A distal enhancer of GATA3 regulates Th2 differentiation and allergic inflammation.

Asthma is a widespread airway disorder where GATA3-dependent Type-2 helper T (Th2) cells and group 2 innate lymphoid cells (ILC2s) play vital roles. Asthma-associated single nucleotide polymorphisms (SNPs) are enriched in a region located 926-970 kb downstream from GATA3 in the 10p14 (hG900). However, it is unknown how hG900 affects the pathogenesis of allergic airway inflammation. To investigate the roles of the asthma-associated GATA3 enhancer region in experimental allergic airway inflammation, we first examined the correlation between GATA3 expression and the activation of the hG900 region was analyzed by flow cytometry and ChIP-qPCR. We found that The activation of enhancers in the hG900 region was strongly correlated to the levels of GATA3 in human peripheral T cell subsets. We next generated mice lacking the mG900 region (mG900KO mice) were generated by the CRISPR-Cas9 system, and the development and function of helper T cells and ILCs in mG900KO mice were analyzed in steady-state conditions and allergic airway inflammation induced by papain or house dust mite (HDM). The deletion of the mG900 did not affect the development of lymphocytes in steady-state conditions or allergic airway inflammation induced by papain. However, mG900KO mice exhibited reduced allergic inflammation and Th2 differentiation in the HDM-induced allergic airway inflammation. The analysis of the chromatin conformation around Gata3 by circular chromosome conformation capture coupled to high-throughput sequencing (4C-seq) revealed that the mG900 region interacted with the transcription start site of Gata3 with an influencing chromatin conformation in Th2 cells. These findings indicate that the mG900 region plays a pivotal role in Th2 differentiation and thus enhances allergic airway inflammation.

Recombinant thrombomodulin attenuates hyper-inflammation and glycocalyx damage in a murine model of Streptococcus pneumoniae-induced sepsis.

PURPOSE: The anticoagulant agent recombinant thrombomodulin (rTM) activates protein C to prevent excessive coagulation and also possibly regulates hyper-inflammation via neutralization of high-mobility-group B1 (HMG-B1). The glycocalyx layer in endothelial cells also plays a pivotal role in preventing septic shock-associated hyperpermeability. The present study examined the effect of rTM in a murine model of Streptococcus pneumoniae-induced sepsis. METHODS: Male C57BL/6N mice were injected intratracheally via midline cervical incision with 2 × 107 CFU of S. pneumoniae (capsular subtype 19A). Control mice were sham-treated identically but injected with saline. rTM (10 mg/kg) was injected intraperitoneally 3 h after septic insult. Blood concentrations of soluble inflammatory mediators (interleukin [IL]-1ß, IL-6, IL-10, and tumor necrosis factor [TNF]-α) were determined using a microarray immunoassay. Serum concentrations of HMG-B1 and syndecan-1, as a parameter of glycocalyx damage, were determined by enzyme-linked immunosorbent assay. The glycocalyx was also evaluated with electron microscopy. The lungs were removed, and digested to cells, which were then stained with a mixture of fluorophore-conjugated antibodies. Anti-mouse primary antibodies included PE-Cy7-conjugated anti-CD31, AlexaFluor 700-conjugated anti-CD45, PerCP-Cy5.5-conjugated anti-CD326, APC-conjugated anti-TNF-α, PE-conjugated anti-IL-6, and PE-conjugated anti-IL-10. A total of 1 × 106 cells per sample were analyzed, and 2 × 105 events were recorded by flow cytometry, and parameters were compared with/without rTM treatment. RESULTS: The blood concentration of TNF-α was significantly reduced 24 h after intratracheal injection in S. pneumoniae-challenged mice treated with rTM (P = 0.016). Levels of IL-10 in the lung endothelium of rTM-treated S. pneumoniae-challenged mice increased significantly 12 h after intratracheal injection (P = 0.03). Intriguingly, serum HMGB-1 and syndecan-1 levels decreased significantly (P = 0.010 and 0.015, respectively) in rTM-treated mice 24 h after intratracheal injection of S. pneumoniae. Electron microscopy indicated that rTM treatment preserved the morphology of the glycocalyx layer in septic mice. CONCLUSIONS: These data suggest that rTM modulates local inflammation in the lung endothelium, thus diminishing systemic inflammation, i.e., hypercytokinemia. Furthermore, rTM treatment reduced serum syndecan-1 levels, thus preventing glycocalyx damage. The use of rTM to treat sepsis caused by bacterial pneumonia could therefore help prevent both excessive inflammation and glycocalyx injury in the lung endothelium.

