γ Production In Vivo.

Upon their recognition of antigens presented by the MHC, T cell proliferation is vital for clonal expansion and the acquisition of effector functions, which are essential for mounting adaptive immune responses. The CD98 heavy chain (CD98hc, Slc3a2) plays a crucial role in the proliferation of both CD4+ and CD8+ T cells, although it is unclear if CD98hc directly regulates the T cell effector functions that are not linked with T cell proliferation in vivo. Here, we demonstrate that CD98hc is required for both CD4+ T cell proliferation and Th1 functional differentiation. T cell-specific deletion of CD98hc did not affect T cell development in the thymus. CD98hc-deficient CD4+ T cells proliferated in vivo more slowly as compared with control T cells. C57BL/6 mice lacking CD98hc in their CD4+ T cells could not control Leishmania major infections due to lowered IFN-γ production, even with massive CD4+ T cell proliferation. CD98hc-deficient CD4+ T cells exhibited lower IFN-γ production compared with wild-type T cells, even when comparing IFN-γ expression in cells that underwent the same number of cell divisions. Therefore, these data indicate that CD98hc is required for CD4+ T cell expansion and functional Th1 differentiation in vivo, and suggest that CD98hc might be a good target for treating Th1-mediated immune disorders.

Branched chain amino acid suppressed insulin-initiated proliferation of human cancer cells through induction of autophagy.

BACKGROUND: Branched chain amino acid (BCAA) dietary supplementation inhibits activation of the insulin-like growth factor (IGF)/IGF-I receptor (IGF-IR) axis in diabetic animal models. However, the in vitro effect of BCAA on human cancer cell lines under hyper-insulinemic conditions remains unclear. MATERIALS AND METHODS: Colon (HCT-116) and hepatic (HepG2) tumor cells were treated with varying concentrations of BCAA with or without fluorouracil (5-FU). The effect of BCAA on insulin-initiated proliferation was determined. Gene and protein expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. RESULTS: BCAA supplementation had no significant effect on cell proliferation and did not show significant synergistic or antagonistic effects with 5-FU. However, BCAA significantly decreased insulin-initiated proliferation of human colon and hepatic cancer cell lines in vitro. BCAA supplementation caused a marked decrease in activated IGF-IR expression and significantly enhanced both mRNA and protein expression of LC3-II and BECN1 (BECLIN-1). CONCLUSION: BCAA could be a useful chemopreventive modality for cancer in hyperinsulinemic conditions.

CD98hc regulates the development of experimental colitis by controlling effector and regulatory CD4(+) T cells.

CD4(+) T cell activation is controlled by signaling through the T cell receptor in addition to various co-receptors, and is also affected by their interactions with effector and regulatory T cells in the microenvironment. Inflammatory bowel diseases (IBD) are caused by the persistent activation and expansion of auto-aggressive CD4(+) T cells that attack intestinal epithelial cells. However, the molecular basis for the persistent activation of CD4(+) T cells in IBD remains unclear. In this study, we investigated how the CD98 heavy chain (CD98hc, Slc3a2) affected the development of colitis in an experimental animal model. Transferring CD98hc-deficient CD4(+)CD25(-) T cells into Rag2(-/-) mice did not cause colitis accompanied by increasing Foxp3(+) inducible regulatory T cells. By comparison, CD98hc-deficient naturally occurring regulatory T cells (nTregs) had a decreased capability to suppress colitis induced by CD4(+)CD25(-) T cells, although CD98hc-deficient mice did not have a defect in the development of nTregs. Blocking CD98hc with an anti-CD98 blocking antibody prevented the development of colitis. Our results indicate that CD98hc regulates the expansion of autoimmune CD4(+) T cells in addition to controlling nTregs functions, which suggests the CD98hc as an important target molecule for establishing strategies for treating colitis.

The ARNT-STAT3 axis regulates the differentiation of intestinal intraepithelial TCRαß⁺CD8αα⁺ cells.

Intestinal intraepithelial T cells contribute to the regulation of inflammatory responses in the intestine; however, the molecular basis for their development and maintenance is unknown. The aryl hydrocarbon receptor complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT) and senses environmental factors, including gut microbiota. Here, we identify ARNT as a critical regulator of the differentiation of TCRαß(+)CD8αα(+) intestinal intraepithelial T cells. Mice deficient in either ARNT or aryl hydrocarbon receptor show a greater than- eight-fold reduction in the number of TCRαß(+)CD8αα(+) intestinal intraepithelial T cells. The number of TCRαß(+)CD8αα(+) intestinal intraepithelial T cells is increased by treatment with an aryl hydrocarbon receptor agonist in germ-free mice and is decreased by antibiotic treatment. The Arnt-deficient precursors of TCRαß(+)CD8αα(+) intestinal intraepithelial T cells express low amounts of STAT3 and fail to differentiate towards the TCRαß(+)CD8αα(+) cell fate after IL-15 stimulation, a deficiency that is overcome by overexpression of Stat3. These data demonstrate that the ARNT-STAT3 axis is a critical regulator of TCRαß(+)CD8αα(+) intestinal intraepithelial T-cell development and differentiation.

