Specific Pathogen-free Conditions Research Articles

Administration of a Bacterial Lysate to the Airway Compartment Is Sufficient to Inhibit Allergen-Induced Lung Eosinophilia in Germ-free Mice.

The nexus between eosinophils and microbes is attracting increasing attention. We previously showed that airway administration of sterile microbial products contained in dust collected from traditional dairy farms virtually abrogated broncho-alveolar lavage (BAL) eosinophilia and other cardinal asthma phenotypes in allergen-sensitized specific pathogen-free (SPF) mice. Interestingly, comparable inhibition of allergen-induced BAL eosinophilia and promotion of airway barrier integrity were found upon administration of a sterile, pharmacological grade bacterial lysate, OM-85, to the airway compartment of allergen-sensitized SPF mice. Here we asked whether intrinsic properties of airway-delivered microbial products were sufficient to inhibit allergic lung inflammation or whether these effects were mediated by reprogramming of the host microbiota. We compared germ-free (GF) mice and offspring of GF mice associated with healthy mouse gut microbiota and maintained under SPF conditions for multiple generations (Ex-GF mice). These mice were treated intra-nasally with OM-85 and evaluated in the OVA and Alternaria models of allergic asthma focusing primarily on BAL eosinophilia. Levels of allergen-induced BAL eosinophilia were comparable in GF and conventionalized Ex-GF mice. Airway administration of the OM-85 bacterial lysate was sufficient to inhibit allergen-induced lung eosinophilia in both Ex-GF and GF mice, suggesting that host microbiota are not required for the protective effects of bacterial products in these models and local airway exposure to microbial products is an effective source of protection. OM-85-dependent inhibition of BAL eosinophilia in GF mice was accompanied by suppression of lung type-2 cytokines and eosinophil-attracting chemokines, suggesting that OM-85 may work at least by decreasing eosinophil lung recruitment.

A194 EARLY LIFE WESTERN-TYPE DIET ACCELERATES THE ONSET OF MURINE IL-10 KO COLITIS

Abstract Background Early life is a critical time for gut microbiome and immune development, including the establishment of proper host-microbe interactions. While exposures to western world environmental factors are associated with inflammatory bowel disease (IBD) susceptibility, the link between environment in early life and later disease onset is unclear. One of the most important western world environmental factors is diet, which can distinctly alter the gut microbiome. We believe an early life western-type diet (WD) can affect disease progression in a murine IL-10 KO colitis model through dysregulated T-cell responses against the microbiome. Aims We aimed to characterize how an early life WD given to IL-10 KO mice would affect colitis development and the gut microbiome. Methods IL-10 KO mice were housed in specific pathogen free conditions and fed normal chow (NC) or WD ad libitum, either between days 10 to 35 (early WD, eWD) or days 35 to 60 (adolescent WD, aWD). Stool was collected from mice at days 35, 56, and 84 for lipocalin-2 (LCN-2) and 16S rDNA sequencing. Mice were sacrificed at day 84, assessing mesenteric lymph node weight, gene expression, colon damage by histopathology, and colon lamina propria leukocyte (LPL) phenotype and cytokine expression by flow cytometry. Results Compared to NC and aWD, the eWD fed IL-10 KO mice displayed increased fecal LCN-2 at days 56 and 84, indicating greater inflammation. At sacrifice, eWD fed mice had larger mesenteric lymph nodes and more significant colon damage by histopathological scoring. Colon LPL preparations showed that eWD fed IL-10 KO mice had higher proportions and absolute numbers of effector and regulatory T-cells. Additionally, higher proportions and absolute numbers of those T-cells were IFNγ+, IL-17A+, and IL-22+. qPCR of distal colon tissue revealed increased gene expression of innate and type 3 proinflammatory cytokines such as Il1b, Tnfα, and Il23, while showing no change in Il6 and Il4. Taxa positively associated with WD were able to establish a persistent niche in eWD fed mice but not in aWD fed mice. An increase in only one specific taxon was associated with eWD at all timepoints while also most strongly correlating with inflammatory markers. Conclusions This data suggests that eWD feeding during gut microbiome and immune development is uniquely capable of increasing long-term susceptibility to intestinal inflammation in IL-10 KO mice. This appears to be associated with persistent colonization of potentially more inflammatory taxa. This work provides insight into the potential role of early life environmental risk factors, i.e. WD, in the later development of IBD. Funding Agencies CAG, CIHRUniversity of Toronto, Taconic Biosciences

Induction of CD8+ immune memory and enhanced inflammation in a skin inflammation model through pre-immunization with inactivated pathogens.

