Amelioration of Autoimmune Diabetes of NOD Mice by Immunomodulating Probiotics.

Type 1 autoimmune diabetes is an autoimmune disease characterized by specific destruction of pancreatic ß-cells producing insulin. Recent studies have shown that gut microbiota and immunity are closely linked to systemic immunity, affecting the balance between pro-inflammatory and regulatory immune responses. Altered gut microbiota may be causally related to the development of immune-mediated diseases, and probiotics have been suggested to have modulatory effects on inflammatory diseases and immune disorders. We studied whether a probiotic combination that has immunomodulatory effects on several inflammatory diseases can reduce the incidence of diabetes in non-obese diabetic (NOD) mice, a classical animal model of human T1D. When Immune Regulation and Tolerance 5 (IRT5), a probiotic combination comprising Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus reuteri, Bifidobacterium bifidium, and Streptococcus thermophiles, was administered 6 times a week for 36 weeks to NOD mice, beginning at 4 weeks of age, the incidence of diabetes was significantly reduced. Insulitis score was also significantly reduced, and ß-cell mass was conversely increased by IRT5 administration. IRT5 administration significantly reduced gut permeability in NOD mice. The proportion of total regulatory T cells was not changed by IRT5 administration; however, the proportion of CCR9+ regulatory T (Treg) cells expressing gut-homing receptor was significantly increased in pancreatic lymph nodes (PLNs) and lamina propria of the small intestine (SI-LP). Type 1 T helper (Th1) skewing was reduced in PLNs by IRT5 administration. IRT5 could be a candidate for an effective probiotic combination, which can be safely administered to inhibit or prevent type 1 diabetes (T1D).

Obesogenic diet-induced gut barrier dysfunction and pathobiont expansion aggravate experimental colitis.

Consumption of a typical Western diet is a risk factor for several disorders. Metabolic syndrome is the most common disease associated with intake of excess fat. However, the incidence of inflammatory bowel disease is also greater in subjects consuming a Western diet, although the mechanism of this phenomenon is not clearly understood. We examined the morphological and functional changes of the intestine, the first site contacting dietary fat, in mice fed a high-fat diet (HFD) inducing obesity. Paneth cell area and production of antimicrobial peptides by Paneth cells were decreased in HFD-fed mice. Goblet cell number and secretion of mucin by goblet cells were also decreased, while intestinal permeability was increased in HFD-fed mice. HFD-fed mice were more susceptible to experimental colitis, and exhibited severe colonic inflammation, accompanied by the expansion of selected pathobionts such as Atopobium sp. and Proteobacteria. Fecal microbiota transplantation transferred the susceptibility to DSS-colitis, and antibiotic treatment abrogated colitis progression. These data suggest that an experimental HFD-induced Paneth cell dysfunction and subsequent intestinal dysbiosis characterized by pathobiont expansion can be predisposing factors to the development of inflammatory bowel disease.

Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis.

Autophagy, which is critical for the proper turnover of organelles such as endoplasmic reticulum and mitochondria, affects diverse aspects of metabolism, and its dysregulation has been incriminated in various metabolic disorders. However, the role of autophagy of myeloid cells in adipose tissue inflammation and type 2 diabetes has not been addressed. We produced mice with myeloid cell-specific deletion of Atg7 (autophagy-related 7), an essential autophagy gene (Atg7 conditional knockout [cKO] mice). While Atg7 cKO mice were metabolically indistinguishable from control mice, they developed diabetes when bred to ob/w mice (Atg7 cKO-ob/ob mice), accompanied by increases in the crown-like structure, inflammatory cytokine expression and inflammasome activation in adipose tissue. Mφs (macrophages) from Atg7 cKO mice showed significantly higher interleukin 1 ß release and inflammasome activation in response to a palmitic acid plus lipopolysaccharide combination. Moreover, a decrease in the NAD(+):NADH ratio and increase in intracellular ROS content after treatment with palmitic acid in combination with lipopolysaccharide were more pronounced in Mφs from Atg7 cKO mice, suggesting that mitochondrial dysfunction in autophagy-deficient Mφs leads to an increase in lipid-induced inflammasome and metabolic deterioration in Atg7 cKO-ob/ob mice. Atg7 cKO mice were more susceptible to experimental colitis, accompanied by increased colonic cytokine expression, T helper 1 skewing and systemic bacterial invasion. These results suggest that autophagy of Mφs is important for the control of inflammasome activation in response to metabolic or extrinsic stress, and autophagy deficiency in Mφs may contribute to the progression of metabolic syndrome associated with lipid injury and colitis.

NKG2D ligation relieves 2B4-mediated NK-cell self-tolerance in mice.

