Stabilization but no functional influence of HIF-1α expression in the intestinal epithelium during Salmonella Typhimurium infection

2022 ◽  
Author(s):  
Laura Robrahn ◽  
Aline Dupont ◽  
Sandra Jumpertz ◽  
Kaiyi Zhang ◽  
Christian H. Holland ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intestinal Epithelium ◽  
Bacterial Infections ◽  
Inflammatory Diseases ◽  
Protein Stabilization ◽  
Inflammatory Factors ◽  
Intestinal Epithelial ◽  
Data Set ◽  
The Impact

The hypoxia-inducible transcription factor 1 (HIF-1) has been shown to enhance microbial killing and to ameliorate the course of bacterial infections. While the impact of HIF-1 on inflammatory diseases of the gut has been studied intensively, its function in bacterial infections of the gastrointestinal tract remains largely elusive. With the help of a publicly available gene expression data set, we could infer significant activation of HIF-1 after oral infection of mice with Salmonella Typhimurium. Immunohistochemistry and western blot analysis confirmed marked HIF-1α protein stabilization, especially in the intestinal epithelium. This prompted us to analyze conditional Hif1a -deficient mice to examine cell type-specific functions of HIF-1 in this model. Our results demonstrate enhanced non-canonical induction of HIF-1 activity upon Salmonella infection in the intestinal epithelium as well as in macrophages. Surprisingly, Hif1a deletion in intestinal epithelial cells did not impact on inflammatory gene expression, bacterial spread or disease outcome. In contrast, Hif1a deletion in myeloid cells enhanced intestinal Cxcl2 expression and reduced the cecal Salmonella load. In vitro , HIF-1α-deficient macrophages showed an overall impaired transcription of mRNA encoding pro-inflammatory factors, however, intracellular survival of Salmonella was not impacted by HIF-1α deficiency.

scholarly journals Conditional deletion of HIF-1α provides new insight regarding the murine response to gastrointestinal infection with Salmonella Typhimurium

2021 ◽  
Author(s):  
Laura Robrahn ◽  
Aline Dupont ◽  
Sandra Jumpertz ◽  
Kaiyi Zhang ◽  
Christian H. Holland ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Bacterial Infections ◽  
Myeloid Cells ◽  
Immune Defense ◽  
Conditional Knockout ◽  
Disease Outcome ◽  
Inflammatory Factors ◽  
Intestinal Epithelial ◽  
Data Set ◽  
The Impact

AbstractThe hypoxia-inducible transcription factor 1 (HIF-1) has been shown to ameliorate different bacterial infections through enhancement of microbial killing. While the impact of HIF-1 on inflammatory diseases of the gut has been studied intensively, its function in bacterial infections of the intestine remains largely elusive. With the help of a publicly available gene expression data set, we could infer significant activation of the HIF-1 transcription factor after oral infection of mice with Salmonella Typhimurium. This prompted us to apply lineage-restricted deletion of the Hif1a locus in mice to examine cell type-specific functions of HIF-1 in this model. We show hypoxia-independent induction of HIF-1 activity upon Salmonella infection in the intestinal epithelium as well as in macrophages. Surprisingly, Hif1a deletion in intestinal epithelial cells impacted neither disease outcome nor inflammatory activity. The conditional knockout of Hif1a in myeloid cells enhanced the mRNA expression of the largely pro-inflammatory chemokine Cxcl2, revealing a potentially inflammatory effect of HIF-1 deficiency in myeloid cells in the gut in vivo. Again, the disease outcome was not affected. In vitro HIF-1-deficient macrophages showed an overall impaired transcription of pro-inflammatory factors, however, Salmonella bypassed direct intracellular, bactericidal HIF-1-dependent mechanisms in a Salmonella pathogenicity island (SPI)-2 independent manner. Taken together, our data suggest that HIF-1 in intestinal epithelial and myeloid cells is either dispensable or compensable in the immune defense against Salmonella Typhimurium.

scholarly journals A206 SUSCEPTIBILITY OF HNF4AΔIEC MICE TO SALMONELLA INFECTIONS

