RACK1 enhances STAT3 stability and promotes T follicular helper cell development and function during blood-stage Plasmodium infection in mice.

CD4+ T cells are central mediators of protective immunity to blood-stage malaria, particularly for their capacity in orchestrating germinal center reaction and generating parasite-specific high-affinity antibodies. T follicular helper (Tfh) cells are predominant CD4+ effector T cell subset implicated in these processes, yet the factors and detailed mechanisms that assist Tfh cell development and function during Plasmodium infection are largely undefined. Here we provide evidence that receptor for activated C kinase 1 (RACK1), an adaptor protein of various intracellular signals, is not only important for CD4+ T cell expansion as previously implied but also plays a prominent role in Tfh cell differentiation and function during blood-stage Plasmodium yoelii 17XNL infection. Consequently, RACK1 in CD4+ T cells contributes significantly to germinal center formation, parasite-specific IgG production, and host resistance to the infection. Mechanistic exploration detects specific interaction of RACK1 with STAT3 in P. yoelii 17XNL-responsive CD4+ T cells, ablation of RACK1 leads to defective STAT3 phosphorylation, accompanied by substantially lower amount of STAT3 protein in CD4+ T cells, whereas retroviral overexpression of RACK1 or STAT3 in RACK1-deficient CD4+ T cells greatly restores STAT3 activity and Bcl-6 expression under the Tfh polarization condition. Further analyses suggest RACK1 positively regulates STAT3 stability by inhibiting the ubiquitin-proteasomal degradation process, thus promoting optimal STAT3 activity and Bcl-6 induction during Tfh cell differentiation. These findings uncover a novel mechanism by which RACK1 participates in posttranslational regulation of STAT3, Tfh cell differentiation, and subsequent development of anti-Plasmodium humoral immunity.

Myeloid neddylation targets IRF7 and promotes host innate immunity against RNA viruses.

Neddylation, an important type of post-translational modification, has been implicated in innate and adapted immunity. But the role of neddylation in innate immune response against RNA viruses remains elusive. Here we report that neddylation promotes RNA virus-induced type I IFN production, especially IFN-α. More importantly, myeloid deficiency of UBA3 or NEDD8 renders mice less resistant to RNA virus infection. Neddylation is essential for RNA virus-triggered activation of Ifna gene promoters. Further exploration has revealed that mammalian IRF7undergoes neddylation, which is enhanced after RNA virus infection. Even though neddylation blockade does not hinder RNA virus-triggered IRF7 expression, IRF7 mutant defective in neddylation exhibits reduced ability to activate Ifna gene promoters. Neddylation blockade impedes RNA virus-induced IRF7 nuclear translocation without hindering its phosphorylation and dimerization with IRF3. By contrast, IRF7 mutant defective in neddylation shows enhanced dimerization with IRF5, an Ifna repressor when interacting with IRF7. In conclusion, our data demonstrate that myeloid neddylation contributes to host anti-viral innate immunity through targeting IRF7 and promoting its transcriptional activity.

RACK1 T50 Phosphorylation by AMPK Potentiates Its Binding with IRF3/7 and Inhibition of Type 1 IFN Production.

The receptor for activated C kinase 1 (RACK1) adaptor protein has been implicated in viral infection. However, whether RACK1 promotes in vivo viral infection in mammals remains unknown. Moreover, it remains elusive how RACK1 is engaged in antiviral innate immune signaling. In this study, we report that myeloid RACK1 deficiency does not affect the development and survival of myeloid cells under resting conditions but renders mice less susceptible to viral infection. RACK1-deficient macrophages produce more IFN-α and IFN-ß in response to both RNA and DNA virus infection. In line with this, RACK1 suppresses transcriptional activation of type 1 IFN gene promoters in response to virus infection. Analysis of virus-mediated signaling indicates that RACK1 inhibits the phosphorylation of IRF3/7. Indeed, RACK1 interacts with IRF3/7, which is enhanced after virus infection. Further exploration indicates that virus infection triggers AMPK activation, which in turn phosphorylates RACK1 at Thr50 RACK1 phosphorylation at Thr50 enhances its interaction with IRF3/7 and thereby limits IRF3/7 phosphorylation. Thus, our results confirm that myeloid RACK1 promotes in vivo viral infection and provide insight into the control of type 1 IFN production in response to virus infection.