Laboratory mice live in specific pathogen-free (SPF) conditions, resulting in an immature immune system comparable to that of newborns rather than adult humans or mice from pet shops. This condition may compromise their translational value. Reintroducing pathogens would lead to the uncontrolled spread of infections and associated diseases, so research facilities should seek safer alternatives. We immunized laboratory mice with a cocktail of pathogens, which were inactivated by ultraviolet irradiation and mixed with the adjuvant AddaVax. This immunization resulted in a higher percentage of CD8+ effector memory T cells compared to untreated mice, although the response was not as robust as in pet shop mice. In a model of skin inflammation, pre-immunization led to an increased skin inflammatory response compared to non-immunized mice. All immunized mice seroconverted to the pathogens in the mixture, while none of the non-immunized mice housed together seroconverted to the pathogens applied to the pre-immunized mice. In conclusion, pre-immunization of mice impacts the immune system, which includes increasing the levels of CD8+ effector memory T cells.

MiR-410 Regulates Helper T Cell Differentiation in Ovalbumin-Induced Asthma through the PI3K-AKT-VEGF Signaling Pathway

Introduction: Asthma has been attributed to Th1/Th2 imbalance and inappropriate Th2 responses to environmental allergens. MicroRNAs (miRNAs), 21 to 23 RNA molecules, are first found in mammals and have been implicated in various biological activities. Our previous study found that miR-410 effectively ameliorates airway inflammation in the ovalbumin (OVA)-induced asthma murine model. However, the role of miR-410 in regulating helper T (Th) cell differentiation is not clear. In the present study, we aimed to explore the regulatory effects of miR-410 on the differentiation of Th cells through both in vivo and in vitro studies. Methods: Dual-luciferase reporter assay was used to find if miR-410 has any direct binding position with VEGF mRNAs. PBMC and CD4+ T cells were isolated and stimulated with OVA. The miR-410 mimics and inhibitors were transfected into CD4+ T cells. The differentiation of Th cells was evaluated by enzyme-linked immunosorbent assay (ELISA) for the concentration of IL-4, IFN-γ, and TGF-β levels in supernatants. Western Blot was used to detect protein expression and phosphorylation of PI3K and AKT. BALB/c mice were kept in a specific pathogen-free condition and received sterile OVA-free food and water. OVA-induced asthmatic mice model was established. ELISA was used to measure the bronchoalveolar lavage fluid (BALF) concentrations of IL-4, IFN-γ, TGF-β, and VEGF. Hematoxylin and eosin staining and immunohistochemical staining were conducted to analyze inflammatory cell infiltration, pathological changes, and the expression of VEGF. Results: Dual-luciferase reporter assay showed that miR-410 has no direct binding position with VEGF mRNAs. In the OVA-primed mononuclear cells compared to normal cells, IFN-γ and TGF-β were decreased while IL-4 and VEGF were increased. This change was reversed while miRNA-410 mimics were transfected into CD4+ T cells. Besides, the OVA-primed CD4+ T cells treated with miR-410 decrease the proliferation of cytokine of Th2 cells as well as phosphorylation of PI3K, and AKT. In OVA-induced asthma mice, IFN-γ and TGF-β were decreased in BALF while the IL-4 and VEGF were increased. OVA-induced mice with asthma treated with miR-410 mimics showed marked reductions in the infiltration of inflammatory cells as well as IL-4 and VEGF in BALF. The immunohistochemical staining of the expression of VEGF also decreased in OVA-induced asthma mice with the instillation of miR-410. Conclusions: In this study, we revealed that miR-410 could regulate the differentiation of Th cells via the PI3K-AKT-VEGF signaling pathway in asthma.

Effects of herbal tea (Platostoma palustre) on the Hyperlipidemia in vivo

Effects of herbal tea (Platostoma palustre) on the Hyperlipidemia in vivo

The promoting effect of modified Dioscorea pills on vascular remodeling in chronic cerebral hypoperfusion via the Ang/Tie signaling pathway.

The objective of this study was to investigate the effect of modified Dioscorea pills (MDP) on microcirculatory remodeling in the hippocampus of rats with chronic cerebral hypoperfusion (CCH) through the angiopoietin (Ang)/tyrosine kinase receptor tyrosine kinase with immunoglobulin-like and EGF-like domains (Ang receptor) 2 (Tie-2) signaling pathways, which may underlie the cognitive improvement observed in CCH rats. Forty male Sprague-Dawley rats raised under specific pathogen-free conditions were randomly divided into three groups: control group (10 rats), model group (15 rats), and MDP group (15 rats). The rats in the model group and MDP group underwent bilateral common carotid artery occlusion using the 2-vessel occlusion (2-VO) method to induce CCH. Rats in the control group underwent the same surgical procedures as those in the model group, except for ligation and occlusion of the carotid arteries. After 1 week of 2-VO, rats in the MDP group were administered MDP condensed decoction intragastrically at a dose of 1 ml/100 g body weight (prepared by the Preparation Room of Hubei Provincial Hospital of Traditional Chinese Medicine) for 45 days, while rats in the other two groups received normal saline intragastrically with the same dose and duration as the MDP group. After the intervention, all rats were euthanized, and brain perfusion was performed to obtain the hippocampal tissue for analysis. Immunohistochemical staining for CD43 was performed to assess microvessel density (MVD); western blot and the reverse transcription-polymerase chain reaction (RT-PCR) were used to analyze the expression of proteins and genes in angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-2, and vascular endothelial growth factor (VEGF) proteins and genes in the hippocampal tissue and compute the Ang-1/Ang-2 ratio. MDP treatment reduced neuronal loss and promoted restoration of the damaged hippocampal structure in CCH rats. The model group showed significantly higher MVD (14.93 ± 1.92) compared to the control group (5.78 ± 1.65) (P < 0.01), whereas MDP treatment further increased MVD (21.19 ± 2.62). Western blot and RT-PCR analysis revealed that CCH significantly increased the expression of Ang-1, Ang-2, Tie-2, and VEGF proteins and genes, while MDP treatment further significantly upregulated the expression of these proteins and genes. In addition, MDP significantly elevated the gene and protein expression of the Ang-1/Ang-2 ratio compared to the control group (P = 0.041, P = 0.029). CCH induces microvascular neogenesis in the hippocampus, and MDP promotes angiogenesis and microcirculation remodeling in CCH rats via the Ang/Tie signaling pathway, which may be an important mechanism for its restorative effects on hippocampal perfusion and improvement of cognitive function in CCH rats.

