Gene Expression Levels Of Cytokines Research Articles

Effects of low ethanol consumption on nonalcoholic steatohepatitis in mice.

Several recent clinical and epidemiological studies have suggested inhibitory effects of light-to-moderate alcohol consumption on nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH); however, these effects have not been confirmed in experimental studies. Therefore, in this study, we examined the effects of small amounts of ethanol consumption on a mouse model of NASH. Nine-week-old male obese mice (db/db mice) were divided into the following groups: control, high-fat, and low-ethanol groups. The control group was provided ad libitum access to a control liquid diet, the high-fat group was provided access to a high-fat liquid diet, and the low-ethanol group was provided access to the high-fat liquid diet supplemented with 0.1% (w/w) ethanol. Eight weeks later, the mice were sacrificed and serum biochemical, histopathological, and molecular analyses were performed. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were significantly lower in the low-ethanol group than in the high-fat group (p=0.033 and 0.037, respectively). Liver histopathological analysis showed that intralobular and portal inflammation was significantly milder in the low-ethanol group than in the high-fat group (p=0.018 and 0.041, respectively). However, no significant differences were observed among the groups in serum insulin and adiponectin levels, hepatic 4-hydroxynonenal (oxidative injury marker) levels, and hepatic cytokine and receptor gene expression levels. In conclusion, the serum transaminase levels and hepatic inflammation in NASH model mice improved after administration of small amounts of ethanol. This study directly demonstrated inhibitory effects of small amounts of ethanol on NASH in mice. The mechanisms underlying these inhibitory effects remain to be elucidated.

Phosphoinositide 3-kinase γ deficiency attenuates kidney injury and fibrosis in angiotensin II-induced hypertension.

We have shown that the CXCL16/CXCR6 axis plays a critical role in recruiting inflammatory cells and bone marrow-derived fibroblasts into the kidney leading to renal injury and fibrosis. However, the underlying signaling mechanisms are not known. In the present study, we examined the role of phosphoinositide-3 kinase γ (PI3Kγ) signaling in the recruitment of inflammatory cells and bone marrow-derived fibroblasts into the kidney and development of renal injury and fibrosis in an experimental model of hypertension induced by angiotensin II. Blood pressure was comparable between wild-type (WT) and PI3Kγ knockout (KO) mice at baseline. Angiotensin II treatment led to an increase in blood pressure that was similar between WT and PI3Kγ KO mice. Compared with WT mice, PI3Kγ KO mice were protected from angiotensin II-induced renal dysfunction and injury and developed less proteinuria. PI3Kγ deficiency suppressed bone marrow-derived fibroblast accumulation and myofibroblast formation in the kidney and inhibited total collagen deposition and extracellular matrix protein production in the kidney in response to angiotensin II. PI3Kγ deficiency inhibited the infiltration of F4/80+ macrophages and CD3+ T cells into the kidney and reduced gene expression levels of pro-inflammatory cytokines in the kidney following angiotensin II treatment. Finally, inhibition of PI3Kγ suppressed CXCL16-induced monocyte migration in vitro. These results indicate that PI3Kγ mediates the influx of macrophages, T cells and bone marrow-derived fibroblasts into the kidney resulting in kidney injury and fibrosis.

463-P: Deterioration of Atherosclerosis via Dysbiosis in ApoE Null Mice Fed with High-Salt Diet