Frequency and function of circulating regulatory T-cells in biliary atresia.

PURPOSE: Although the impairment of regulatory T-cells (Tregs) has been shown in the liver or portal area of biliary atresia (BA) the frequency and function of circulating Tregs in BA patients is poorly understood. We aimed to investigate the frequency and function of circulating Tregs in BA patients. METHODS: Peripheral blood mononuclear cells were collected from 25 BA patients and 24 controls. Treg frequency was measured by flow cytometry; function was determined by T-cell proliferation assay. We also assessed the association between Treg frequency/function and clinical parameters in BA cases. RESULTS: There was no significant difference between the two groups in both frequency (BA: 3.4%; control: 3.2%; p = 0.97) and function (BA: 22.0%; control: 7.5%; p = 0.23) of Tregs. We further focused on 13 preoperative BA patients and 14 age-matched controls. Neither Treg frequency nor function were significantly different (frequency: BA: 4.6%; control: 3.4%; p = 0.38, function: BA: 2.7%; control: 7.6%; p = 0.89). There was no association between Treg frequency/function and clinical parameters. CONCLUSION: Neither the frequency nor function of circulating Tregs was affected in BA patients, suggesting the negative role of circulating Tregs in the pathogenesis of BA. Further investigation of local Treg profiles is warranted.

Development of chronic allergic responses by dampening Bcl6-mediated suppressor activity in memory T helper 2 cells.

Mice deficient in the transcriptional repressor B-cell CLL/lymphoma 6 (Bcl6) exhibit similar T helper 2 (TH2) immune responses as patients with allergic diseases. However, the molecular mechanisms underlying Bcl6-directed regulation of TH2 cytokine genes remain unclear. We identified multiple Bcl6/STAT binding sites (BSs) in TH2 cytokine gene loci. We found that Bcl6 is modestly associated with the BSs, and it had no significant effect on cytokine production in newly differentiated TH2 cells. Contrarily, in memory TH2 (mTH2) cells derived from adaptively transferred TH2 effectors, Bcl6 outcompeted STAT5 for binding to TH2 cytokine gene loci, particularly Interleukin4 (Il4) loci, and attenuated GATA binding protein 3 (GATA3) binding to highly conserved intron enhancer regions in mTH2 cells. Bcl6 suppressed cytokine production epigenetically in mTH2 cells to negatively tune histone acetylation at TH2 cytokine gene loci, including Il4 loci. In addition, IL-33, a pro-TH2 cytokine, diminished Bcl6's association with loci to which GATA3 recruitment was inversely augmented, resulting in altered IL-4, but not IL-5 and IL-13, production in mTH2 cells but no altered production in newly differentiated TH2 cells. Use of a murine asthma model that generates high levels of pro-TH2 cytokines, such as IL-33, suggested that the suppressive function of Bcl6 in mTH2 cells is abolished in severe asthma. These findings indicate a role of the interaction between TH2-promoting factors and Bcl6 in promoting appropriate IL-4 production in mTH2 cells and suggest that chronic allergic diseases involve the TH2-promoting factor-mediated functional breakdown of Bcl6, resulting in allergy exacerbation.

Deletion of Eqtn in mice reduces male fertility and sperm-egg adhesion.

A number of sperm proteins are involved in the processes from gamete adhesion to fusion, but the underlying mechanism is still unclear. Here, we established a mouse mutant, the EQUATORIN-knockout (EQTN-KO, Eqtn - / - ) mouse model and found that the EQTN-KO males have reduced fertility and sperm-egg adhesion, while the EQTN-KO females are fertile. Eqtn - / - sperm were normal in morphology and motility. Eqtn - / - -Tg (Acr-Egfp) sperm, which were produced as the acrosome reporter by crossing Eqtn - / - with Eqtn +/+ -Tg(Acr-Egfp) mice, traveled to the oviduct ampulla and penetrated the egg zona pellucida of WT females. However, Eqtn - / - males mated with WT females showed significant reduction in both fertility and the number of sperm attached to the zona-free oocyte. Sperm IZUMO1 and egg CD9 behaved normally in Eqtn - / - sperm when they were fertilized with WT egg. Another acrosomal protein, SPESP1, behaved aberrantly in Eqtn - / - sperm during the acrosome reaction. The fertility impairment of EQTN/SPESP1-double KO males lacking Eqtn and Spesp1 (Eqtn/Spesp1 - / - ) was more severe compared with that of Eqtn - / - males. Eqtn - / - -Tg (Eqtn) males, which were generated to rescue Eqtn - / - males, restored the reduced fertility.