Reduced diversity and imbalance of fecal microbiota in patients with ulcerative colitis.

BACKGROUND: Clinical observations and experimental colitis models have indicated the importance of intestinal bacteria in the etiology of ulcerative colitis (UC), but a causative bacterial agent has not been identified. AIM: To determine how intestinal bacteria are associated with UC, fecal microbiota and other components were compared for UC patients and healthy adults. METHODS: Fresh feces were collected from 48 UC patients. Fecal microbiota were analyzed by use of terminal-restriction fragment length polymorphism (T-RFLP), real-time PCR, and culture. The concentrations of organic acids, indole, and ammonia, and pH and moisture, which are indicators of the intestinal environment, were measured and compared with healthy control data. RESULTS: T-RFLP data divided the UC patients into four clusters; one cluster was obtained for healthy subjects. The diversity of fecal microbiota was significantly lower in UC patients. There were significantly fewer Bacteroides and Clostridium subcluster XIVab, and the amount of Enterococcus was higher in UC patients than in healthy subjects. The fecal concentration of organic acids was significantly lower in UC patients who were in remission. CONCLUSION: UC patients have imbalances in the intestinal environment-less diversity of fecal microbiota, lower levels of major anaerobic bacteria (Bacteroides and Clostridium subcluster XIVab), and a lower concentration of organic acids.

Manipulation of CD98 resolves type 1 diabetes in nonobese diabetic mice.

The interplay of CD4(+) and CD8(+) T cells targeting autoantigens is responsible for the progression of a number of autoimmune diseases, including type 1 diabetes mellitus (T1D). Understanding the molecular mechanisms that regulate T cell activation is crucial for designing effective therapies for autoimmune diseases. We probed a panel of Abs with T cell-modulating activity and identified a mAb specific for the H chain of CD98 (CD98hc) that was able to suppress T cell proliferation. The anti-CD98hc mAb also inhibited Ag-specific proliferation and the acquisition of effector function by CD4(+) and CD8(+) T cells in vitro and in vivo. Injection of the anti-CD98hc mAb completely prevented the onset of cyclophosphamide-induced diabetes in NOD mice. Treatment of diabetic NOD mice with anti-CD98hc reversed the diabetic state to normal levels, coincident with decreased proliferation of CD4(+) T cells. Furthermore, treatment of diabetic NOD mice with CD98hc small interfering RNA resolved T1D. These data indicate that strategies targeting CD98hc might have clinical application for treating T1D and other T cell-mediated autoimmune diseases.

Notch2 regulates the development of marginal zone B cells through Fos.

B cells are classified into several subsets depending on their functions, marker expression pattern and localization. Marginal zone B (MZB) cells are a distinct lineage from follicular B cells, and regulate host defenses against blood-borne pathogens. Notch2/RBP-J signaling regulates the development of MZB cells by interacting with delta-like 1 ligand, although the target genes for Notch2 signaling remain unclear. We identified Fos as an upregulated gene in LPS-stimulated B cells that received Notch2 signaling. Fos is expressed in CD21(high)CD23(low) MZB cells at a higher level compared to CD21(Int)CD23(high) follicular B cells. Deleting the Notch2 gene in CD19(+) B cells decreased Fos expression in B cells. Overexpression of Fos in Notch2-deficient B cells or bone marrow cells partially restored MZB development. Fos promoter activity was upregulated by Notch2 signaling, indicating that Notch2 directly controls Fos transcription associated with MZB development. These data identify Fos as one of the target genes for Notch2 signaling that is crucial for MZB development.

In vitro cytotoxic effect of ethanol extract prepared from sporophyll of Undaria pinnatifida on human colorectal cancer cells.