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 78-79
Author(s):  
D Pupo Gómez ◽  
V Reyes-Nicolas ◽  
G Marrero ◽  
N Perreault ◽  
A Menendez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Salmonella Typhimurium ◽  
Intestinal Epithelium ◽  
Bacterial Infections ◽  
Mutant Mice ◽  
Epithelial Barrier ◽  
Epithelial Barrier Function ◽  
Efficacy Analysis ◽  
Salmonella Infections ◽  
The Impact

Abstract Background Inflammatory bowel diseases (IBD) are a group of chronic disorders that affect more than 233 000 Canadians and for which there is not yet an effective treatment. In addition, IBD is a multifactorial disease depending on genetic, immune and environmental dysregulations. The gastrointestinal epithelium plays an important role as a barrier that protects against antigens and bacterial products that are in the lumen. It is well recognized that a defect in the integrity of the barrier and its functions may be involved in the development of these diseases. On the other hand, our laboratory has shown that the conditional deletion of HNF4alpha nuclear receptor (Hnf4a) in the intestinal epithelium of mice can lead to the development of chronic inflammation of the intestine. However, the impact of the loss of this transcriptional factor on the epithelial barrier is still controversial. Aims To evaluate the impact of Hnf4a deletion on the epithelial barrier during bacterial infections. Methods We used a tamoxifen-inducible Cre-loxP system to delete the Hnf4a gene in the intestinal epithelium of 2-month-old mice, that were then infected with an attenuated strain of Salmonella typhimurium (SB1003) during 4 days. S. typhimurium loads were determined in cecum and colon content, and in liver, spleen tissues by plating homogenates on LB agar supplemented with streptomycin. Also, histological examinations and gene expression of selected targets were assessed between mutant (Hnf4aΔIEC) and control mice. Results The tamoxifen-inducible Cre-loxP system was able to delete intestinal Hnf4a gene expression with almost 100% of efficacy. Analysis by qPCR showed that the infection caused significant changes on the response of different infection responsive components (Relmβ, Muc2 and IL-33) in mutant mice. In addition, morphological analyses revealed an increase in the infiltration of immune cells and the number of goblets cells, indicative of an increase in the susceptibility to Salmonella typhimurium (SB1003) infection of the mutant mice. Conclusions Altogether, our results suggest that Hnf4a could be involved or play an important role as a modulator of the intestinal epithelial barrier function during Salmonella typhimurium (SB1003) infection. Therefore, understanding the mechanisms involved in this process could allow the development of better therapies for IBD. Funding Agencies CIHR

scholarly journals The benefit of combining curcumin, bromelain and harpagophytum to reduce inflammation in osteoarthritic synovial cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sybille Brochard ◽  
Julien Pontin ◽  
Benoit Bernay ◽  
Karim Boumediene ◽  
Thierry Conrozier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Proteomic Analysis ◽  
Complex Disease ◽  
Inflammatory Diseases ◽  
Biological Action ◽  
Inflammatory Factors ◽  
Synovial Cells ◽  
Mrna Levels ◽  
Bioinformatics Analyses

Abstract Background Osteoarthritis (OA) is the most common form of arthritis, affecting millions of people worldwide and characterised by joint pain and inflammation. It is a complex disease involving inflammatory factors and affecting the whole joint, including the synovial membrane. Since drug combination is widely used to treat chronic inflammatory diseases, a similar strategy of designing plant-derived natural products to reduce inflammation in OA joints may be of interest. In this study, we characterised the response of OA synovial cells to lipopolysaccharide (LPS) and investigated the biological action of the combination of curcumin, bromelain and harpagophytum in this original in vitro model of osteoarthritis. Methods Firstly, human synovial cells from OA patients were stimulated with LPS and proteomic analysis was performed. Bioinformatics analyses were performed using Cytoscape App and SkeletalVis databases. Additionally, cells were treated with curcumin, bromelain and harpagophytum alone or with the three vegetal compounds together. The gene expression involved in inflammation, pain or catabolism was determined by RT-PCR. The release of the encoded proteins by these genes and of prostaglandin E2 (PGE2) were also assayed by ELISA. Results Proteomic analysis demonstrated that LPS induces the expression of numerous proteins involved in the OA process in human OA synovial cells. In particular, it stimulates inflammation through the production of pro-inflammatory cytokines (Interleukin-6, IL-6), catabolism through an increase of metalloproteases (MMP-1, MMP-3, MMP-13), and the production of pain-mediating neurotrophins (Nerve Growth Factor, NGF). These increases were observed in terms of mRNA levels and protein release. LPS also increases the amount of PGE2, another inflammation and pain mediator. At the doses tested, vegetal extracts had little effect: only curcumin slightly counteracted the effects of LPS on NGF and MMP-13 mRNA, and PGE2, IL-6 and MMP-13 release. In contrast, the combination of curcumin with bromelain and harpagophytum reversed lots of effects of LPS in human OA synovial cells. It significantly reduced the gene expression and/or the release of proteins involved in catabolism (MMP-3 and -13), inflammation (IL-6) and pain (PGE2 and NGF). Conclusion We have shown that the stimulation of human OA synovial cells with LPS can induce protein changes similar to inflamed OA synovial tissues. In addition, using this model, we demonstrated that the combination of three vegetal compounds, namely curcumin, bromelain and harpagophytum, have anti-inflammatory and anti-catabolic effects in synovial cells and may thus reduce OA progression and related pain.