Suppression of T Cell Autophagy Results in Decreased Viability and Function of T Cells Through Accelerated Apoptosis in a Murine Sepsis Model.

OBJECTIVE: While type 1 programmed cell death (apoptosis) of T cells leads to immunosuppression in sepsis, a crosstalk between apoptosis and autophagy (type 2 programmed cell death) has not been shown. The aim of this study is to elucidate the details of the interaction between autophagy and immunosuppression. DESIGN: Laboratory investigation in the murine sepsis model. SETTING: University laboratory. SUBJECTS: Six- to 8-week-old male mice. INTERVENTIONS: We investigated the kinetics of autophagy in T cells from spleen in a cecal ligation and puncture model with green fluorescent protein-microtubule-associated protein light chain 3 transgenic mice. We analyzed apoptosis, mitochondrial homeostasis and cytokine production in T cells, and survival rate after cecal ligation and puncture using T cell-specific autophagy-deficient mice. MEASUREMENTS AND MAIN RESULTS: We observed an increase of autophagosomes, which was assessed by flow cytometry. However, an autophagy process in CD4 T cells during sepsis was insufficient including the accumulation of p62. On the other hand, a blockade of autophagy accelerated T cell apoptosis compared with the control mice, augmenting the gene expression of Bcl-2-like 11 and programmed cell death 1. Furthermore, mitochondrial accumulation in T cells occurred via a blockade of autophagy during sepsis. In addition, interleukin-10 production in CD4 T cells from the cecal ligation and puncture-operated knockout mice was markedly increased. Consequently, deficiency of autophagy in T cells significantly decreased the survival rate in the murine sepsis model. CONCLUSIONS: We demonstrated that blocking autophagy accelerated apoptosis and increased mortality in concordance with the insufficient autophagy process in CD4 T cells in the murine sepsis model, suggesting that T cell autophagy plays a protective role against apoptosis and immunosuppression in sepsis.

Dual Strands of Pre-miR-149 Inhibit Cancer Cell Migration and Invasion through Targeting FOXM1 in Renal Cell Carcinoma.

Our recent studies revealed that dual strands of certain pre-microRNAs, e.g., pre-miR-144, pre-miR-145, and pre-miR-150, act as antitumor microRNAs (miRNAs) in several cancers. The involvement of passenger strands of miRNAs in cancer pathogenesis is a novel concept in miRNA research. The analysis of a miRNA expression signature in clear cell renal cell carcinoma (ccRCC) has revealed that the guide strand of pre-miR-149 is significantly downregulated in cancer tissues. The aims of this study were to investigate the functional significance of miR-149's guide strand (miR-149-5p) and passenger strand (miR-149-3p), and to identify the oncogenic genes regulated by these miRNAs in ccRCC cells. The ectopic expression of these miRNAs significantly inhibited cancer cell migration and invasion in ccRCC cells. Forkhead box protein M1 (FOXM1) was directly regulated by miR-149-5p and miR-149-3p in ccRCC cells. Knockdown studies using si-FOXM1 showed that the expression of FOXM1 enhanced RCC cell aggressiveness. Interestingly, the analysis of a large number of patients in the The Cancer Genome Atlas (TCGA) database (n = 260) demonstrated that patients with high FOXM1 expression had significantly shorter survival than did those with low FOXM1 expression (p = 1.5 × 10⁻6). Taken together, dual strands of pre-miR-149 (miR-149-5p and miR-149-3p) acted as antitumor miRNAs through the targeting of FOXM1 in ccRCC cells.

Nepro is localized in the nucleolus and essential for preimplantation development in mice.