IL-1β transgenic mouse model of inflammation driven esophageal and oral squamous cell carcinoma

Chronic inflammation is integral to the development of esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), although the latter has not been associated with reflux esophagitis. The L2-IL-1β transgenic mice, expressing human interleukin (IL)-1β in the oral, esophageal and forestomach squamous epithelia feature chronic inflammation and a stepwise development of Barrett’s esophagus-like metaplasia, dysplasia and adenocarcinoma at the squamo-columnar junction. However, the functional consequences of IL-1β-mediated chronic inflammation in the oral and esophageal squamous epithelia remain elusive. We report for the first time that in addition to the previously described Barrett’s esophagus-like metaplasia, the L2-IL-1β mice also develop squamous epithelial dysplasia with progression to squamous cell carcinoma (SCC) in the esophagus and the tongue. L2-IL-1β showed age-dependent progression of squamous dysplasia to SCC with approximately 40% (n = 49) and 23.5% (n = 17) incidence rates for esophageal and tongue invasive SCC respectively, by 12–15 months of age. Interestingly, SCC development and progression in L2-IL-1β was similar in both Germ Free (GF) and Specific Pathogen Free (SPF) conditions. Immunohistochemistry revealed a T cell predominant inflammatory profile with enhanced expression of Ki67, Sox2 and the DNA double-strand break marker, γ-H2AX, in the dysplastic squamous epithelia of L2-IL-1β mice. Pro-inflammatory cytokines, immunomodulatory players, chemoattractants for inflammatory cells (T cells, neutrophils, eosinophils, and macrophages) and oxidative damage marker, iNOS, were significantly increased in the esophageal and tongue tissues of L2-IL-1β mice. Our recent findings have expanded the translational utility of the IL-1β mouse model to aid in further characterization of the key pathways of inflammation driven BE and EAC as well as ESCC and Oral SCC.

Sequential Early-Life Infections Alter Peripheral Blood Transcriptomics in Aging Female Mice but Not the Response to De Novo Infection with Influenza Virus or M. tuberculosis.

To determine the impact of accumulating Ag exposure on immunity in the aging mouse, and to develop a model more relevant to humans who are exposed to multiple pathogens during life, we sequentially infected young female mice with four distinct pathogens at 8-wk intervals: murine γ-herpesvirus 68, Sendai virus, murine CMV, and Heligmosomoides polygyrus. Mock-infected mice received PBS. After aging the sequentially infected and mock-infected mice to 18-25 mo under specific pathogen-free conditions, we analyzed multiple immune parameters. We assessed transcriptional activity in peripheral blood, T cell phenotype, the diversity of influenza epitopes recognized by CD8 T cells, and the response of the animals to infection with influenza virus and Mycobacterium tuberculosis. Our data show enhanced transcriptional activation in sequentially infected aged mice, with changes in some CD8 T cell subsets. However, there was no measurable difference in the response of mock-infected and sequentially infected aged mice to de novo infection with either influenza virus or M. tuberculosis at 18-21 mo. Unexpectedly, a single experiment in which 25-mo-old female mice were challenged with influenza virus revealed a significantly higher survival rate for sequentially infected (80%) versus mock-infected (20%) mice. These data suggest that although exposure to a variety of pathogen challenges in the mouse model does not overtly impact cellular markers of immunity in aged female mice following de novo respiratory infection, subtle changes may emerge in other compartments or with increasing age.

Spontaneous episodic inflammation in the intestines of mice lacking HNF4A is driven by microbiota and associated with early life microbiota alterations.