scholarly journals Cell–cell coupling and DNA methylation abnormal phenotypes in the after-hours mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Federico Tinarelli ◽  
Elena Ivanova ◽  
Ilaria Colombi ◽  
Erica Barini ◽  
Edoardo Balzani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Neuronal Networks ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Neuronal Cultures ◽  
Functional Impairments ◽  
After Hours ◽  
The Impact

Abstract Background DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. Methods In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. Results We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. Conclusions The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.

scholarly journals Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

2021 ◽  
Vol 22 (3) ◽  
pp. 1222
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Inmaculada Parrilla ◽  
Heriberto Rodriguez-Martinez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Survival Rates ◽  
Control Group ◽  
P Value ◽  
Transcriptome Profile ◽  
Embryo Viability ◽  
The Impact

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

scholarly journals Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Mandy Rauschner ◽  
Luisa Lange ◽  
Thea Hüsing ◽  
Sarah Reime ◽  
Alexander Nolze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Western Blot ◽  
Tumor Cell ◽  
Low Ph ◽  
Strong Increase ◽  
Acidic Ph ◽  
The Impact

Abstract Background The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. Methods Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. Results NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. Conclusions These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

scholarly journals Down-regulation of SNHG16 alleviates the acute lung injury in sepsis rats through miR-128-3p/HMGB3 axis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Junli Sun ◽  
Keke Xin ◽  
Chenghui Leng ◽  
Jianlin Ge
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Western Blot ◽  
Cell Viability ◽  
Inflammatory Diseases ◽  
Cecal Ligation ◽  
Inflammatory Factors ◽  
Lung Epithelial

Abstract Background Long noncoding RNAs contribute to various inflammatory diseases, including sepsis. We explore the role of small nucleolar RNA host gene 16 (SNHG16) in sepsis-mediated acute lung injury (ALI) and inflammation. Methods A sepsis-induced ALI rat model was constructed by the cecal ligation and perforation method. The profiles of SNHG16, miR-128-3p, and high-mobility group box 3 (HMGB3) were monitored by quantitative reverse transcription PCR and Western blot. The pathologic changes of lung tissues were evaluated by Hematoxylin–Eosin staining, immunohistochemistry, and dry and wet method. Meanwhile, the pro-inflammatory factors and proteins were determined by ELISA and Western blot. In contrast, a sepsis model in BEAS-2B was induced with lipopolysaccharide (LPS) to verify the effects of SNHG16/miR-128-3p/HMGB3 on lung epithelial cell viability and apoptosis. Results As a result, SNHG16 and HMGB3 were up-regulated, while miR-128-3p was down-regulated in sepsis-induced ALI both in vivo and in vitro. Inhibiting SNHG16 reduced the apoptosis and inflammation in the sepsis-induced ALI model. Overexpressing SNHG16 promoted LPS-mediated lung epithelial apoptosis and inhibited cell viability and inflammation, while miR-128-3p had the opposite effects. Mechanistically, SNHG16 targeted miR-128-3p and attenuated its expression, while miR-128-3p targeted the 3′ untranslated region of HMGB3. Conclusions Overall, down-regulating SNHG16 alleviated the sepsis-mediated ALI by regulating miR-128-3p/HMGB3.