We generated knockout (KO) mice of Nepro, which has been shown to be necessary to maintain neural progenitor cells downstream of Notch in the mouse developing neocortex by using knockdown experiments, to explore its function in embryogenesis. Nepro KO embryos were morphologically indistinguishable from wild type (WT) embryos until the morula stage but failed in blastocyst formation, and many cells of the KO embryos resulted in apoptosis. We found that Nepro was localized in the nucleolus at the blastocyst stage. The number of nucleolus precursor bodies (NPBs) and nucleoli per nucleus was significantly higher in Nepro KO embryos compared with WT embryos later than the 2-cell stage. Furthermore, at the morula stage, whereas 18S rRNA and ribosomal protein S6 (rpS6), which are components of the ribosome, were distributed to the cytoplasm in WT embryos, they were mainly localized in the nucleoli in Nepro KO embryos. In addition, in Nepro KO embryos, the amount of the mitochondria-associated p53 protein increased, and Cytochrome c was distributed in the cytoplasm. These findings indicate that Nepro is a nucleolus-associated protein, and its loss leads to the apoptosis before blastocyst formation in mice.

Stage-specific GATA3 induction promotes ILC2 development after lineage commitment.

Group 2 innate lymphoid cells (ILC2s) are a subset of innate lymphocytes that produce type 2 cytokines, including IL-4, IL-5, and IL-13. GATA3 is a critical transcription factor for ILC2 development at multiple stages. However, when and how GATA3 is induced to the levels required for ILC2 development remains unclear. Herein, we identify ILC2-specific GATA3-related tandem super-enhancers (G3SE) that induce high GATA3 in ILC2-committed precursors. G3SE-deficient mice exhibit ILC2 deficiency in the bone marrow, lung, liver, and small intestine with minimal impact on other ILC lineages or Th2 cells. Single-cell RNA-sequencing and subsequent flow cytometry analysis show that GATA3 induction mechanism, which is required for entering the ILC2 stage, is lost in IL-17RB+PD-1- late ILC2-committed precursor stage in G3SE-deficient mice. Cnot6l, part of the CCR4-NOT deadenylase complex, is a possible GATA3 target during ILC2 development. Our findings implicate a stage-specific regulatory mechanism for GATA3 expression during ILC2 development.

Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy.

Spermatids must precisely integrate specific molecules into structurally supported domains that develop during spermatogenesis. Once established, the architecture of the acrosome contributes to the acrosome reaction, which occurs prior to gamete interaction in mammals. The present study aims to clarify the morphology associated with the integration of the mouse fertilization-related acrosomal protein equatorin (mEQT) into the developing acrosome. EQT mRNA was first detected by in situ hybridization in round spermatids but disappeared in early elongating spermatids. The molecular size of mEQT was approximately 65 kDa in the testis. Developmentally, EQT protein was first detected on the nascent acrosomal membrane in round spermatids at approximately step 3, was actively integrated into the acrosomal membranes of round spermatids in the following step and then participated in acrosome remodeling in elongating spermatids. This process was clearly visualized by high-resolution fluorescence microscopy and super-resolution stimulated emission depletion nanoscopy by using newly generated C-terminally green-fluorescent-protein-tagged mEQT transgenic mice. Immunogold electron microscopy revealed that mEQT was anchored to the acrosomal membrane, with the epitope region observed as lying 5-70 nm away from the membrane and was associated with the electron-dense acrosomal matrix. This new information about the process of mEQT integration into the acrosome during spermatogenesis should provide a better understanding of the mechanisms underlying not only acrosome biogenesis but also fertilization and male infertility.

Kinetics and protective role of autophagy in a mouse cecal ligation and puncture-induced sepsis.