The inflammatory bowel diseases (IBD) occur in genetically susceptible individuals who mount inappropriate immune responses to their microbiota leading to chronic intestinal inflammation. Whereas IBD clinical presentation is well described, how interactions between microbiota and host genotype impact early subclinical stages of the disease remains unclear. The transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) has been associated with human IBD, and deletion of Hnf4a in intestinal epithelial cells (IECs) in mice (Hnf4aΔIEC) leads to spontaneous colonic inflammation by 6-12 mo of age. Here, we tested if pathology in Hnf4aΔIEC mice begins earlier in life and if microbiota contribute to that process. Longitudinal analysis revealed that Hnf4aΔIEC mice reared in specific pathogen-free (SPF) conditions develop episodic elevated fecal lipocalin 2 (Lcn2) and loose stools beginning by 4-5 wk of age. Lifetime cumulative Lcn2 levels correlated with histopathological features of colitis at 12 mo. Antibiotic and gnotobiotic tests showed that these phenotypes in Hnf4aΔIEC mice were dependent on microbiota. Fecal 16S rRNA gene sequencing in SPF Hnf4aΔIEC and control mice disclosed that genotype significantly contributed to differences in microbiota composition by 12 mo, and longitudinal analysis of the Hnf4aΔIEC mice with the highest lifetime cumulative Lcn2 revealed that microbial community differences emerged early in life when elevated fecal Lcn2 was first detected. These microbiota differences included enrichment of a novel phylogroup of Akkermansia muciniphila in Hnf4aΔIEC mice. We conclude that HNF4A functions in IEC to shape composition of the gut microbiota and protect against episodic inflammation induced by microbiota throughout the lifespan. IMPORTANCE The inflammatory bowel diseases (IBD), characterized by chronic inflammation of the intestine, affect millions of people around the world. Although significant advances have been made in the clinical management of IBD, the early subclinical stages of IBD are not well defined and are difficult to study in humans. This work explores the subclinical stages of disease in mice lacking the IBD-associated transcription factor HNF4A in the intestinal epithelium. Whereas these mice do not develop overt disease until late in adulthood, we find that they display episodic intestinal inflammation, loose stools, and microbiota changes beginning in very early life stages. Using germ-free and antibiotic-treatment experiments, we reveal that intestinal inflammation in these mice was dependent on the presence of microbiota. These results suggest that interactions between host genotype and microbiota can drive early subclinical pathologies that precede the overt onset of IBD and describe a mouse model to explore those important processes.

The gut microbiome regulates astrocyte reaction to Aβ amyloidosis through microglial dependent and independent mechanisms

BackgroundPrevious studies show that antibiotic-mediated (abx) alteration of the gut microbiome (GMB) results in a reduction of amyloid beta (Aβ) plaques and proinflammatory microglial phenotype in male APPPS1-21 mice. However, the effect of GMB perturbation on astrocyte phenotypes and microglial-astrocyte communication in the context of amyloidosis has not been examined.MethodsTo study whether the GMB modulates astrocyte phenotype in the context of amyloidosis, APPPS1-21 male and female mice were treated with broad-spectrum abx leading to GMB perturbation. GFAP + astrocytes, plaque-associated astrocytes (PAA), PAA morphological parameters, and astrocyte complement component C3 levels were quantified using a combination of immunohistochemistry, immunoblotting, widefield microscopy, and confocal microscopy. Furthermore, these same astrocyte phenotypes were assessed in abx-treated APPPS1-21 male mice that received either fecal matter transplant (FMT) from untreated APPPS1-21 male donors to restore their microbiome or vehicle control. To assess complete absence of the GMB on astrocyte phenotypes, the same astrocyte phenotypes were quantified in APPPS1-21 male mice raised in germ-free (GF) or specific-pathogen free conditions (SPF). Lastly, we assessed whether microglia are necessary for abx-induced astrocyte phenotypes by depleting microglia in APPPS1-21 male mice via treatment with a colony-stimulating factor 1 receptor (CSF1R) inhibitor (PLX5622) and vehicle control or PLX5622 and abx.ResultsHerein, we demonstrate that postnatal treatment of male APPPS1-21 mice with broad-spectrum abx leading to GMB perturbation reduces GFAP + reactive astrocytes and PAAs, suggesting that the GMB plays a role in regulating reactive astrocyte induction and recruitment to Aβ plaques. Additionally, we show that compared to controls, PAAs in abx-treated male APPPS1-21 mice exhibit an altered morphology with increased number and length of processes and reduced astrocytic complement C3, consistent with a homeostatic phenotype. GFAP + astrocyte reduction, PAA reduction, astrocyte morphological changes, and C3 levels are restored when abx-treated mice are subject to FMT from untreated APPPS1-21 male donor mice. Next, we found that APPPS1-21 male mice raised in GF conditions have similar astrocyte phenotypes as abx-treated male APPPS1-21 male mice. Correlational analysis revealed that pathogenic bacteria depleted by abx correlate with GFAP + astrocytosis, PAAs, and astrocyte morphological changes. Finally, we determined that abx-mediated reduction in GFAP + astrocytosis, PAAs, and astrocytic C3 expression is independent of microglia. However, abx-induced astrocyte morphological alterations are dependent on the presence of microglia, suggesting that there is both microglial independent and dependent GMB control of reactive astrocyte phenotypes.ConclusionsWe show for the first time, in the context of amyloidosis, that the GMB plays an important role in controlling reactive astrocyte induction, morphology, and astrocyte recruitment to Aβ plaques. GMB regulation of these astrocytic phenotypes is both independent and dependent on microglia.

