Integrin ß3-Mediated Cell Senescence Associates with Gut Inflammation and Intestinal Degeneration in Models of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by memory loss and personality changes that ultimately lead to dementia. Currently, 50 million people worldwide suffer from dementia related to AD, and the pathogenesis underlying AD pathology and cognitive decline is unknown. While AD is primarily a neurological disease of the brain, individuals with AD often experience intestinal disorders, and gut abnormalities have been implicated as a major risk factor in the development of AD and relevant dementia. However, the mechanisms that mediate gut injury and contribute to the vicious cycle between gut abnormalities and brain injury in AD remain unknown. In the present study, a bioinformatics analysis was performed on the proteomics data of variously aged AD mouse colon tissues. We found that levels of integrin ß3 and ß-galactosidase (ß-gal), two markers of cellular senescence, increased with age in the colonic tissue of mice with AD. The advanced artificial intelligence (AI)-based prediction of AD risk also demonstrated the association between integrin ß3 and ß-gal and AD phenotypes. Moreover, we showed that elevated integrin ß3 levels were accompanied by senescence phenotypes and immune cell accumulation in AD mouse colonic tissue. Further, integrin ß3 genetic downregulation abolished upregulated senescence markers and inflammatory responses in colonic epithelial cells in conditions associated with AD. We provide a new understanding of the molecular actions underpinning inflammatory responses during AD and suggest integrin ß3 may function as novel target mediating gut abnormalities in this disease.

Vγ6+ γδ T cells are critical for protection against infection by Escherichia coli in mice.

γδ T cells producing IL-17A (γδTh17 cells) are known to be involved in peritonitis induced by Escherichia coli infection in mice. In vivo treatment with Vγ6-specific mAb (1C10-1F7) significantly hampered resolution of E. coli infection. Thus, Vγ6+ γδTh17 cells mainly contributed to protection against E. coli infection.

Nitric Oxide Is Involved in Activation of Toll-Like Receptor 4 Signaling through Tyrosine Nitration of Src Homology Protein Tyrosine Phosphatase 2 in Murine Dextran Sulfate-Induced Colitis.

Ulcerative colitis is characterized by colonic mucosal bleeding and ulceration, often with repeated active and remission stages. One factor in ulcerative colitis development is increased susceptibility to commensal bacteria and lipopolysaccharide (LPS). LPS activates macrophages to release nitric oxide (NO) through Toll-like receptor 4 (TLR4) signaling. However, whether NO is beneficial or detrimental to colitis remains controversial. In this study, we investigated whether NO enhances the development of colitis in mice treated with dextran sulfate sodium (DSS) and inflammation in cells treated with low-dose LPS. An NO donor, NOC18, induced colitis and increased CD14 protein and nitrotyrosine levels in colonic macrophages from mice treated with DSS for 7 d (molecular weight: 5000). In the mouse peritoneal macrophage cell line RAW264.7 stimulated with 3 ng/mL LPS, NO activated the CD14-TLR4-nuclear factor kappa B (NF-κB) axis. Low-dose LPS stimulation did not change the levels of signal transducer and activator of transcription (STAT) 3 phosphorylation, CD14, inducible NO synthase, interleukin (IL)-6, or NF-κB. In addition, low-dose LPS increased phosphorylation of src homology protein tyrosine phosphatase 2 (SHP2), a negative regulator of STAT3 phosphorylation. However, NO decreased SHP2 phosphorylation and significantly activated the downstream signaling molecules. NO increased SHP2 nitration in LPS-stimulated RAW264.7 cells and DSS-treated mice. These results indicate that SHP2 nitration in macrophages might be involved in activation of the CD14-TLR4-NF-κB axis through STAT3 signaling in mice with DSS-induced colitis.

In vivo redox imaging of dextran sodium sulfate-induced colitis in mice using Overhauser-enhanced magnetic resonance imaging.