Electrochemical Biosensor with Enhanced Antifouling Capability Based on Amyloid-like Bovine Serum Albumin and a Conducting Polymer for Ultrasensitive Detection of Proteins in Human Serum.

Biofouling has been a substantial burden on biomarker analysis in complex biological media, leading to poor sensitivity and selectivity or even malfunction of the sensing devices. In this work, an electrochemical biosensor with excellent antifouling ability and high stability was fabricated based on amyloid-like bovine serum albumin (AL-BSA) crosslinked with the conducting polymer polyaniline (PANI). Compared with the crosslinked conventional bovine serum albumin (BSA), the crosslinked AL-BSA exhibited enhanced antifouling capability, and it was able to form an effective antifouling film within a significantly short reaction time. With further immobilization of immunoglobulin M (IgM) antibodies onto the prepared AL-BSA surface via the formation of amide bonds, an electrochemical biosensor capable of assaying IgM in human serum samples with superior selectivity and sensitivity was constructed. The biosensor exhibited excellent antifouling performance even in 100% human serum, a low limit of detection down to 2.32 pg mL-1, and acceptable accuracy for real sample analysis compared with the standard enzyme-linked immunosorbent assay for IgM detection. This strategy of using AL-BSA to construct antifouling sensing interfaces provided a reliable diagnostic method for the detection of a series of protein biomarkers in complex biological media.

pH-Responsive Biomimetic Polymeric Micelles as Lymph Node-Targeting Vaccines for Enhanced Antitumor Immune Responses.

Lymph nodes are proposed as the intriguing target in cancer immunotherapy, and cellular immunity is vital for vaccines to fight against cancer. However, inefficient delivery of vaccines to lymph nodes and deficient lysosomal escape of antigens result in weak cellular immunity, which restrains the strength of vaccines in inducing antitumor immune responses. Hence, dendritic cell membrane (DCM)/histidine-modified stearic acid-grafted chitosan (HCtSA)/ovalbumin (OVA) micelles, as pH-responsive biomimetic vaccines, were fabricated to target lymph nodes and induce cellular immunity for enhanced antitumor immune responses. DCM/HCtSA/OVA micelles exhibited pH-dependent antigen release behavior, which resulted in efficient escape of antigens from dendritic cell (DC) lysosomes. Besides, DCM/HCtSA/OVA micelles accumulated and reserved in the lymph nodes, which ensured effective uptake by DCs. Importantly, DCM/HCtSA/OVA micelles induced potent T cell immune responses, promoted secretion of antitumor-related cytokines, and notably inhibited tumor growth. Overall, DCM/HCtSA/OVA micelles exhibit great potential in targeted immunotherapy and can provide guidance for the design of vaccines.

Negative Surface Shielded Polymeric Micelles with Colloidal Stability for Intracellular Endosomal/Lysosomal Escape.

The critical process and step in achieving effective antitumor therapies is facilitating endosomal escape, which can enhance the intracellular target delivery of therapeutics. However, the normally adopted approaches tend to result in colloidal instability as a result of the inevitable interactions between the resulting positively charged surfaces of micelles and proteins in vivo. Herein, negatively charged surface shielded polymeric micelles, consisting of polymethylacrylamide derivatives and hydrophilic chitosan ( Mw = 18.8 kDa) linked by 3,3'-dithiodipropionic, are constructed. Until the pH decreases to less than 4.5, the DOX-loaded polymeric micelles (CSO-SS-PDPA/DOX) retain a negative surface charge as a result of the abundant amide groups, which could resist formation of the protein "corona" as visualized by transmission electron microscopy. Robust endosomal escape within tens of minutes due to protonated amine groups and specific redox-responsive drug release is visualized by confocal microscopy. The superior therapeutic efficacy in both 3D tumor spheroids and MCF-7 bearing mice further suggested that the prepared CSO-SS-PDPA/DOX is a promising approach for maintaining colloidal stability while achieving intracellular endosomal/lysosomal escape, which opens new opportunities for drug delivery.