Recent studies reported that the impairment of gut microbiota (Dysbiosis) cause impairments of gut metabolisms, which induce chronic inflammation in the body. High salt intake not only increases blood pressure, but also causes Dysbiosis. However, the detailed mechanism has not been clarified yet. Here we investigate the mechanism of the atherosclerosis progression through dysbiosis caused by high salt diet. 16-week-old male Apolipoprotein E-deficient (ApoE-/-) mice were fed normal breeding diet (ND group), high-fat high-sucrose diet (HFHSD group), and high-fat high-sucrose high-salt diet (HFHSDNaCl8%, high salt group) for 8 weeks. The concentration of salt in ND and HFHSD is set as 2%. Immune cells in the aorta were extracted and evaluated by FACS CANTII. The metagenome was evaluated by the 16S target sequencing method. Lipidome in the aorta, serum, or intestinal contents were evaluated and quantified by GCMS system (Agilent 7890B/5977B). In high salt group, gene expression levels of inflammatory cytokines and chemokines were upregulated, and M1 macrophages and Group 2 innate lymphoid cells in atherosclerotic lesions increased. The prevalence of Firmicutes decreased and the prevalence of Bacteroidetes increased. Lipidome in the aorta, serum and intestinal contents were significantly different among the HFHSD group and high salt group, although the lipids administered were the same. High salt intake increases M1 macrophages and Group 2 innate lymphoid cells by altering intestinal bacterial lipid metabolisms, and could deteriorate chronic inflammation of arteriosclerosis. Disclosure T. Kimura: None. Y. Hashimoto: None. T. Senmaru: None. E. Ushigome: None. M. Hamaguchi: None. M. Yamazaki: None. M. Fukui: None.

Anthocyanin-Containing Purple Potatoes Ameliorate DSS-Induced Colitis in Mice

Abstract Objectives Ulcerative colitis (UC), a major form of inflammatory bowel disease (IBD), is on the rise worldwide. Approximately 3 million people suffer from IBD in the US alone, but the current therapeutic options (e.g., corticosteroids) come with adverse side effects including reduced ability to fight infections. Thus, there is a critical need for developing effective, safe and evidence-based food products with anti-inflammatory activity. This study aimed to evaluate the anti-inflammatory potential of purple-fleshed potato anthocyanins using a dextran sodium sulfate (DSS) murine model of colitis. Methods Four weeks old male C57BL/6 mice were randomly assigned to control (AIN-93 G diet), P15 (15% purple-fleshed potato) and P25 (25% purple-fleshed potato) diets for eight weeks. Colitis was induced by 2% DSS administration in drinking water for six days. Intestinal permeability was measured using FITC-dextran. Serum and colonic myeloperoxidase (MPO) levels were measured using ELISA and hexadecyltrimethylammonium bromide method, respectively. Flagellin was quantified using human embryonic kidney (HEK)-Blue-mTLR5 cells. RT-PCR was used to analyze the relative gene expression levels of cytokines and pathogenic bacteria abundance. Results 1) Purple- fleshed potato supplementation suppressed (P ≤ 0.05) the DSS-induced reduction in body weight and colon length as well as the increase in spleen and liver weight. 2) P25 alone suppressed (P ≤ 0.05) the elevation in the intestinal permeability and serum MPO levels in DSS-exposed mice. 3) The colonic mRNA expression levels of pro-inflammatory cytokines IL-6, IL-17 and TNF-α were attenuated in mice consuming P25 diet. 4) However, both purple potato diets suppressed the relative abundance of specific pathogenic bacteria such as Enterobacteriaceae and pks + E. coli. However, flagellin levels were significantly (P ≤ 0.05) lower in mice consuming the P25 diet alone. Conclusions Purple-fleshed potato diet consumption dose-dependently ameliorated the DSS-induced elevation in gut permeability, oxidative stress and inflammation and pathogenic gut bacteria. Collectively, our study results suggest that purple-fleshed potatoes have the potential to be used as a functional food in the amelioration of UC symptoms. Funding Sources USDA-NIFA awards 2016-67,017-29,285 and 2019-67,017-29,258.