INTRODUCTION: It is not well understood whether the process of autophagy is accelerated or blocked in sepsis, and whether it is beneficial or harmful to the immune defense mechanism over a time course during sepsis. Our aim was to determine both the kinetics and the role of autophagy in sepsis. METHODS: We examined autophagosome and autolysosome formation in a cecal ligation and puncture (CLP) mouse model of sepsis (in C57BL/6N mice and GFP-LC3 transgenic mice), using western blotting, immunofluorescence, and electron microscopy. We also investigated the effect of chloroquine inhibition of autophagy on these processes. RESULTS: Autophagy, as demonstrated by increased LC3-II/LC3-I ratios, is induced in the liver, heart, and spleen over 24 h after CLP. In the liver, autophagosome formation peaks at 6 h and declines by 24 h. Immunofluorescent localization of GFP-LC3 dots (alone and with lysosome-associated membrane protein type 1 (LAMP1)), as well as electron microscopic examination, demonstrate that both autophagosomes and autolysosomes are increased after CLP, suggesting that intact autophagy mechanisms operate in the liver in this model. Furthermore, inhibition of autophagy process by chloroquine administration immediately after CLP resulted in elevated serum transaminase levels and a significant increase in mortality. CONCLUSIONS: All autophagy-related processes are properly activated in the liver in a mouse model of sepsis; autophagy appears to play a protective role in septic animals.

Tumor suppressive microRNA-133a regulates novel targets: moesin contributes to cancer cell proliferation and invasion in head and neck squamous cell carcinoma.

Recently, many studies suggest that microRNAs (miRNAs) contribute to the development, invasion and metastasis of various types of human cancers. Our recent study revealed that expression of microRNA-133a (miR-133a) was significantly reduced in head and neck squamous cell carcinoma (HNSCC) and that restoration of miR-133a inhibited cell proliferation, migration and invasion in HNSCC cell lines, suggesting that miR-133a function as a tumor suppressor. Genome-wide gene expression analysis of miR-133a transfectants and TargetScan database showed that moesin (MSN) was a promising candidate of miR-133a target gene. MSN is a member of the ERM (ezrin, radixin and moesin) protein family and ERM function as cross-linkers between plasma membrane and actin-based cytoskeleton. The functions of MSN in cancers are controversial in previous reports. In this study, we focused on MSN and investigated whether MSN was regulated by tumor suppressive miR-133a and contributed to HNSCC oncogenesis. Restoration of miR-133a in HNSCC cell lines (FaDu, HSC3, IMC-3 and SAS) suppressed the MSN expression both in mRNA and protein level. Silencing study of MSN in HNSCC cell lines demonstrated significant inhibitions of cell proliferation, migration and invasion activities in si-MSN transfectants. In clinical specimen with HNSCC, the expression level of MSN was significantly up-regulated in cancer tissues compared to adjacent non-cancerous tissues. These data suggest that MSN may function as oncogene and is regulated by tumor suppressive miR-133a. Our analysis data of novel tumor-suppressive miR-133a-mediated cancer pathways could provide new insights into the potential mechanisms of HNSCC oncogenesis.

Tumor suppressive microRNA-375 regulates oncogene AEG-1/MTDH in head and neck squamous cell carcinoma (HNSCC).

Our microRNA (miRNA) expression signatures of hypopharyngeal squamous cell carcinoma, maxillary sinus squamous cell carcinoma and esophageal squamous cell carcinoma revealed that miR-375 was significantly reduced in cancer tissues compared with normal epithelium. In this study, we focused on the functional significance of miR-375 in cancer cells and identification of miR-375-regulated novel cancer networks in head and neck squamous cell carcinoma (HNSCC). Restoration of miR-375 showed significant inhibition of cell proliferation and induction of cell apoptosis in SAS and FaDu cell lines, suggesting that miR-375 functions as a tumor suppressor. We adopted genome-wide gene expression analysis to search for miR-375-regulated molecular targets. Gene expression data and luciferase reporter assays revealed that AEG-1/MTDH was directly regulated by miR-375. Cancer cell proliferation was significantly inhibited in HNSCC cells transfected with si-AEG-1/MTDH. In addition, expression levels of AEG-1/MTDH were significantly upregulated in cancer tissues. Therefore, AEG-1/MTDH may function as an oncogene in HNSCC. The identification of novel tumor suppressive miRNA and its regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC oncogenesis.

Genetic background-dependent abnormalities of the enteric nervous system and intestinal function in Kif26a-deficient mice.