In ulcerative colitis, an inflammatory bowel disease of unknown cause, diagnosis of the degree and location of colitis at an early stage is required to control the symptoms. Changes in redox status, including the production of reactive oxygen and nitrogen species (RONS), have been associated with ulcerative colitis in humans and dextran sodium sulfate (DSS)-induced colitis in rodents. In this study, the in vivo redox status of colons of DSS-induced colitis mice were monitored by Overhauser-enhanced magnetic resonance imaging (OMRI), and the relationship between redox status and colitis development was investigated. Colitis was induced by administering 5% DSS in drinking water to male Slc:ICR mice, which are a strain classified as closed colony outbred mice (5-week-old, 25-30 g). On the 3rd day of the DSS challenge, when no symptoms of colitis were displayed, the contrast decays of 15N-CmP and 14N-CxP tended to show enhancement in the whole colon and were not altered by DMSO. On the 5th day of the DSS challenge, with histological damage of the rectum being displayed, the contrast decay of 15N-CmP was significantly enhanced not only in the rectum, but also in the proximal colon, and this was suppressed by DMSO. On the 7th day of the DSS challenge, with the mice displaying severe colitis symptoms, the image contrasts of 15N-labeled 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (15N-CmP) and 14N-labeled 3-carboxyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (14N-CxP) showed much faster decay than those of healthy mice, while the increased decays of both probes were restored by the membrane-permeable reactive oxygen species (ROS) scavenger dimethyl sulfoxide (DMSO). Image differencing between the decay rate images of 15N-CmP and 14N-CxP showed the DSS-induced redox imbalance spreading over the whole colon, and a histogram of the difference image showed a smaller peak and broader distribution with the DSS treatment. These data indicate that ROS are produced intracellularly in the distal and proximal colon in the initiation stage of DSS-induced colitis, and that ROS are produced intracellularly and extracellularly in the advanced stage of DSS-induced colitis.

In Vivo Imaging of the Intra- and Extracellular Redox Status in Rat Stomach with Indomethacin-Induced Gastric Ulcers Using Overhauser-Enhanced Magnetic Resonance Imaging.

AIMS: Repeated use of nonsteroidal anti-inflammatory drugs can induce changes in the redox status, including production of reactive oxygen species (ROS), but the specific details of these changes remain unknown. Overhauser-enhanced magnetic resonance imaging (OMRI) has been used in vivo to monitor the redox status in several diseases and map tissue oxygen concentrations. We monitored the intra- and extracellular redox status in the stomach of rats with indomethacin-induced gastric ulcers using OMRI and investigated the relationship with gastric mucosal damage. RESULTS: One hour after oral administration of indomethacin (30 mg/kg), OMRI measurements in the stomach were made following nitroxyl probe administration. OMRI with the membrane-permeable nitroxyl probe, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPOL), demonstrated a redox change toward oxidation, which was reversed by a membrane-permeable antioxidant. Conversely, imaging with the impermeable probe, 4-trimethylammonium-2,2,6,6-tetramethyl-piperidine-1-oxyl (CAT-1), demonstrated little redox change. Redox imbalance imaging of a live rat stomach with indomethacin-induced gastric ulcers was produced by dual imaging of 15N-labeled TEMPOL and 14N-labeled CAT-1, in addition to imaging with another membrane-permeable 15N-labeled probe, 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL), and 14N-labeled CAT-1. Pretreatment with MC-PROXYL suppressed gastric mucosal damage, whereas pretreatment with CAT-1 did not suppress ulcer formation. INNOVATION: OMRI combined with a dual probe is a less invasive imaging technique for evaluation of intracellular ROS production contributing to the formation of gastric ulcers in the stomach of indomethacin-treated rats, which cannot be done with other methods. CONCLUSION: This method may be a very powerful tool for characterizing the pathogenesis of various diseases and may have medical applications.

Development of a new monoclonal antibody specific to mouse Vγ6 chain.

There are seven Vγ gene segments in the TCR γ chain loci of mice. We developed monoclonal antibodies (mAbs) specific to the Vγ6 chain (Heilig & Tonegawa nomenclature). By immunizing Vγ4/6 KO mice with complementarity-determining region peptides in Vγ6 chains, we generated three hybridomas. These hybridomas produced mAbs capable of cell surface staining of Vγ6/Vδ1 gene-transfected T-cell line lacking TCR as well as of Vγ1- Vγ4- Vγ5- Vγ7- γδ T cells and the CD3high TCRδint γδ T cells in various organs. The location of Vγ6+ γδ T cells, which peaked in the newborn thymus, was associated with mTEC. In vivo administration of clone 1C10-1F7 mAb impaired protection against Klebsiella pneumoniae infection but ameliorated psoriasis-like dermatitis induced by imiquimod treatment. These new mAbs are useful to elucidate the development, location, and functions of Vγ6 γδ T cells in mice.