Role of Gut Microbiota in Anti-Colitic Effects of Color-Fleshed Potatoes

Abstract Objectives The prevalence of ulcerative colitis (UC), a chronic inflammatory bowel disease, is on the rise with ∼700,000 patients in the US alone in 2018. Gut bacterial dysbiosis plays an important role in ulcerative colitis. We have recently shown that anthocyanin-containing potatoes exert anti-inflammatory activity in colitic mice. However, no information is available on whether gut bacteria play a role in the anti-colitic activity of color-fleshed potatoes. This study examined the anti-colitic activity of red/purple-fleshed potatoes in mice with intact and antibiotic-ablated microbiome. Methods We used DSS-induced murine (C57BL6) colitis model with and without the administration of antibiotics in drinking water for nine weeks. Mice were randomly assigned to the control (AIN-93G diet), DSS (AIN-93G diet), DSS + PP (20% purple potato) and DSS + RP (20% red potato) groups. After eight weeks, mice were treated with 2% DSS in their drinking water for five days to induce colitis. Intestinal permeability was measured using FITC-dextran. Serum myeloperoxidase (MPO) levels were measured using ELISA. RT-PCR was used to analyze the relative gene expression levels of cytokines and bacterial abundance. Results Administration of antibiotics resulted in a 95% reduction in gut bacterial load. Antibiotics administration did not alter food intake, water intake, and weight gain. Antibiotic-treated mice had five times greater cecum weight, a hallmark of germ-free mice, compared to no-antibiotic mice. In antibiotic mice, DSS-induced splenomegaly, elevated gut permeability (serum levels of FITC-dextran), and reduced colon length and weight were more pronounced compared to no antibiotic mice. Purple- or red-fleshed potato supplementation (20% w/w) ameliorated (P ≤ 0.05) DSS-induced reduction in colon length, elevation in permeability, spleen weight and MPO levels in no antibiotic mice only. Moreover, purple-fleshed potato supplementation alone improved the ZO-1 and MUC-2 gene expression levels in no-antibiotic mice, but not in microbiota-ablated mice. Conclusions In summary, these results suggest that purple-fleshed potatoes are more potent compared to red-fleshed potatoes and the gut microbiome is critical for the anti-colitic activity of anthocyanin-containing potatoes. Funding Sources USDA-NIFA awards 2016-67,017-29,285 and 2019-67,017-29,258.

Decreasing of S100A4 in bovine endometritis in vivo and in vitro

Endometritis is a prevalent reproductive disease in dairy cows, and is a superficial inflammation of the endometrium. S100 calcium-binding protein A4 (S100A4) is suggested to be implicated in the progression of inflammation. However, to our knowledge, no study has reported the changes of S100A4 during bovine endometritis. The objective of this study was to investigate S100A4 gene expression and protein levels in the uterus with endometritis in dairy cows. Vaginal mucus samples were collected for diagnosis of the severity degree of endometritis and the detection of S100A4 protein content. Blood samples and endometrial biopsies were collected and divided into the control (CN), mild endometrtis (M), and severe endometritis (S) groups according to the characteristics of the vaginal mucus type. The isolated bovine endometrial epithelial cells (BEECs) were challenged with E. coli (2 × 106 CFU/mL, 2 × 107 CFU/mL) or lipopolysaccharide (LPS, 3 and 10 μg/mL) as an inflammatory model. RT-qPCR was used to detect the gene expression levels of S100A4 and cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumour necrosis factor-alpha (TNF-α), in tissues or cells. Enzyme-linked immunosorbent assay (ELISA) was used for S100A4 protein level detection in tissues, cells, cell supernatant, vaginal mucus, and serum samples. The results showed that S100A4 gene and protein levels decreased in bovine endometrium with endometritis and in E. coli- or LPS-stimulated BEECs. We failed to detect S100A4 in the cell supernatant, vaginal mucus, and serum samples. This study suggested that S100A4 is a pathogenesis-related protein of endometritis, and decreased expression of S100A4 may pave the way for the development of endometritis in dairy cows.

Bioactive Components of Polyphenol-Rich and Non-Polyphenol-Rich Cranberry Fruit Extracts and Their Chemopreventive Effects on Colitis-Associated Colon Cancer.

Cranberries contain various constituents relevant to human health. Our previous study demonstrated the chemopreventive effects of whole cranberry against colon cancer in mice. In order to determine the role of different cranberry secondary metabolites in inhibiting colon cancer, cranberry ethyl acetate extract (EAE) and polyphenol extract (PPE) were obtained. The free-radical scavenging activities and chemical composition of the cranberry extracts were determined. EAE consisted of triterpenes and sterols and a trace amount of proanthocyanidins. PPE mainly contained polyphenol with a trace amount of triterpenes. The chemopreventive effects of orally administered EAE and PPE on colitis-associated colon carcinogenesis were determined in mice. Dietary EAE and PPE significantly suppressed tumor metrics without noticeable adverse effects. Gene expression levels of key proinflammatory cytokines were also attenuated by EAE and PPE in the mouse colon. In conclusion, the novel cranberry extracts may offer an efficacious and safe means to prevent colonic tumorigenesis in humans.