Advances in Anti-inflammatory Activity, Mechanism and Therapeutic Application of Ursolic Acid

2021 ◽  
Vol 21 ◽  
Author(s):  
Mingzhu Luan ◽  
Huiyun Wang ◽  
Jiazhen Wang ◽  
Xiaofan Zhang ◽  
Fenglan Zhao ◽  
...  
Keyword(s):  
Ursolic Acid ◽  
Inflammatory Diseases ◽  
Kidney Diseases ◽  
Inflammatory Activity ◽  
Inflammatory Factors ◽  
Inflammatory Stimuli ◽  
Bowel Diseases ◽  
Anti Inflammatory

: In vivo and in vitro studies reveal that ursolic acid (UA) is able to counteract endogenous and exogenous inflammatory stimuli, and has favorable anti-inflammatory effects. The anti-inflammatory mechanisms mainly include decreasing the release of histamine in mast cells, suppressing the activities of lipoxygenase, cyclooxygenase and phospholipase, and reducing the production of nitric oxide and reactive oxygen species, blocking the activation of signal pathway, down-regulating the expression of inflammatory factors, and inhibiting the activities of elastase and complement. These mechanisms can open up new avenues for the scientific community to develop or improve novel therapeutic approaches to tackle inflammatory diseases such as arthritis, atherosclerosis, neuroinflammation, liver diseases, kidney diseases, diabetes, dermatitis, bowel diseases, cancer. The anti-inflammatory activity, the anti-inflammatory mechanism of ursolic acid and its therapeutic applications are reviewed in this paper.

scholarly journals Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

2021 ◽  
Author(s):  
Jozsef Bodis ◽  
Endre Sulyok ◽  
Akos Varnagy ◽  
Viktória Prémusz ◽  
Krisztina Godony ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
The Impact ◽  
Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

scholarly journals C5a and C5aR1 are key drivers of microvascular platelet aggregation in clinical entities spanning from aHUS to COVID-19

2021 ◽  
Author(s):  
Sistiana Aiello ◽  
Sara Gastoldi ◽  
Miriam Galbusera ◽  
Piero Luigi Ruggenenti ◽  
Valentina Portalupi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Bacterial Infections ◽  
Viral Infections ◽  
Ex Vivo ◽  
Inflammatory Diseases ◽  
Thrombus Formation ◽  
Alternative Pathway ◽  
Atypical Hemolytic Uremic Syndrome ◽  
Uremic Syndrome

Unrestrained activation of the complement system till the terminal products, C5a and C5b-9, plays a pathogenetic role in acute and chronic inflammatory diseases. In endothelial cells, complement hyperactivation may translate into cell dysfunction, favoring thrombus formation. The aim of this study was to investigate the role of the C5a/C5aR1 axis as opposite to C5b-9 in inducing endothelial dysfunction and loss of anti-thrombogenic properties. In vitro and ex vivo assays with serum from patients with atypical hemolytic uremic syndrome (aHUS) -a prototype rare disease of complement-mediated microvascular thrombosis due to genetically determined alternative pathway dysregulation- and cultured microvascular endothelial cells, demonstrated that the C5a/C5aR1 axis is a key player of endothelial thromboresistance loss. C5a added to normal human serum, fully recapitulated the pro-thrombotic effects of aHUS serum. Mechanistic studies showed that C5a caused RalA-mediated exocytosis of vWF and P-selectin from Weibel-Palade bodies, which favored further vWF binding on the endothelium and platelet adhesion and aggregation. In patients with severe COVID-19 -who suffered from acute activation of complement triggered by SARS-CoV-2 infection- we found the same C5a-dependent pathogenic mechanisms. These results highlight C5a/C5aR1 as a common pro-thrombogenic effector spanning from genetic rare diseases to viral infections, and may have clinical implications. Selective C5a/C5aR1 blockade could have advantages over C5 inhibition, since the former preserves the formation of C5b-9 that is critical to control bacterial infections that often develop as comorbidities in severely ill patients. (Clinicaltrials.gov identifier NCT02464891)

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