The Kif26a protein-coding gene has been identified as a negative regulator of the GDNF-Ret signaling pathway in enteric neurons. The aim of this study was to investigate the influence of genetic background on the phenotype of Kif26a-deficient (KO, -/-) mice. KO mice with both C57BL/6 and BALB/c genetic backgrounds were established. Survival rates and megacolon development were compared between these two strains of KO mice. Functional bowel assessments and enteric neuron histopathology were performed in the deficient mice. KO mice with the BALB/c genetic background survived more than 400 days without evidence of megacolon, while all C57BL/6 KO mice developed megacolon and died within 30 days. Local enteric neuron hyperplasia in the colon and functional bowel abnormalities were observed in BALB/c KO mice. These results indicated that megacolon and enteric neuron hyperplasia in KO mice are influenced by the genetic background. BALB/c KO mice may represent a viable model for functional gastrointestinal diseases such as chronic constipation, facilitating studies on the underlying mechanisms and providing a foundation for the development of treatments.

miR-145, miR-133a and miR-133b: Tumor-suppressive miRNAs target FSCN1 in esophageal squamous cell carcinoma.

MicroRNAs (miRNAs), noncoding RNAs 21-25 nucleotides in length, regulate gene expression primarily at the posttranscriptional level. Growing evidence suggests that miRNAs are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. A search for miRNAs with a tumor-suppressive function in esophageal squamous cell carcinoma (ESCC) was performed using the miRNA expression signatures obtained from ESCC clinical specimens. A subset of 15 miRNAs was significantly downregulated in ESCC. A comparison of miRNA signatures from ESCC and our previous report identified 4 miRNAs that are downregulated in common (miR-145, miR-30a-3p, miR-133a and miR-133b), suggesting that these miRNAs are candidate tumor suppressors. Gain-of-function analysis revealed that 3 transfectants (miR-145, miR-133a and miR-133b) inhibit cell proliferation and cell invasion in ESCC cells. These miRNAs (miR-145, miR-133a and miR-133b), which have conserved sequences in the 3'UTR of FSCN1 (actin-binding protein, Fascin homolog 1), inhibited FSCN1 expression. The signal from a luciferase reporter assay was significantly decreased at 2 miR-145 target sites and 1 miR-133a/b site, suggesting both miRNAs directly regulate FSCN1. An FSCN1 loss-of-function assay found significant cell growth and invasion inhibition, implying an FSCN1 is associated with ESCC carcinogenesis. The identification of tumor-suppressive miRNAs, miR-145, miR-133a and miR-133b, directly control oncogenic FSCN1 gene. These signal pathways of ESCC could provide new insights into potential mechanisms of ESCC carcinogenesis.

Development of Noninvasive in Vivo Approach to Assess Vascular Permeability in Inflammation Using Fluorescence Imaging.

INTRODUCTION: In vivo fluorescence imaging can quantify vascular permeability without requiring sacrifice of animals. However, use of this noninvasive approach for vascular permeability assessment in remote organ injury caused by systemic inflammatory disease has not been reported. METHODS: Evans blue (EB) and Genhance 750 fluorescent dye were mixed and injected into mice. The lung as a remote organ and the footpad as a noninvasive observational site were assessed in a cecal ligation and puncture (CLP)-induced systemic inflammation mouse model and compared with sham and hydrocortisone pretreated (CLP + HC) mouse models. Extraction of EB in harvested tissues was assessed as a conventional indicator of vascular permeability. Fluorescent intensities in the footpad or harvested lung were assessed and their correlation was analyzed to investigate this novel, noninvasive approach for estimation of lung vascular permeability. RESULTS: Fluorescent intensity in the footpad and harvested lung in the CLP group was significantly higher than in the other groups (footpad, sham vs. CLP, P < 0.0001; CLP vs. CLP + HC, P = 0.0004; sham vs. CLP + HC, P = 0.058; lung, sham vs. CLP, P < 0.0001; CLP vs. CLP + HC, P < 0.0001; sham vs. CLP + HC, P = 0.060). The fluorescent intensity in the footpad was strongly correlated with that in the lung (r = 0.95). CONCLUSIONS: This fluorescent technique may be useful for vascular permeability assessment based on EB quantification. Footpad fluorescent intensity was strongly correlated with that in the lung, and may be a suitable indicator in noninvasive estimation of lung vascular permeability.

Influence of autophagy on acute kidney injury in a murine cecal ligation and puncture sepsis model.