Baicalin alleviates deoxynivalenol-induced intestinal inflammation and oxidative stress damage by inhibiting NF-κB and increasing mTOR signaling pathways in piglets

This study was conducted to evaluate the intestinal protective effects of baicalin (BAI) in deoxynivalenol (DON)-treated piglets. A total of 320 weaned piglets were randomly allotted to 1 of 4 treatments with 8 replication pens per treatment and 10 piglets per pen. The treatments were basal diet (NC), basal diet + 0.1% baicalin (BAI), basal diet + 4 mg/kg DON (DON), and basal diet + 4 mg/kg DON + 0.1% BAI (DON + BAI). The experiment was conducted for 14 days. BAI supplementation alleviated the impairment of growth performance and the disorder of serum biochemical parameters in DON-challenged piglets. BAI supplementation also alleviated DON-induced negative effects, decreasing protein and gene expression levels of pro-inflammatory cytokines in the serum and intestinal tissue and increasing antioxidant capacity in the serum. BAI increased villus height and villus height/crypt depth but decreased the protein expression levels of nuclear factor kappa B (NF-κB), as determined by immunohistochemical analysis, in the ileum and jejunum. Moreover, we found that BAI inhibited NF-κB and increased mTOR protein and gene expression levels in the serum and intestinal tissues. Collectively, BAI alleviates intestinal inflammatory and oxidative damage by inhibiting NF-κB and increasing mTOR signaling to modulate downstream inflammatory and oxidative responses after DON challenge.

Trehalose Does Not Improve Alcohol‐Impaired TFEB and Liver Injury in Mice

Alcoholic liver disease (ALD) is one of the major causes of chronic liver disease worldwide. We previously demonstrated that chronic plus acute alcohol binge (“Gao‐binge”) impaired hepatic transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, resulted in insufficient autophagy that contributes to Gao‐binge alcohol‐induced liver injury in mice. Trehalose, a disaccharide, has been shown to activate TFEB and protects against diet‐induced non‐alcoholic fatty liver disease in mice. The aim of the present study was to investigate whether trehalose would reverse impaired TFEB and insufficient autophagy induced by Gao‐binge alcohol and in turn protect against Gao‐binge alcohol‐induced liver injury. We found that trehalose increased TFEB nuclear translocation, elevated levels of LC3‐II and lysosomal proteins in cultured AML12 cells and in mouse livers, confirming the activation of TFEB by trehalose. Surprisingly, trehalose failed to activate TFEB in Gao‐binge alcohol‐treated mouse livers. As a result, trehalose failed to protect against Gao‐binge alcohol‐induced liver injury and steatosis. However, Gao‐binge alcohol increased infiltration of hepatic neutrophils and levels of inflammatory cytokine gene expression, which were attenuated by trehalose, suggesting trehalose inhibits alcohol‐induced inflammation independent of TFEB activation. In conclusion, our results indicate that trehalose fails to improve the impaired TFEB and liver injury but ameliorates hepatic inflammation induced by Gao‐binge alcohol.Support or Funding InformationNIH R01 AA020518, R01 DK102142, U01 AA024733 AND P30GM118247 (W.X.D).

Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression.

The role of thioredoxin-1 (TRX), a small redox-active protein with antioxidant effects, during hyperoxic lung injury in newborns remains undetermined. We investigated TRX impact on hyperoxic lung injury in newborn TRX transgenic (TRX-Tg) and wildtype (WT) mice exposed to 21% or 95% O2 for four days, after which some mice were allowed to recover in room air for up to 14 days. Lung morphology was assessed by hematoxylin/eosin and elastin staining, as well as immunostaining for macrophages. The gene expression levels of proinflammatory cytokines were evaluated using quantitative real-time polymerase chain reaction. During recovery from hyperoxia, TRX-Tg mice exhibited an improved mean linear intercept length and increased number of secondary septa in lungs compared with the WT mice. Neonatal hyperoxia enhanced the mRNA expression levels of proinflammatory cytokines in the lungs of both TRX-Tg and WT mice. However, interleukin-6, monocyte chemoattractant protein-1, and chemokine (C-X-C motif) ligand 2 mRNA expression levels were reduced in the lungs of TRX-Tg mice compared with the WT mice during recovery from hyperoxia. Furthermore, TRX-Tg mice exhibited reduced macrophage infiltration in lungs during recovery. These results suggest that in newborn mice TRX ameliorates hyperoxic lung injury during recovery likely through the suppression of proinflammatory cytokines.