Along with MHC class I (MHCI), 2B4 provides nonredundant NK-cell inhibition in mice. The immunoregulatory role of 2B4 has been increasingly appreciated in models of tumor and viral infection, however, the interactions among 2B4, MHCI, and other activating NK-cell receptors remain uncertain. Here, we dissect the influence of two distinct inhibitory pathways in modulating NK-cell-mediated control of tumors expressing strong activating ligands, including RAE-1γ. In vitro cytotoxicity and in vivo peritoneal clearance assays using MHCI(+) CD48(+) (RMA-neo), MHCI(+) CD48(+) RAE-1γ (RMA-RAE-1γ), MHCI(-) CD48(+) (RMA-S-neo), and MHCI(-) CD48(+) RAE-1γ (RMA-S-RAE-1γ) tumor lines demonstrated that NKG2D activation supersedes the inhibitory effect of both 2B4- and MHCI-mediated immune-tolerance systems. Furthermore, 2B4KO mice subcutaneously challenged with RMA-neo and RMA-S-neo exhibited reduced tumor growth and significantly prolonged survival compared with WT mice, implying that 2B4 is constitutively engaged in the NK-cell tolerance mechanism in vivo. Nevertheless, the inhibitory effect of 2B4 is significantly attenuated when NK cells encountered highly stressed tumor cells expressing RAE-1γ, resulting in an immune response shift toward NK-cell activation and tumor regression. Therefore, our data highlight the importance of the 2B4-mediated inhibitory system as an alternate self-tolerance mechanism, whose role can be modulated by the strength of activating receptor signaling within the tumor microenvironment.

An increase in the Akkermansia spp. population induced by metformin treatment improves glucose homeostasis in diet-induced obese mice.

BACKGROUND: Recent evidence indicates that the composition of the gut microbiota contributes to the development of metabolic disorders by affecting the physiology and metabolism of the host. Metformin is one of the most widely prescribed type 2 diabetes (T2D) therapeutic agents. OBJECTIVE: To determine whether the antidiabetic effect of metformin is related to alterations of intestinal microbial composition. DESIGN: C57BL/6 mice, fed either a normal-chow diet or a high-fat diet (HFD), were treated with metformin for 6 weeks. The effect of metformin on the composition of the gut microbiota was assessed by analysing 16S rRNA gene sequences with 454 pyrosequencing. Adipose tissue inflammation was examined by flow cytometric analysis of the immune cells present in visceral adipose tissue (VAT). RESULTS: Metformin treatment significantly improved the glycaemic profile of HFD-fed mice. HFD-fed mice treated with metformin showed a higher abundance of the mucin-degrading bacterium Akkermansia than HFD-fed control mice. In addition, the number of mucin-producing goblet cells was significantly increased by metformin treatment (p<0.0001). Oral administration of Akkermansia muciniphila to HFD-fed mice without metformin significantly enhanced glucose tolerance and attenuated adipose tissue inflammation by inducing Foxp3 regulatory T cells (Tregs) in the VAT. CONCLUSIONS: Modulation of the gut microbiota (by an increase in the Akkermansia spp. population) may contribute to the antidiabetic effects of metformin, thereby providing a new mechanism for the therapeutic effect of metformin in patients with T2D. This suggests that pharmacological manipulation of the gut microbiota in favour of Akkermansia may be a potential treatment for T2D.

Role of TLR2 in the pathogenesis of autoimmune diabetes and its therapeutic implication.

Recently, a couple of articles suggested the possibility that apoptosis of pancreatic ß-cells induces inflammatory/immune responses to ß-cells. Such a theory is based on the assumption that apoptotic cells can, under certain circumstances, induce immune responses, inflammatory and autoimmune disorders, which is in contrast to the dogma that apoptotic cells result in immunosuppression and necrotic cells provoke inflammation/immunity. We observed that late apoptotic ß-cells with secondary necrosis elicited inflammatory responses in macrophages through the toll-like receptor 2 (TLR2)/MyD88/nuclear factor-κB signalling pathway. Late apoptotic cells also induced TLR2-dependent maturation of dendritic cells and then activation of autoreactive T-cells. TLR2 knockout mice showed defective priming of diabetogenic T-cells by apoptotic ß-cells in the pancreatic lymph nodes. Furthermore, TLR2 deficiency conferred a significant protection against type 1 diabetes (T1D) and insulitis in T1D animal models. These findings present evidence suggesting that apoptosis of pancreatic ß-cells could be one of the initial events in T1D and provide a novel strategy for therapeutic or preventive intervention in T1D.

Inhibition of autoimmune diabetes by TLR2 tolerance.