Hepatitis C Virus Genotype Analyses in Chronic Hepatitis C Patients and Individuals With Spontaneous Virus Clearance Using a Newly Developed Serotyping Assay.

BACKGROUND: We developed a novel HCV serotyping assay and detected the genotypes in chronic hepatitis C (CHC) patients and individuals with spontaneous viral clearance (SVC). METHODS: Nine hundred and ninety-seven patients were enrolled in a previous study; their samples were genotyped originally using the molecular assays. Among them, 190 patients achieved sustained virological response; the post-treatment samples were also serotyped. Moreover, 326 samples from follow-up cohorts were serotyped, among whom 66 were from SVC individuals, and 260 from CHC patients. RESULTS: Nine hundred and fifty-eight out of 997 samples were available for serotyping, among which 29 samples generated indeterminate serotyping results. The consistency between the genotyping and serotyping assays was 91.50% (850/929). The specificity and sensitivity were 98.45% and 88.77% for genotype 1, 96.42% and 93.97% for genotype 2, and 94.15% and 80.52% for non-genotype 1 or 2. However, only 41 of 60 genotype-6 samples were correctly serotyped. Little difference was found in the 190 paired serotyping results. No difference existed in the genotype distribution between the SVC and CHC groups (P = 0.08). CONCLUSIONS: The assay provides an accurate alternative for determining HCV genotypes, whereas it is not recommended for detecting genotype 6. Furthermore, it facilitates identifying the genotypes in SVC individuals. HCV genotype has little impact on SVC.

Vitronectin: a promising breast cancer serum biomarker for early diagnosis of breast cancer in patients.

Breast cancer is the most common cancer in women worldwide, identification of new biomarkers for early diagnosis and detection will improve the clinical outcome of breast cancer patients. In the present study, we determined serum levels of vitronectin (VN) in 93 breast cancer patients, 30 benign breast lesions, 9 precancerous lesions, and 30 healthy individuals by enzyme-linked immunosorbent assays. Serum VN level was significantly higher in patients with stage 0-I primary breast cancer than in healthy individuals, patients with benign breast lesion or precancerous lesions, as well as those with breast cancer of higher stages. Serum VN level was significantly and negatively correlated with tumor size, lymph node status, and clinical stage (p < 0.05 in all cases). In addition, VN displayed higher area under curve (AUC) value (0.73, 95 % confidence interval (CI) [0.62-0.84]) than carcinoembryonic antigen (CEA) (0.64, 95 % CI [0.52-0.77]) and cancer antigen 15-3 (CA 15-3) (0.69, 95 % CI [0.58-0.81]) when used to distinguish stage 0-I cancer and normal control. Importantly, the combined use of three biomarkers yielded an improvement in receiver operating characteristic curve with an AUC of 0.83, 95 % CI [0.74-0.92]. Taken together, our current study showed for the first time that serum VN is a promising biomarker for early diagnosis of breast cancer when combined with CEA and CA15-3.

Non-invasive detection of hepatocellular carcinoma serum metabolic profile through surface-enhanced Raman spectroscopy.