Androgen Suppresses Hyperosmolarity-Induced Inflammatory Mediators in Human Corneal Epithelial Cells.

To investigative the effects of sex steroids on hyperosmolar stress-induced proinflammatory cytokine expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-8, and IL-6, and on the mitogen-activated protein kinase pathway in immortalized human corneal epithelial cells (hCECs). Immortalized hCECs were cultured with keratinocyte-free medium until reaching 80% confluency with either 10 M dihidrotestosteron (DHT) or 10 M 17-β-estradiol, and then, the medium was changed to hyperosmolar for various time points. After hyperosmolar treatment, a real-time polymerase chain reaction was performed to show the TNF-α, IL-8, and IL-6 gene expression levels in hCECs. In addition, the treated cells were lysed, and Western blot analysis was applied for phosphorylated and nonphosphorylated forms of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 kinase, and c-Jun N-terminal kinase 1/2 (JNK1/2). hCECs viability was measured with Annexin V/propidium iodide. Pretreatment with 10 M DHT or 17-β-estradiol inhibited the high osmolarity-induced expression of TNF-α, IL-8, and IL-6. The upregulation of p-ERK, p-JNK, and p-p38 with high osmolarity was inhibited partially by DHT, but 17-β-estradiol pretreatment only affected p-p38 for a short time interval. In addition, DHT increased cell viability of hCECs under hyperosmolar conditions. Our results demonstrated that DHT and 17-β-estradiol decreased the proinflammatory cytokine gene expression levels which were stimulated by high osmolarity in immortalized hCECs. The mitogen-activated protein kinase signaling pathway is partially involved in the regulatory effects of DHT on hCECs. These findings may contribute to the etiologic role and therapeutic implications of sex steroids in certain ocular surface diseases.

Chemical hypoxia induces pro-inflammatory signals in fat-laden hepatocytes and contributes to cellular crosstalk with Kupffer cells through extracellular vesicles

BackgroundObstructive sleep apnea syndrome (OSAS) is associated to intermittent hypoxia (IH) and is an aggravating factor of non-alcoholic fatty liver disease (NAFLD). We investigated the effects of hypoxia in both in vitro and in vivo models of NAFLD. MethodsPrimary rat hepatocytes treated with free fatty acids (FFA) were subjected to chemically induced hypoxia (CH) using the hypoxia-inducible factor-1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Triglyceride (TG) content, mitochondrial superoxide production, cell death rates, cytokine and inflammasome components gene expression and protein levels of cleaved caspase-1 were assessed. Also, Kupffer cells (KC) were treated with conditioned medium (CM) and extracellular vehicles (EVs) from hypoxic fat-laden hepatic cells. The choline deficient L-amino acid defined (CDAA)-feeding model used to assess the effects of IH on experimental NAFLD in vivo. ResultsHypoxia induced HIF-1α in cells and animals. Hepatocytes exposed to FFA and CoCl2 exhibited increased TG content and higher cell death rates as well as increased mitochondrial superoxide production and mRNA levels of pro-inflammatory cytokines and of inflammasome-components interleukin-1β, NLRP3 and ASC. Protein levels of cleaved caspase-1 increased in CH-exposed hepatocytes. CM and EVs from hypoxic fat-laden hepatic cells evoked a pro-inflammatory phenotype in KC. Livers from CDAA-fed mice exposed to IH exhibited increased mRNA levels of pro-inflammatory and inflammasome genes and increased levels of cleaved caspase-1. ConclusionHypoxia promotes inflammatory signals including inflammasome/caspase-1 activation in fat-laden hepatocytes and contributes to cellular crosstalk with KC by release of EVs. These mechanisms may underlie the aggravating effect of OSAS on NAFLD. [Abstract word count: 257].