P-266 Study on the mechanisms of ovol1/2 during the morula-to-blastocyst transition

Abstract Study question Screen the key gene groups that determine the development of blastocyst. Explore the effect of ovol1/2 gene on early embryonic development. Summary answer We founded that there were insufficient activation of the Major ZGA genes in the AE group,and focused on candidate gene ovol1/2. What is known already At present, the clinical success rate of assisted reproductive technology (ART) is less than 50%. A large number of embryos cultured in vitro can not develop to blastocyst, and there are cleavage or morula stage arrest. The developmental arrest of preimplantation embryos is an important reason for treatment failure of infertile patients. However, the cause of early embryonic development arrest is not clear, and there is also a lack of effective methods to improve embryo quality. Study design, size, duration In this study, fertilized embryos were obtained through ICSI and a single cell from eight-cell embryo was biopsied through micromanipulation for single-cell RNA sequencing (scRNA-seq). Subsequently, we tracked the developmental potential of remaining cells and divided them into high-quality blastocyst(Bla) group and high-quality eight-cell failed blastocyst formation(arrest embryo, AE) group. In addition,we employed a RNAi approach to study the function of candidate gene ovol1/2, by injecting ovol1/2-targeting small interfering RNAs (siovol1/2) into wild-type (WT) zygotes. Participants/materials, setting, methods The study collected 21 human blastocysts and 5 arrest embryos. Human immature oocytes from ICSI treatments were clinically discarded and donated by women after signing informed consent by donors. C57B6 background mouse strains were used in this study. Mice were maintained under specific pathogen free conditions in a controlled environment of 20–22 °C,with a 12/12 h light/dark cycle, 50–70% humidity, and food and water provided. Main results and the role of chance The results showed that the gene expression of embryos in AE group was significantly different from that of Bla group: 79.5% of differential expression genes were down-regulated, including 48.3% of the Major ZGA gene. Then we focused on the candidate gene ovol1/2. When ovol1/2 were knocked down alone, there was no significant difference in the development rate of embryos from 2-cell to blastocyst between knockdown group and normal control group. When ovol1/2 were knocked down at the same time, there was no significant difference in the development rate of embryos among three groups before the morula stage. But in the morula developed to the blastocyst stage, there were significant differences among three groups of embryos: about 73% of embryos in the normal control group formed blastocyst; about 39% of the embryos in the low-dose knockdown group; the high-dose knockdown group embryos were all arrested in the morula stage. Further investigation revealed that the number of inner cell mass(ICM) cells in low-dose knockdown group did not change significantly, while the number of trophoblastic ectoderm(TE) cells significantly decreased. In addition, the TE functions of low-dose knockdown group such as blastocyst cavity size, paved area, hatching and adhesion rate were damaged. Limitations, reasons for caution Further research of the mechanisms is needed. Wider implications of the findings This is significant to understand molecular mechanisms of early embryonic development, and will provide important theoretical basis for clinical diagnosis and intervention of embryonic development arrest. Our study provides clues for the causes of abnormal blastocyst formation and new ideas for possible clinical improvements in the success rate of ART. Trial registration number not applicable

Intestinal epithelial transcription factor HNF4A protects against microbiota dependent colitis and expansion of Akkermansia muciniphila

Development of the inflammatory bowel diseases (IBD) has been linked to altered homeostatic relationships between the intestine and the microbiota it harbors. However early preclinical stages of IBD remain poorly understood, especially with respect to host-microbiota interactions. The nuclear receptor transcription factor Hepatocyte nuclear factor 4 alpha (HNF4A) has important roles in intestinal epithelial cell (IEC) physiology, and our recent studies in zebrafish and mice revealed that HNF4A activity in IEC is suppressed by intestinal microbiota. Variants at HNF4A have been associated with human IBD, and IEC-specific knockout of Hnf4a in mice ( Hnf4a ΔIEC ) leads to spontaneous colonic inflammation by 6-12 months. We hypothesized that intestinal pathology in Hnf4a ΔIEC mice may begin earlier in life and that microbiota may contribute to that process. Longitudinal analysis revealed that Hnf4a ΔIEC mice reared in specific pathogen-free (SPF) conditions develop fecal lipocalin 2 (LCN2) flares and episodic loose stools beginning as early as 4-5 weeks of age. Lifetime cumulative LCN2 levels correlated with histopathological features of colitis at 12 months of age. Antibiotic and gnotobiotic tests showed that these phenotypes in Hnf4a ΔIEC mice were dependent on the presence of microbiota. Fecal 16S rRNA gene sequencing in an SPF cohort of Hnf4a ΔIEC mice and controls at 12 months of age disclosed that genotype was a significant contributor to alterations in microbiota composition. Longitudinal analysis in a subset of inflamed Hnf4a ΔIEC and wild-type mice revealed that microbial community differences emerged at early life stages when fecal LCN2 flares first arose. These microbiota differences included a salient increase in the relative abundance of Akkermansia muciniphila in a subset of Hnf4a ΔIEC mice. We isolated an A. muciniphila strain from an aged, inflamed Hnf4a ΔIEC mouse, and its genome sequence revealed it to be a member of a new Akkermansia phylogroup with genetic features distinct from the known phylogroups. We conclude that HNF4A functions in IEC throughout the lifespan to protect against episodic flares of inflammation induced by microbiota while also shaping microbiota assembly. These results establish Hnf4a ΔIEC mice as a useful model for investigating preclinical pathology and host-microbiota interactions that precede overt IBD onset. NIH P01-DK094779 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of herbal tea (Platostoma palustre) on blood glucose regulation in vivo