We have reported that apoptotic ß cells undergoing secondary necrosis, called "late apoptotic (LA) ß cells," stimulated APCs and induced diabetogenic T cell priming through TLR2, which might be one of the initial events in autoimmune diabetes. Indeed, diabetogenic T cell priming and the development of autoimmune diabetes were significantly inhibited in TLR2-null NOD mice, suggesting the possibility that TLR2 blockade could be used to inhibit autoimmune diabetes. Because prolonged TLR stimulation can induce TLR tolerance, we investigated whether repeated TLR2 administration affects responses to LA ß cells and inhibits autoimmune diabetes in NOD mice by inducing TLR2 tolerance. Treatment of primary peritoneal macrophages with a TLR2 agonist, Pam3CSK(4), suppressed cytokine release in response to LA insulinoma cells or further TLR2 stimulation. The expression of signal transducer IRAK-1 and -4 proteins was decreased by repeated TLR2 stimulation, whereas expression of IRAK-M, an inhibitory signal transducer, was enhanced. Chronic Pam3CSK(4) administration inhibited the development of diabetes in NOD mice. Diabetogenic T cell priming by dendritic cells and upregulation of costimulatory molecules on dendritic cells by in vitro stimulation were attenuated by Pam3CSK(4) administration in vivo. Pam3CSK(4) inhibited diabetes after adoptive transfer of diabetogenic T cells or recurrence of diabetes after islet transplantation by pre-existing sensitized T cells. These results showed that TLR2 tolerance can be achieved by prolonged treatment with TLR2 agonists, which could inhibit priming of naive T cells, as well as the activity of sensitized T cells. TLR2 modulation could be used as a novel therapeutic modality against autoimmune diabetes.

A possible mechanism of impaired NK cytotoxicity in cancer patients: down-regulation of DAP10 by TGF-beta1.

AIMS AND BACKGROUND: Elevated TGF-BETA1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients. However, the molecular mechanism of immunosuppression by TGF-BETA1 is not yet clarified. METHODS: IL-2-activated human NK cells were cultured with TGF-BETA1. Protein levels of NKG2D and DAP10 were examined by FACS or immunoblot analyses. Real-time RTPCR was performed to quantify the transcription levels. MAPK inhibitors were used to investigate intracellular signaling. RESULTS: TGF-BETA1 down-regulated total and surface NKG2D, which was partially dependent on transcriptional regulation. TGF-BETA1 treatment of human NK cells resulted in significant changes in both transcriptional and translational levels of DAP10. Moreover, treatment with bafilomycin A1 or folimycin restored total NKG2D levels in TGF-BETA1-treated NK cells. The impaired NKG2D down-modulation by TGF-BETA1 was not associated with activation of the MAPK signaling pathway. CONCLUSIONS: TGF-BETA1 down-modulates surface NKG2D expression by controlling the transcriptional and translational levels of DAP10.

Presence of membrane-bound TGF-beta1 and its regulation by IL-2-activated immune cell-derived IFN-gamma in head and neck squamous cell carcinoma cell lines.

The presence of membrane-bound TGF-beta1 (mTGF-beta1) has been recently observed in regulatory T cells, but only a few studies have reported the same phenomenon in cancer cells. In this study, we investigate the regulation of mTGF-beta1 expression in five head and neck squamous cell carcinoma cell lines using FACS analysis. Through blocking Ab and exogenous cytokine treatment experiments, we found that expression of mTGF-beta1 is significantly induced by the activated immune cell-derived factor IFN-gamma. In addition, IFN-gamma and TNF-alpha are shown to have a synergistic effect on mTGF-beta1 expression. Moreover, we found that exogenous TNF-alpha induces endogenous TNF-alpha mRNA expression in an autocrine loop. In contrast to previous reports, we confirm that, in this model, mTGF-beta1 is neither a rebound form of once-secreted TGF-beta1 nor an activated form of its precursor membrane latency-associated peptide. Inhibitors of transcription (actinomycin D), translation (cycloheximide), or membrane translocation (brefeldin A) effectively block the induction of mTGF-beta1, which suggests that induction of mTGF-beta1 by IFN-gamma and/or TNF-alpha occurs through de novo synthesis. These findings suggest that some cancer cells can detect immune activating cytokines, such as IFN-gamma and TNF-alpha, and actively block antitumor immunity by induction of mTGF-beta1.

Association of CD47 with natural killer cell-mediated cytotoxicity of head-and-neck squamous cell carcinoma lines.

OBJECTIVE: Integrin-associated protein (CD47) binds specifically to the inhibitory receptor signal-regulatory protein. This study was designed to evaluate the role of CD47 in natural killer (NK) cell-mediated cytotoxicity against cancer cells. METHODS: Head-and-neck squamous cell carcinoma (HNSCC) cell lines were analyzed for the expression of CD47 and susceptibility to NK cell-mediated killing. Cytolytic activity was assessed by (51)Cr-specific release assays and by measuring cytokine production. RESULTS: HNSCC cell lines that had high CD47 expression showed lower levels of NK cytotoxicity than those with low CD47 expression. After pre-treating cells with neutralizing major histocompatibility complex (MHC) class I or anti-CD47 antibodies, NK cell-mediated cytotoxicity against HNSCC cell lines increased. In addition, when CD47 cDNA was transfected into Caco-2 cells, NK cell-mediated cytotoxicity decreased. CONCLUSION: These findings suggest that CD47 may play an inhibitory role in NK cell-mediated cytotoxicity against cancer cells, implying a possible mechanism of immune escape in human cancer.