Selected cytokine expression in dogs with alergic conjunctivitis: Correlation with disease activity

IntroductionCanine allergic conjunctivitis (cAC) is described as the most frequent ocular manifestation associated with canine atopic dermatitis (cAD). ObjectivesClinical and immunological characterization of cAD through IL-6, TNF-α and IL-12 mRNA expression quantification in canine conjunctivae. ProceduresTwenty client-owned dogs with both cAC and cAD and twenty-one healthy controls were enrolled and clinician assessed CADESI-04 and grade of ocular signs were calculated. Conjunctival biopsies were performed on all animals and relative quantification of the interleukins mRNA expression performed by qRT-PCR. The correlation between cytokine gene expression and cAC score was evaluated, as well as CADESI-04 values. ResultsThe qRT-PCR showed a significant gene upregulation of respectively 291.48 (p = 1.306e-09) and 4.85 (p = .00033) folds on IL-6 and IL-12 in dogs with allergic conjunctivitis compared to the control group. Regarding the average expression of TNF-α there were no statistical significant differences between both groups (p = .18). Higher cAC scores were associated with enhanced gene expression of TNF-α and IL-12. No correlation was found between the cytokine gene expression levels and the CADESI-04 values. ConclusionAn increase of IL6 and IL12 in cAC was found in the studied population. These two cytokines may be potential immunotherapy targets cAC classification.

CpG Oligonucleotides Protect Mice From Alphavirus Encephalitis: Role of NK Cells, Interferons, and TNF.

Arboviruses including alphavirus are responsible for most emerging infectious diseases worldwide. Recent outbreaks of chikungunya virus serve as a stark reminder to their pathogenic potential. There are no vaccines or therapeutics currently available to contain alphavirus outbreaks. In this study we evaluated the effect of immunomodulatory CpG ODN on the clinical progression of neurotropic Sindbis virus infection. Neonatal C57Bl-6 mice challenged with Sindbis virus AR339 (25 PFU Subcutaneous) infect neurons in the CNS leading to the development of ataxia, seizures, paralysis, and death. We show that systemic administration of CpG ODN modulates the cytokine and chemokine gene expression levels in the CNS and ultimately protects neonatal mice from lethal neurotropic infection. The protection conferred by CpG ODN is controlled by innate immune response and T and B cells were dispensable. Further, protection required Type I, Type II interferons, and TNF as well as functional NK cells, but did not involve iNOS. This study confirms that administration of innate immune modulators can be used as a strategy to boost host innate immune responses and protect against neurotropic viruses reducing their pathogenic footprint.

CD14 and ALPK1 Affect Expression of Tight Junction Components and Proinflammatory Mediators upon Bacterial Stimulation in a Colonic 3D Organoid Model.

Cd14 and Alpk1 both encode pathogen recognition receptors and are known candidate genes for affecting severity in inflammatory bowel diseases. CD14 acts as a coreceptor for bacterial lipopolysaccharide (LPS), while ALPK1 senses ADP-D-glycero-beta-D-manno-heptose, a metabolic intermediate of LPS biosynthesis. Intestinal barrier integrity can be influenced by CD14, whereas to date, the role of ALPK1 in maintaining barrier function remains unknown. We used colon-derived 3D organoids, first characterised for growth, proliferation, stem cell markers, and expression of tight junction (TJ) components using qPCR and immunohistochemistry. They showed characteristic crypt stem cells, apical shedding of dead cells, and TJ formation. Afterwards, organoids of different genotypes (WT, Il10−/−, Cd14−/−, and Alpk1−/−) were then stimulated with either LPS or Escherichia coli Nissle 1917 (EcN). Gene expression and protein levels of cytokines and TJ components were analysed. WT organoids increased expression of Tnfα and tight junction components. Cd14−/− organoids expressed significantly less Tnfα and Ocln after LPS stimulation than WT organoids but reacted similarly to WT organoids after EcN stimulation. In contrast, compared to WT, Alpk1−/− organoids showed decreased expression of different TJ and cytokine genes in response to EcN but not LPS. However, Western blotting revealed an effect of ALPK1 on TJ protein levels. These findings demonstrate that Cd14, but not Alpk1, alters the response to LPS stimulation in colonic epithelial cells, whereas Alpk1 is involved in the response upon bacterial challenge.