Platostoma palustre jelly is a traditional food. Platostoma palustre has been used as folk medicine and is effective against heat-shock, hypertension and diabetes. Therefore, the aim of in vivo study was to determine the effects of herbal tea (Platostoma palustre) on blood glucose regulation. The commercial herbal tea (Platostoma palustre) was kindly provided by Yueta Agricultural Biotechnology Inc. Adult male 18 Institute of Cancer Research (ICR) mice [8 weeks old; body weight (BW) between 31-33 g] with specific pathogen-free conditions were used for this study. All ICR mice were divided respectively the normal control group (n = 6), the negative control group (n = 6), and the herbal tea group (n = 6). The ICR mice (the negative control group and the herbal tea group) were intraperitoneally injected with streptozotocin (65 mg/kg BW) and nicotinamide (230 mg/kg BW) for inducing the symptoms of hyperglycemia. In the herbal tea group, the herbal tea (10 mL/kg BW) was administrated to ICR mice by gavage. To monitor the blood glucose levels in ICR mice, blood was obtained from the tail of ICR mice, and blood glucose levels were determined using the external glucometer. Blood glucose measurements were conducted once in ICR mice before the experiment and were detected once per month after the experiment. Additionally, the change of ICR mice’ BW, the blood glucose of ICR mice, and the observation of ICR mice’ behavior were monitored and detected during the experiment. The results of this experiment showed · Weight change: the weight gain of the negative control group and the herbal tea group were significantly lower than those of the normal control group and there was no significant difference between the negative control group and the herbal tea group. · Fasting blood glucose and postprandial blood glucose: the fasting blood glucose of the negative control group was significantly higher than that of the normal control group, while the herbal tea group had a tendency to lower the fasting blood glucose, but there was no significant difference compared with the negative control group. The postprandial blood glucose level results showed that the negative control group was significantly higher than the normal control group. The postprandial blood glucose level of the herbal tea group was between the normal control group and the negative control group at the 4th week administration with the herbal tea. At the 8th week administration with the herbal tea, there was a significant difference compared with the negative control group. · Oral glucose tolerance test (OGTT): 4 to 12 weeks after hyperglycemia was induced, the blood glucose levels of the negative control group at each OGTT test point were significantly higher than those of the normal control group. The blood glucose level at the test point tended to decrease, but there was no significant difference from the negative control group. By calculating the area under the OGTT curve, it was found that the area under the curve of the negative control group was significantly higher than that of the normal control group, while the results of the herbal tea group at the 8th and 12th weeks were significantly lower than those of the negative control group. Taken all results together, the hyperglycemia was successfully induced in the experimental ICR mice. After administrating with the herbal tea, the fasting blood glucose level of the ICR mice tended to improve. In addition, a significant improvement was seen in the results of postprandial blood sugar and oral glucose tolerance test effectiveness. Therefore, based on the results of this experiment, it is speculated that drinking the herbal tea for 3 months has considerable potential for blood glucose regulation, which can be used as the basis for the development of related products of the herbal tea in the future.

Natural Microbial Exposure from the Earliest Natural Time Point Enhances Immune Development by Expanding Immune Cell Progenitors and Mature Immune Cells.

Microbial experience fundamentally shapes immunity, particularly during the perinatal period when the immune system is underdeveloped, and novel microbial encounters are common. Most animal models are raised in specific pathogen-free (SPF) conditions with relatively uniform microbial communities. How SPF housing conditions alter early-life immune development relative to natural microbial exposure (NME) has not been thoroughly investigated. In this article, we compare immune development in SPF-raised mice with mice born from immunologically experienced mothers in microbially diverse environments. NME induced broad immune cell expansion, including naive cells, suggesting mechanisms besides activation-induced proliferation contribute to the increase in immune cell numbers. We found NME conditions also expanded immune cell progenitor cell populations in the bone marrow, suggesting microbial experience enhances immune development at the earliest stages of immune cell differentiation. Multiple immune functions characteristically impaired in infants were also enhanced by NME, including T cell memory and Th1 polarization, B cell class switching and Ab production, proinflammatory cytokine expression, and bacterial clearance after Listeria monocytogenes challenge. Collectively, our studies reveal numerous impairments in immune development in SPF conditions relative to natural immune development.

Absence of gut microbiota impairs depletion of Paneth cells but not goblet cells in germ-free Atoh1lox/lox VilCreERT2 mice.