The present study aims to identify distinctive Raman spectrum metabolic peaks to predict hepatocellular carcinoma (HCC). We performed a label-free, non-invasive surface-enhanced Raman spectroscopy (SERS) test on 230 serum samples including 47 HCC, 60 normal controls (NC), 68 breast cancer (BC) and 55 lung cancer (LC) by mixing Au@AgNRs with serum directly. Based on the observed SERS spectra, discriminative metabolites including tryptophan, phenylalanine, and etc. were found in HCC, when compared with BC, LC, and NC (P<0.05 in all). Common metabolites-proline, valine, adenine and thymine were found in HCC, BC and LC with compared to NC group (P<0.05). Importantly, Raman spectra of HCC serum biomarker AFP were firstly detected to analyze the HCC prominent peak. Orthogonal partial least squares discriminant analysis was adopted to assess the diagnostic accuracy; area under curve value of HCC is 0.991. This study provides new insights into the HCC metabolites detection through Raman spectroscopy.

IgG, IgM and IgA antibodies against the novel polyprotein in active tuberculosis.

BACKGROUND: The present study was aimed to evaluate whether IgG, IgM and IgA antibodies levels detected against a novel Mycobacterium tuberculosis polyprotein 38 F-64 F (with 38 F being the abbreviation for 38kD-ESAT6-CFP10 and 64 F for Mtb8.4-MPT64-TB16.3-Mtb8) are suitable for diagnosing active tuberculosis, and for monitoring the efficacy of chemotherapy on TB patients. METHODS: In this study, a total of 371 active TB patients without treatment were selected and categorized into S+/C+group (n=143), S-/C+group (n=106) or S-/C- group (n=122). A series of serum samples were collected from 82 active TB patients who had undergone anti-TB chemotherapy for 0-6 months at one month interval. Humoral responses (IgG, IgM and IgA) were determined for the novel Mycobacterium tuberculosis polyprotein using indirect ELISA methods in all of serum samples. RESULTS: For S+/C+, S-/C+and S-/C- active tuberculosis patients before anti-TB chemotherapy, the sensitivities of tests based on IgG were 65.7%, 46.2% and 52.5% respectively; the sensitivities based on IgM were 21.7%, 24.5% and 18.9%; and the sensitivities based on IgA were 25.2%, 17.9% and 23.8%. By combination of three isotypes, for all active tuberculosis patients, the test sensitivity increased to 70.4% with the specificity being 91.5%. After anti-TB chemotherapy, there were no significant differences between groups with different courses of anti-TB chemotherapy. CONCLUSIONS: The novel Mycobacterium tuberculosis polyprotein 38 F-64 F represents potential antigen suitable for measuring IgG, IgM and IgA antibodies. However, the serodiagnostic test based on the 38 F-64 F polyprotein appears unsuitable for monitoring the efficacy of chemotherapy.

Discovery of novel diaryl substituted isoquinolin-1(2H)-one derivatives as hypoxia-inducible factor-1 signaling inhibitors for the treatment of rheumatoid arthritis.

Since synovial hypoxic microenvironment significantly promotes the pathological progress of rheumatoid arthritis (RA), hypoxia-inducible factor 1 (HIF-1) has been emerged as a promising target for the development of novel therapeutic agents for RA treatment. In this study, we designed and synthesized a series of diaryl substituted isoquinolin-1(2H)-one derivatives as HIF-1 signaling inhibitors using scaffold-hopping strategy. By modifying the substituents on N-atom and 6-position of isoquinolin-1-one, we discovered compound 17q with the most potent activities against HIF-1 (IC50 = 0.55 µM) in a hypoxia-reactive element (HRE) luciferase reporter assay. Further pharmacological studies revealed that 17q concentration-dependently blocked hypoxia-induced HIF-1α protein accumulation, reduced inflammation response, inhibited cellular invasiveness and promoted VHL-dependent HIF-1α degradation in human RA synovial cell line. Moreover, 17q improved the pathological injury of ankle joints, decreased angiogenesis and attenuated inflammation response in the adjuvant-induced arthritis (AIA) rat model, indicating the promising therapeutic potential of compound 17q as an effective HIF-1 inhibitor for RA therapy.

Use of immuno-dominant epitope derived from genotype 4 as a diagnostic reagent for detecting the antibodies against Hepatitis E Virus.