Abstract 108: Sex Differences in Stroke-induced Lymphocyte Responses in the Gut Epithelia

Introduction: Stroke is a sexually dimorphic disease. Notably, emerging evidence suggests that stroke leads to gut dysbiosis and pathological shifts in the gut microbiota. However, little is known about how the gut epithelial surface maintains homeostasis after stroke. In this study, we isolated intraepithelial lymphocytes (IELs), which are mainly CD8 + T cells and do not need priming against antigens, as well as intestinal epithelial cells (IECs). We examined their responses to stroke-induced gut epithelial damage. Methods: C57BL/6 (2-3 months) mice of both sexes were subjected to either 60-minute middle cerebral artery occlusion or sham surgery. At day 7 after stroke, IECs and IELs, which are intercalated between IECs, were isolated from mouse colonic tissues using Percoll density gradients. RNA was isolated from the cells and amplified to quantitate expression levels of cell-specific genes for antibacterial proteins and chemotactic cytokines. Tissues were then used for histological analysis by hematoxylin and eosin staining and bacterial fluorescence in situ hybridization (FISH). Results: Stroke caused significant colonic damage in male mice, compared to female mice. Bacterial FISH demonstrated microbial encroachment into the host gut epithelium in males after stroke; whereas female mice maintained spatial segregation between the host gut and bacteria. Stroke increased expression levels of pro-inflammatory cytokine genes in male IELs including IL-1β (28.3 fold increase, P <0.05), KC or CXCL1 (5.8 fold increase, P <0.05) and MIP2α or CXCL2 (9.9 fold increase, P <0.05) after stroke compared with sham mice. Additionally, both IELs and IECs of male stroke mice exhibited an up-regulation of antimicrobial protein gene, regenerating islet-derived protein 3 (Reg3γ, P <0.05). This pattern of up-regulation, however, was not present in female mice. Conclusions: This is the first study that demonstrates robust sex differences in lymphocyte responses at the gut mucosal surface between males and females following stroke. The closer proximity of the gut microbiota with the host may direct gene expression related to microbial responses in the injured epithelium after brain injury. Our study provides novel insight into host-microbial homeostasis in stroke.

Vitamin D Receptor Activation in Liver Macrophages Protects Against Hepatic Endoplasmic Reticulum Stress in Mice.

Hepatic endoplasmic reticulum (ER) stress, whether triggered by intrinsic or extrinsic factors, can be resolved by the unfolded protein response (UPR). Sustained UPR activation leads to cell death and inflammatory response and contributes to liver disease progression. Hepatic tissue macrophages are key players in orchestrating liver inflammation, and ER stress can enhance macrophage activation. However, it is not well defined how the interplay between ER stress and inflammation is regulated during hepatic stress response. Here we demonstrate that vitamin D receptor (VDR) activation mitigates hepatic ER stress response, whereas VDR knockout mice undergo persistent UPR activation and apoptosis in response to chemical ER stress inducer. Moreover, VDR deficiency promotes hepatic macrophage infiltration and increases gene expression and systematic levels of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor α. VDR expression is induced in hepatic macrophages by ER stress, and VDR plays a dual regulatory role in macrophages by protecting against ER stress and promoting anti-inflammatory polarization. Co-culture with VDR-activated bone marrow-derived macrophages suppresses UPR target genes in primary hepatocytes treated with ER stress inducers. Thus, the immunomodulatory functions of VDR in macrophages are critical in hepatic ER stress resolution in mice. VDR signaling in macrophages regulates a shift between proinflammatory and anti-inflammatory activation during ER stress-induced inflammation to promote hepatic ER stress resolution.