Mouse atonal homolog 1 (Math1/Atoh1) is a basic helix-loop-helix transcription factor important for the differentiation of secretory cells within the intestinal epithelium. The analysis of Paneth depletion efficiency on Math1lox/loxVilCreERT2 (Math1ΔIEC) mice treatment with tamoxifen in the presence or absence of intestinal microbiota showed a failure on Paneth cell depletion in germ-free mice as compared with specific pathogen-free (SPF) mice. However, goblet cells were efficiently depleted in Math1ΔIEC germ-free mice. The gene expression of Math1 was significantly reduced in the ileum of germ-free Math1ΔIEC mice 5 days after tamoxifen injection as compared with germ-free control, but its protein expression was still detectable in the nuclei of epithelial cells in the crypts. Germ-free mice showed low proliferative ileal crypts and apoptotic cells that were mainly detected in the tip of the villus, consistent with a slow turnover rate of epithelial cells. Although Paneth cells were not depleted in germ-free Math1ΔIEC mice for the first 7 wk after the last tamoxifen injection, far already from the 5 days time-laps observed in SPF conditions, an incomplete depletion of Paneth cells was observed 14 wk after the last tamoxifen injection. Colonization of germ-free mice restored the phenotype observed in SPF mice, highlighting the regulatory role of gut microbes in our model. We conclude that absence of intestinal microbiota in Math1ΔIEC mice is associated with reduced epithelial cell renewal and delays the depletion of preexisting Paneth cells.NEW & NOTEWORTHY Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1lox/loxVilCreERT2 mouse model.

The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked tothe induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for theregulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to theunderstanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

DIACC2010, Sole-in-Class Selective Inhibitor of Kinesin KIF20A, Has Potent Preclinical Efficacy in Acute Myeloid Leukemia

Context: Despite recent therapeutic advances in Acute Myeloid Leukemia (AML) and the decent proportion of complete remission after induction chemotherapy, relapse rate is still high and long-term survival does not exceed 20%. The therapeutic arsenal of AML could benefit from differentiated strategies. Mitotic kinesins are essential regulators of cancer cell replication and migration. The microtubule associated motor protein KIF20A (also called MKlp2), a member of the kinesin-6 family, plays an essential role during cytokinesis and is also involved in the fission of RAB6-positive vesicles from Golgi membrane, therefore contributing to intracellular vesicular trafficking. KIF20A plays a critical role in the development and progression of many cancers, and its high expression is associated with disease progression and poor survival outcome. More specifically, immature hematopoietic cells, exhibit high KIF20A expression, whereas mature peripheral blood cells do not. Herein we describe the preclinical anti-leukemic efficacy of DIACC2010, sole-in-class selective KIF20A inhibitor. Methods: We used cell lines (U-937, KG-1, CEM, KASUMI, SKM-1, MOLM-14, HL-60, MV-4-11, THP1) provided by ATCC and a Cell line-Derived Xenograft models to demonstrate the anti-leukemic activity od DIAC2010. Briefly, NOD.Cg-Prkdc scid/J (NSG) mice were obtained from Charles River France and maintained under specific pathogen-free conditions. Animals were injected via tail vein with 0.2 106 viable human leukemia cells. Mice were treated with 0.9% NaCl or Diacc2010 (200 mg/kg IV; once a week). Daily monitoring of mice for symptoms of disease determined the time of killing for animals with signs of distress. In addition, primary AML samples were obtained after informed consent. Cells were transplanted to NSG mice, collected after the 3rd transplantation and drug tests were performed ex vivo. All animal experiments were performed in compliance with the laws and in agreement with the French Guidelines for animal handling and approved by animal ethics committees (Agreement no. APAFIS#6743). Golgi imaging was performed using anti-GM130 antibody. Cell cycle, Caspase-Glo 3/7 and Annexin V/PI tests were performed according to the manufacturer's protocols. Results: DIACC2010 demonstrated potent and consistent cytotoxic activity in vitro against a panel of 9 human AML cell lines, with median IC50 of 40 nM (range 10-77 nM). In the same experimental conditions, cytarabine (CYTA) had median IC50 of 207 nM (range 4-1580 nM). DIACC2010 was subsequently evaluated in vivo in xenograft models of AML cell lines. DIACC2010 significantly improved overall survival as compared to CYTA in all models, with dose-dependent efficacy as shown in Figure A ( MOLM14 cells. Next, DIACC2010 cytotoxic activity was evaluated in vitro on 8 primary AML patient samples, IC50 ranged from 57 nM to 728 nM across the tested samples. DIACC2010 antitumoral activity is currently evaluated in Patients Derived Xenograft model. In parallel, no in vitro toxicity of DIACC2010 was observed on human normal cells such as peripheral blood mononuclear cells (PBMC) and primary hepatocytes (IC50 >50 µM). On a mechanistic standpoint, DIACC2010-exposed AML cells displayed characteristic Golgi scattering, observed within 1 hour of treatment and majoring at 6 hours confirming target engagement (Figure B). In parallel, we observed a strong block in cell cycle in G2/M after 6 hours of treatment (45% of cells versus 21% in the control condition). This was associated with a high caspase activation that occurred mainly after 24 hours of treatment. This cascade led to cell death assessed by annexin V/PI between 24 and 48 hours after treatment initiation. Nevertheless, caspase inhibitors ZVAD-fmk and Emricasan partially rescued DIACC2010 cell death, although these inhibitors significantly rescued caspase activation induced by Venetoclax, a BCL-2 inhibitor triggering intrinsic apoptosis commonly used in AML treatment. We ruled out necroptosis and ferroptosis as cell death pathways triggered by DIACC2010 using pharmacological inhibitors. These data strongly suggests that DIACC2010 induces a non-redundant cell death pathway that could be advantageously used to kill resistant AML cells. Conclusion: Altogether, these results confirm the relevance of KIF20A-directed therapeutic approaches and support the development of DIACC2010 for the treatment of AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A gut-oral microbiome-driven axis controls oropharyngeal candidiasis through retinoic acid.