S100A4 Protein Is Essential for the Development of Mature Microfold Cells in Peyer's Patches.

Intestinal microfold cells (M cells) in Peyer's patches are a special subset of epithelial cells that initiate mucosal immune responses through uptake of luminal antigens. Although the cytokine receptor activator of nuclear factor-κB ligand (RANKL) expressed on mesenchymal cells triggers differentiation into M cells, other environmental cues remain unknown. Here, we show that the metastasis-promoting protein S100A4 is required for development of mature M cells. S100A4-producing cells are a heterogenous cell population including lysozyme-expressing dendritic cells and group 3 innate lymphoid cells. We found that in the absence of DOCK8, a Cdc42 activator critical for interstitial leukocyte migration, S100A4-producing cells are reduced in the subepithelial dome, resulting in a maturation defect of M cells. While S100A4 promotes differentiation into mature M cells in organoid culture, genetic inactivation of S100a4 prevents the development of mature M cells in mice. Thus, S100A4 is a key environmental cue that regulates M cell differentiation in collaboration with RANKL.

Involvement of nitric oxide with activation of Toll-like receptor 4 signaling in mice with dextran sodium sulfate-induced colitis.

Ulcerative colitis is an inflammatory bowel disease characterized by acute inflammation, ulceration, and bleeding of the colonic mucosa. Its cause remains unknown. Increases in adhesion molecules in vascular endothelium, and activated neutrophils releasing injurious molecules such as reactive oxygen species, are reportedly associated with the pathogenesis of dextran sodium sulfate (DSS)-induced colitis. Nitric oxide (NO) production derived from inducible NO synthase (iNOS) via activation of nuclear factor κB (NF-κB) has been reported. It is also reported that stimulation of Toll-like receptor 4 (TLR4) by lipopolysaccharide can activate NF-κB. In this study, we investigated the involvement of NO production in activation of the TLR4/NF-κB signaling pathway in mice with DSS-induced colitis. The addition of 5% DSS to the drinking water of male ICR mice resulted in increases in TLR4 protein in colon tissue and NF-κB p65 subunit in the nuclear fraction on day 3, increases in colonic tumor necrosis factor-α on day 4, and increases in P-selectin, intercellular adhesion molecule-1, NO2(-)/NO3(-), and nitrotyrosine in colonic mucosa on day 5. These activated inflammatory mediators and pathology of colitis were completely suppressed by treatment with a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, as well as an iNOS inhibitor, aminoguanidine. Conversely, a NO-releasing compound, NOC-18, increased TLR4 levels and nuclear translocation of NF-κB p65 and exacerbated mucosal damage induced by DSS challenge. These data suggest that increases in TLR4 expression induced by drinking DSS-treated water might be directly or indirectly associated with NO overproduction.

The detrimental effect of nitric oxide on tissue is associated with inflammatory events in the vascular endothelium and neutrophils in mice with dextran sodium sulfate-induced colitis.

Nitric oxide (NO) is thought to be a key molecule in the progression of ulcerative colitis and experimental colitis induced by dextran sodium sulfate (DSS). However, the detrimental effect of DSS-induced NO production on the colonic mucosa is incompletely understood. Increases in the expression of adhesion molecules in the vascular endothelium and activated neutrophils (thereby releasing injurious molecules such as reactive oxygen species) are reportedly associated with the pathogenesis of DSS-induced colitis. We investigated if the detrimental effect of NO production on the colonic mucosa was attributable to the activation of neutrophil infiltration by NO in mice with DSS-induced colitis. NO(2)(-)/NO(3)(-) content in the middle and distal colon was increased on days 5 and 7, but alterations in the proximal colon were not observed. Myeloperoxidase (MPO) activity and expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) were significantly increased in the entire colon, whereas TNF-α levels were significantly increased only in the middle and distal colon on day 7. The pathology of colitis and increases in colonic MPO activity, P-selectin, ICAM-1, and TNF-α levels were suppressed by the inducible NO synthase (iNOS)-specific inhibitor aminoguanidine and NO scavenger c-PTIO, whereas all but TNF-α levels were increased by the non-specific NOS inhibitor L-NAME. These findings suggest that iNOS-derived NO increases TNF-α levels in the middle and distal colon and increased TNF-α levels induce expression of P-selectin and ICAM-1, thereby promoting the infiltration of activated neutrophils, which leads to damage to colonic tissue.