Brown seaweed Undaria pinnatifida (Harvey) Suringar is popular as a foodstuff, and used for medical care in East Asian countries. The major components of this seaweed are shown to benefit hypertension and hyperlipidemia, and considered to reduce the risks of infarction and ischemic diseases. Furthermore, the intake of dietary fiber of seaweeds is considered to prevent the production and proliferation of cancer in the gastrointestinal tract. The direct effect of an ethanol extract prepared from Undaria pinnatifida sporophyll (mekabu) on HCT116 human colorectal cancer cells was examined, and the mekabu extract was shown to induce the non-oxidative apoptotic damage to the cells, thus resulting in the reduction of their viabilities in a concentration-dependent manner. Moreover, the cytotoxic effects of carcinostatic drugs, such as 5-fluorouracil (5-FU) and irinotecan (CPT-11), were observed only in the medium containing sera, while the mekabu extract could effectively reduce the cell viabilities even in the serum-free medium. These findings suggest that the mekabu extract may contain a potential active substance inducing the non-oxidative apoptotic cell death probably through a mechanism different from those of 5-FU and CPT-11, and hence mekabu is possibly useful as an auxiliary drug to the chemotherapy of colorectal cancer.

Effects of fermented brown rice on the intestinal environments in healthy adult.

PURPOSE: The aim of this study is to investigate the prebiotic effects of brown rice fermented by Aspergillus oryzae (FBRA) on the intestinal environment in vitro and in healthy adults. METHODS: Fresh fecal slurries from six healthy adults were incubated with FBRA to confirm prebiotic potentials of FBRA. Another thirty-six healthy adults were randomly allocated to 2 groups for the clinical study. Subjects consumed 21.0 g/day of either FBRA or control food for 2 weeks, followed by a 12-week intermission and then 2-week ingestion vice versa. Main outcome measures were bifidobacterial numbers and organic acid concentration in feces. Sub outcome measures were fecal microbiota, fecal environments and bowel function. RESULTS: Incubation of fecal slurries with FBRA in vitro resulted in increased organic acids with individual-specific patterns. Bifidobacterial numbers were increased during incubation. In the clinical study, all participants safely completed this study. FBRA had little effect on fecal number of bifidobacteria, concentrations of organic acids or putrefactive metabolites, fecal pH, or fecal microbiota. CONCLUSION: FBRA has the potentials as a prebiotic, however, we could not detect its effects on the intestinal environment in vivo. The results in a clinical study indicated that FBRA could be safely used for healthy adults.

Histone deacetylase inhibitors promote neurosteroid-mediated cell differentiation and enhance serotonin-stimulated brain-derived neurotrophic factor gene expression in rat C6 glioma cells.

Progesterone treatment has previously been reported to promote the differentiation of glial cells probably through the production of 5alpha-reduced neurosteroids, resulting in the enhancement of serotonin-stimulated brain-derived neurotrophic factor (BDNF) gene expression, which is considered to contribute to the survival, regeneration, and plasticity of neuronal cells in the brain and hence has been suggested to improve mood disorders and other symptoms in depressive patients. Based on these previous observations, the effects on glial cells of histone deacetylase (HDAC) inhibitors, which are known as agents promoting cell differentiation, were examined using rat C6 glioma cells as a model for in vitro studies. Consequently, trichostatin A (TSA), sodium butyrate (NaB), and valproic acid (VPA) stimulated glial fibrillary acidic protein (GFAP) gene expression, and their stimulatory effects on GFAP gene expression were inhibited by treatment of these cells with finasteride, an inhibitor of the enzyme producing 5alpha-reduced neurosteroids. In addition, HDAC inhibitors enhanced serotonin-stimulated BDNF gene expression, the enhancement of which could be abolished by the inhibition of 5alpha-reduced neurosteroid production in the glioma cells. These results suggest that HDAC inhibitors may be able to promote the differentiation of rat C6 glioma cells through the production of 5alpha-reduced neurosteroids, resulting in the enhancement of serotonin-stimulated BDNF gene expression as a consequence of promoting their differentiation, indicating the possibility that differentiated glial cells may be implicated in preserving the integrity of neural networks as well as improving the function of neuronal cells in the brain.

Production of apoptosis-inducing substances from soybean protein by Clostridium butyricum: characterization of their toxic effects on human colon carcinoma cells.

Microbial metabolism of soybean constituents is known to produce novel active substances as a chemopreventive agent during the fermentation, and enterobacteria are expected to produce chemopreventive agents as a consequence of metabolizing soybean constituents in the intestinal tract. Then, the conditioned medium was prepared by culturing an enterobacterium Clostridium butyricum (C. butyricum) with soybean protein, and its direct effect on human colon carcinoma HCT116 cells was examined. The conditioned medium was shown to induce the cell death, and suggested to contain novel apoptosis-inducing substances. Thus, enterobacteria are predicted to produce anti-tumor substances from food-derived proteins within the intestinal tract.