A side effect of antibiotics is outgrowth of the opportunistic fungus Candida albicans in the oropharynx (oropharyngeal candidiasis, OPC). IL-17 signaling is vital for immunity to OPC, but how the microbiome impacts antifungal immunity is not well understood. Mice in standard specific pathogen-free (SPF) conditions are resistant to OPC, whereas we show that germ-free (GF) or antibiotic-treated mice are susceptible. Oral type 17 cells and IL-17-dependent responses were impaired in antibiotic-treated and GF mice. Susceptibility could be rescued in GF mice by mono-colonization with segmented filamentous bacterium (SFB), an intestine-specific constituent of the microbiota. SFB protection was accompanied by restoration of oral IL-17+CD4+ T cells and gene signatures characteristic of IL-17 signaling. Additionally, RNA-Seq revealed induction of genes in the retinoic acid (RA) and RA receptor-α (RARα) pathway. Administration of RA rescued immunity to OPC in microbiome-depleted or GF mice, while RAR inhibition caused susceptibility in immunocompetent animals. Surprisingly, immunity to OPC was independent of serum amyloids. Moreover, RAR inhibition did not alter oral type 17 cytokine levels. Thus, mono-colonization with a component of the intestinal microflora confers protection against OPC by type 17 and RA/RARα, which act in parallel to promote antifungal immunity. In principle, manipulation of the microbiome could be harnessed to maintain antifungal immunity.

Myeloperoxidase deficiency impacts the composition of the gut microbiome: a case for variable disease penetrance?

Abstract The most abundant proteolytic enzyme found in neutrophilic granules, myeloperoxidase (MPO) is a heme peroxidase with microbicidal activity. While MPO inhibition has therapeutic promise for treatment of inflammatory illness, differential disease penetrance has been observed in MPO-deficient mice during various disease models. In our studies using the DSS-colitis mouse model, MPO-deficient mice under conventional specific pathogen-free (SPF) conditions experienced similar disease penetrance to co-housed, wildtype controls. In contrast, when housed in restricted SPF conditions lacking norovirus and Helicobacter species, MPO-deficient mice experienced more severe colitis while wildtype mice were unchanged. From these data, we hypothesized that an interplay between MPO (microbicidal activity) and the composition of the gut microbiome impacts disease penetrance of colitis. To investigate, we performed 16S rRNA-sequencing on the intestines of MPO-deficient and wildtype mice housed in restricted SPF conditions and found a proportionate increase in Ruminococcus gnavus, which is associated with intestinal dysbiosis. Correspondingly, we found proportionate decreases in the gut beneficial bacteria Akkermansia muciniphilia and Prevotella sp. These data show that MPO does affect the composition of the gut bacteria, and supports an interplay between inflammatory mediators, the microbiome, and altered disease penetrance.

Th17-driven immunity to oral candidiasis is dependent on the microbiome and can be triggered by mono-colonization with segmented filamentous bacteria.

Abstract The opportunistic infection oropharyngeal candidiasis (OPC; oral thrush) is a common oral infection among the immunocompromised. The oral cavity is a major entry portal to the body and oral health impacts several aspects of host immunity. While much is now known about microbiome dependent Th17 induction and protection against intestinal candidiasis and other Th17 driven responses, our understanding of the role of the microbiome in oral Th17 induction and oral antifungal immunity is rather rudimentary. We demonstrate a direct role for the microbiome in triggering protection against OPC. While wildtype (WT) mice raised under specific pathogen free (SPF) conditions are resistant to OPC, WT germ free (GF) mice succumbed to infection. Reconstitution of WT-GF mice with segmented filamentous bacteria (SFB) was sufficient to restore protection against OPC with associated increase in Th17 cells, neutrophils, and antimicrobial responses in the oral mucosa. Thus, SFB mono-colonization in WT-GF mice triggers oral mucosal Th17 induction to protect against oral thrush and reveals a role of the microbiome in promoting oral mucosal immunity. Supported by R37-DE022550