BACKGROUND: Despite the genotype 4 has become the dominant cause of hepatitis E disease in China, none antigen derived from genotype 4 of hepatitis E virus (HEV) was used in current commercial anti-HEV immunoassay, and the serological reactivity of antigen derive from genotype 4 is not well-charactered. METHODS: We expressed and purified the 4 main immuno-dominant epitopes derived from genotype 1 and 4 including ORF2 (410-621aa) of genotype 4, ORF3 (47-114aa) of genotype 4, ORF2 (396-606aa) of genotype 1 and ORF3 (56-123aa) of genotype 4. RESULTS: The ORF2 of genotype 4 displayed good diagnostics performance according to ROC analysis using in-house panel, and the immunoassays based the ORF2 of genotype 4 was then developed to detect the anti-HEV IgG antibodies and evaluated further in 530 anti-HEV IgG positive specimens and 380 negative specimens. The sensitivity and the specificity is 98.1% (520/530) and 94.7% (360/380) for immunoassay based on ORF2 of genotype 4, 96.6% (512/530) and 92.6% (352/380) for commercial immunoassay based on genotype 1. It is noted that all of the positive samples will be detected by combing two assays together. The anti-HEV immunoassays based on genotype 4 are in accordance with Chinese anti-HEV national standard,and show an good agreement of 95.8% with commercial assay (kappa=0.913, P=0.014). CONCLUSIONS: The immunoassay based on ORF2G4 displays good performance, and combining assay based on genotype 1 together with genotype 4 will benefit the HEV diagnosis in large scale samples.

Alpha-aminoisobutyric acid incorporated peptides for the construction of electrochemical biosensors with high stability and low fouling in serum.

An effective strategy to construct low fouling electrochemical biosensors for assaying serum biomarkers was proposed based on specially designed α-aminoisobutyric acid (Aib) incorporated peptides. The Aib-peptides were designed to be of antifouling properties, and at the same to incorporate Aib residues in their interior to enhance the hydrolytic stability. In order to construct the electrochemical biosensor, two kinds of Aib-peptides labelled with biotin were modified on the electrode surface: One with cysteine terminal for easy attachment to the electrode modified with gold nanoparticles, the other with unique terminal peptide sequence for specific binding of immunoglobulin G (IgG), and they were connected through the streptavidin-biotin affinity system. Owing to the interposition of Aib residues, the peptides as well as the constructed biosensors showed excellent antifouling performances and enhanced stability against enzymatic degradation in serum. Furthermore, the IgG biosensor constructed with the Aib-peptides displayed a very low detection limit (29.5 pg mL-1) and a broad linear range (100 pg mL-1 - 10 µg mL-1), and it was able to assay IgG in clinical human sera with decent accuracy and reliability. This strategy provides a new path for the construction of stable antifouling biosensors based on functional peptides for practical biomarker assaying in real clinical samples.

An electrochemical biosensor for HER2 detection in complex biological media based on two antifouling materials of designed recognizing peptide and PEG.

A simple tactic for electrochemical determination of a typical biomarker for breast cancer, human epidermal growth factor receptor 2 (HER2), was presented via the construction of a low fouling sensing interface functionalized with polyethylene glycol (PEG) and peptide. The HER2 biosensor was developed based on an electrode modified by the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) and Au nanoparticles (AuNPs) as the sensing substrate, and followed by the immobilization of an antifouling PEG and a peptide with both recognizing and antifouling properties. Thanks to the combined antifouling effect of the PEG and peptide, and the specific recognizing ability of the peptide to the target HER2, the developed electrochemical biosensor exhibited strong antifouling performances in complex biofluids, such as human blood and serum, and it was capable of assaying target HER2 within a very wide linear range (1.0 pg mL-1 to 1.0 µg mL-1), with an ultralow limit of detection (0.44 pg mL-1). The combination of two kinds of antifouling biomaterials (PEG and peptide) offered an effective strategy for the development of low fouling sensing platforms suitable for practical assay in complex biotic environments.