The role of autophagy in the maintenance of renal homeostasis during sepsis is not well understood. We therefore aimed to determine the influence of autophagy on kidney during sepsis using a murine sepsis model, i.e. cecal ligation and puncture (CLP). In CLP treated animals, the number of autolysosomes observed by electron microscopy increased over time. The number of autophagosomes in CLP animals decreased relative sham operated controls at 24 hrs after CLP, indicating that autophagy flux is already diminishing by that time. Moreover, CLP induced an increase in LC3-II/LC3-I ratio at 6-8 hrs, demonstrated in western blots, as well as an increase in GFP-LC3 dots at 6-8 hrs and 24 hrs, using immunofluorescence and anti-LC3 and LAMP1 antibodies on tissue sections from GFP-LC3 transgenic mice. LC3-II/LC3-I ratio and the number of co-localized GFP-LC3 dots and LAMP1 signals (GFP LC3 + LAMP1 dots) in CLP mice at 24 hrs were significantly reduced compared with data obtained at 6-8 hrs. Notably, acceleration of autophagy by rapamycin resulted in improvement of renal function that was associated with improvement in the histologic severity of tubular epithelial injury in CLP treated animals. Autophagy in the kidney was significantly slowed in the kidney during the acute phase of sepsis; nonetheless, autophagy in kidney appears to play a protective role against sepsis.

Blocking Liver Autophagy Accelerates Apoptosis and Mitochondrial Injury in Hepatocytes and Reduces Time to Mortality in a Murine Sepsis Model.

Autophagy plays an important role in cell survival, sequestering, and degrading a wide variety of substrates. Although an increase of autophagosomes in liver has been reported in sepsis patients as well as in septic mice, the influence of autophagy on liver injury, the interaction between autophagy, and other types of cell death in sepsis remain unclear. The aim of this study was to elucidate the contribution of liver autophagy to the pathophysiology of sepsis. We performed a cecal ligation and puncture on liver-specific autophagy-deficient (Alb-Cre/Atg5) mice (6-8-week-old male). When compared with controls (C57BL/6), we found a significant accumulation of p62 in the liver and demonstrated a greater number of cleaved caspase-3 immunoreactive hepatocytes in these knockout (KO) mice. Additionally, we confirmed a significant increase in autophagic vacuoles in the control mice relative to KO mice; in contrast, cell shrinkage and nuclear fragmentation (morphological characteristics of apoptosis) were preferentially seen in the KO mice by transmission electron microscopy. Severe mitochondrial damage was also prominent in KO mice, relative to controls, associated with an increase of reactive oxygen species in hepatocytes. Serum aspartate transaminase levels (P = 0.005) and serum interleukin-6 levels (P = 0.020) were significantly increased in the KO mice compared with controls. Deficiency of autophagy in liver significantly decreased survival in the murine sepsis model (P = 0.025). In conclusion, blocking liver autophagy accelerates time to mortality in the murine sepsis model, suggesting that liver autophagy plays a protective role for organ failure through degradation of damaged mitochondria, as well as prevention of apoptosis.

Allergic TH2 Response Governed by B-Cell Lymphoma 6 Function in Naturally Occurring Memory Phenotype CD4+ T Cells.

Transcriptional repressor B-cell lymphoma 6 (Bcl6) appears to regulate TH2 immune responses in allergies, but its precise role is unclear. We previously reported that Bcl6 suppressed IL-4 production in naïve CD4+ T cell-derived memory TH2 cells. To investigate Bcl6 function in allergic responses in naturally occurring memory phenotype CD4+ T (MPT) cells and their derived TH2 (MPTH2) cells, Bcl6-manipulated mice, highly conserved intron enhancer (hcIE)-deficient mice, and reporter mice for conserved noncoding sequence 2 (CNS2) 3' distal enhancer region were used to elucidate Bcl6 function in MPT cells. The molecular mechanisms of Bcl6-mediated TH2 cytokine gene regulation were elucidated using cellular and molecular approaches. Bcl6 function in MPT cells was determined using adoptive transfer to naïve mice, which were assessed for allergic airway inflammation. Bcl6 suppressed IL-4 production in MPT and MPTH2 cells by suppressing CNS2 enhancer activity. Bcl6 downregulated Il4 expression in MPTH2 cells, but not MPT cells, by suppressing hcIE activity. The inhibitory functions of Bcl6 in MPT and MPTH2 cells attenuated allergic responses. Bcl6 is a critical regulator of IL-4 production by MPT and MPTH2 cells in TH2 immune responses related to the pathogenesis of allergies.