Elevated TGF-beta1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients.

NK cell function in cancer patients is severely impaired, but the mechanism underlying this impairment is not clearly understood. In this study we show evidence that TGF-beta1 secreted by tumors is responsible for the poor NK lytic activity via down-regulating an NK-activating receptor, NKG2D. The plasma level of TGF-beta1 in human lung cancer or colorectal cancer patients was elevated compared with that in normal volunteers, and this elevation was inversely correlated with surface expression of NKG2D on NK cells in these patients. Incubation of NK cells with plasma obtained from cancer patients specifically down-modulated surface NKG2D expression, whereas addition of neutralizing anti-TGF-beta1 mAbs completely restored surface NKG2D expression. Likewise, incubation of NK cells and lymphokine-activated killer cells with TGF-beta1 resulted in dramatic reduction of surface NKG2D expression associated with impaired NK cytotoxicity. Modulation of NKG2D by TGF-beta1 was specific, as expression of other NK receptors, CD94/NKG2A, CD44, CD16, 2B4, or CD56, was not affected by TGF-beta1. Impaired NK cytotoxicity by TGF-beta1 was not due to alteration of lytic moieties, such as perforin or Fas, or apoptotic pathway, but, rather, appeared to be due to lack of NKG2D expression. Taken together, our data suggest that impaired NK function in cancer patients can be attributed to down-modulation of activating receptors, such as NKG2D, via secretion of TGF-beta1.

YC-1: a potential anticancer drug targeting hypoxia-inducible factor 1.

BACKGROUND: Hypoxia-inducible factor 1 alpha (HIF-1alpha), a component of HIF-1, is expressed in human tumors and renders cells able to survive and grow under hypoxic (low-oxygen) conditions. YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, an agent developed for circulatory disorders that inhibits platelet aggregation and vascular contraction, inhibits HIF-1 activity in vitro. We tested whether YC-1 inhibits HIF-1 and tumor growth in vivo. METHODS: Hep3B hepatoma, NCI-H87 stomach carcinoma, Caki-1 renal carcinoma, SiHa cervical carcinoma, and SK-N-MC neuroblastoma cells were grown as xenografts in immunodeficient mice (69 mice total). After the tumors were 100-150 mm(3), mice received daily intraperitoneal injections of vehicle or YC-1 (30 microg/g) for 2 weeks. HIF-1 alpha protein levels and vascularity in tumors were assessed by immunohistochemistry, and the expression of HIF-1-inducible genes (vascular endothelial growth factor, aldolase, and enolase) was assessed by reverse transcription-polymerase chain reaction. All statistical tests were two-sided. RESULTS: Compared with tumors from vehicle-treated mice, tumors from YC-1-treated mice were statistically significantly smaller (P<.01 for all comparisons), expressed lower levels of HIF-1 alpha (P<.01 for all comparisons), were less vascularized (P<.01 for all comparisons), and expressed lower levels of HIF-1-inducible genes, regardless of tumor type. CONCLUSIONS: The inhibition of HIF-1 alpha activity in tumors from YC-1-treated mice is associated with blocked angiogenesis and an inhibition of tumor growth. YC-1 has the potential to become the first antiangiogenic anticancer agent to target HIF-1 alpha.

Reduced expression of coxsackievirus and adenovirus receptor (CAR) in tumor tissue compared to normal epithelium in head and neck squamous cell carcinoma patients.

Even though adenoviral vector is widely used in gene therapy of squamous cell carcinoma of the head and neck (SCCHN), the expression level of Coxsackievirus and adenovirus receptor (CAR) in SCCNH is not clearly defined. To identify this variability, the expression of CAR was measured using SCCHN cell lines and compared with transfection efficiency. It was found by RT-PCR and Western blot analysis that CAR levels varied in SCCHN cell lines. FACS analysis and adenovirus infection assay revealed that there was a good correlation between the level of CAR expression and the transfection efficiency. To identify the actual CAR expression patterns of human SCCHN tissues in vivo, immunohistochemical staining was undertaken on frozen biopsies of six SCCHN patients. In all the patients examined, the normal tissues showed much stronger staining for CAR than the tumor tissues. These results demonstrate that the level of CAR expression of a tumor should be evaluated before clinical application of adenoviral vector for gene therapy in SCCHN.