Investigation of New Benzimidazole Derivative Compounds’ Effects on A549 Cell Line

Abstract Chronic inflammation is a common indication of several diseases, e.g. asthma, chronic obstructive pulmonary disease (COPD), atherosclerosis, etc. Benzimidazole derivatives are preferable compounds to design new analgesic and anti-inflammatory substances due to their unique biological features. We aimed to investigate the effect of a newly synthesized benzimidazole derivative, ORT-83, on A549 human lung adenocarcinoma cell line. ORT-83 was synthesized, and a non-cytotoxic concentration of ORT-83 on A549 cells was detected with MTT assay. To analyze the anti-inflammatory effect of ORT-83, an inflammatory cell culture model was established by stimulating A549 cell line with IL1-β (10 ng/ml). After 2 hours of treatment with IL1-β to induce inflammation, A549 cells were exposed to ORT-83 (0.78 µg/ml) for 24 hours. Thereafter gene expression analyses were performed with qRT-PCR. We found that ORT-83 significantly suppressed the gene expression levels of the proinflammatory cytokines; IL-6, NFkB, and TNF-α. However, the increased levels of IL-10 (2.8 folds) by IL-1β induction did not change after ORT-83 and/or dexamethasone (Dex: positive control) treatments. While Dex; a COX-2 inhibitor, reduced the COX-2 expression level in inflammatory cells from 10.03 folds to 0.71 folds, ORT-83 reduced its level to 4.37 folds. iNOS expression levels did not change in any experimental groups. In conclusion, we showed that ORT-83 exerted its anti-inflammatory effects by repressing the gene expression of proinflammatory cytokines in the inflammation-induced A549 cell line. Although ORT-83 had a weaker COX-2 inhibitory effect compared to Dex, it was shown to be still a strong anti-inflammatory compound.

Sika deer (Cervus nippon) velvet antler ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis-like clinical signs in a rodent model

Context Deer velvet is a rarely used component in traditional Chinese medicine and has beneficial effects against several diseases. As a substance that covers the bone and cartilage of immature antlers, deer velvet is a natural cytokine ‘storeroom’ that is rich in protein and proteoglycans. Recently, proteoglycans have been shown to have beneficial effects against inflammation. Aims To determine whether antler extract possesses therapeutic effects in a mouse model of atopic dermatitis (AD) and to explore the underlying mechanisms of action. Methods BALB/c mice were randomly divided into the following groups: control, AD, and AD + antler groups. We established an in vivo AD model by repeatedly exposing the ears of mice to Dermatophagoides farinae extract (house dust-mite extract) and 2,4-dinitrochlorobenzene once per week for 4 weeks. On the day after induction, ear thickness was measured. Antler extract (100 mg/kg) was administered orally once a day for 26 days. After 4 weeks of treatment with antler extract, the epidermal and dermal ear thickness, mast-cell infiltration, spleen weight, and lymph-node weight were measured. In addition, the mRNA levels of several pathogenic cytokines in the ears were measured. The concentrations of IL-4, IL-5, IL-10, IL-31 and IL-17 mRNA in the skin lesions of each group were measured by quantitative polymerase chain reaction. Key results Epidermal and dermal ear thickness, mast-cell infiltration, lymph-node weight, and gene expression levels of pathogenic cytokines in ear tissue were diminished following oral administration of antler extract, unlike in the control group. Conclusions The results of the present study strongly suggest that antler extract exhibits therapeutic activity against atopic dermatitis via regulation of inflammatory response. Implications Further exploration of the mechanisms of action of antler extract will be important for clinical application.

The expression level of cytokine genes in the cases of native heart valves in infectious endocarditis

The expression level of IL1B, IL6, IL8, IL10, IL12A, IL12B, IL18, IL23, IL33, CCL2, and IL1RL1 has been investigated using biopsies of native mitral, aortic, and tricuspid valves obtained during surgical correction of acquired defect from 25 patients with infectious endocarditis. Biopsies of native mitral and aortic valve cusps from 12 patients who underwent surgical correction of acquired heart disease of non-infectious etiology were used as control. We used quantitative PCR with fluorescent dye SYBR Green for determination of the cytokine gene expression level. This study revealed that genes could be subdivided into three groups: (i) genes with increased expression (IL1B, IL6, and IL8); (ii) genes with reduced expression (IL33 and IL1RL1); (iii) genes with unchanged expression (IL12A, IL18, IL23, and CCL2). The IL8 gene expression was characterized by the most pronounced increase (9.83 times versus control), while the IL1RL1 gene demonstrated the most pronounced decrease in its expression (4.17 times). Expression of IL10 and IL12B genes was negligible in all samples.