Fluoxetine partially alleviates inflammation in the kidney of socially stressed male C57 BL/6 mice.

Stress-related illnesses are linked to the onset and progression of renal diseases and depressive disorders. To investigate stress-induced changes in the renal transcriptome associated with the development of depressive behaviors, we generated here a chronic social defeat stress (CSDS) model of C57 BL/6 male mice and then performed RNA sequencing of the kidneys to obtain an inflammation-related transcriptome. Administration of the antidepressant drug fluoxetine (10 mg·kg-1 ·day-1 ) during CSDS induction could partially alleviate renal inflammation and reverse CSDS-induced depression-like behaviors. Moreover, fluoxetine also modulated gene expression of stress-related hormone receptors, including prolactin and melanin-concentrating hormone. These results suggest that CSDS can induce gene expression changes associated with inflammation in the kidney of C57 BL/6 male mice, and this inflammation can be treated effectively by fluoxetine.

Antifouling peptides combined with recognizing DNA probes for ultralow fouling electrochemical detection of cancer biomarkers in human bodily fluids.

Herein, a universal strategy for the construction of highly sensitive and low fouling biosensors was proposed based on antifouling peptides conjugated with recognizing DNA probes. The peptide-DNA conjugate was formed through a reagent-free click reaction between a typical DNA aptamer modified with 5'-dibenzocyclooctyne (DBCO) and the designed antifouling peptide terminated with biotin and the azide group at its two ends. With the assistance of streptavidin (SA), the electrochemical biosensor was constructed via immobilization of the straight peptides and peptide-DNA conjugates in sequence onto the electrode surface modified with electrodeposited poly(3,4-ethylenedioxythiophene) (PEDOT) and gold nanoparticles (AuNPs). The prepared biosensor exhibited excellent antifouling performances in various human bodily fluids such as serum, sweat and urine, with a wide linear response range for CA125 from 0.01 U mL-1 to 1000 U mL-1, and a low limit of detection of 0.003 U mL-1. Combining the advantages of the antifouling peptide and recognizing DNA probe, this sensing strategy was capable of assaying CA125 in undiluted human serum, and it also offered a highly promising way for the development of different antifouling biosensors through the conjugation of antifouling peptides with various DNA probes.

Hepatitis C Virus Core Protein Promotes the Metastasis of Human Hepatocytes by Activating the MAPK/ERK/PEA3-SRF/c-Fos/MMPs Axis.

BACKGROUND AND AIM: Previous studies have shown that the hepatitis C virus (HCV) core protein plays an important role in the metastasis of hepatocellular carcinoma (HCC) cells. This study aimed to identify the potential mechanism of HCV core protein in HCC. METHODS: A transcription factor microarray analysis was performed to identify the factors regulated by the HCV core protein. A comprehensive bioinformatics analysis approach was utilized to predict the functions, regulatory signaling pathways and downstream target genes of the differentially regulated transcription factors. Dual-luciferase assays, qPCR, Western blotting, ERK pathway inhibition experiments and siRNA knockdown experiments were performed to verify the effects of the HCV core protein on PEA3, SRF and c-Fos, as well asthe underlying mechanism. The migration/invasion assay and scratch assay served to confirm the metastasis-promoting mechanism of the HCV core protein. RESULTS: The results demonstrated that altered expression of PEA3, SRF and c-Fos mediated by the HCV core protein were associated with the MAPK/ERK pathway. c-Fos was a downstream target protein of PEA3 and SRF. Knockdown of PEA3-SRF/c-Fos expression and ERK pathway components suppressed the migration and invasion activity of hepatocytes by affecting MMP2 and MMP9 expression. CONCLUSION: We provided preliminary evidence that the role of the HCV core protein in promoting metastasis is at least partially dependent on the activation of the MAPK/ERK/PEA3-SRF/c-Fos/MMP2/MMP9 axis. These findings reveal a novel mechanism by which the HCV core protein promotes HCC metastasis and may provide new therapeutic targets for patients with metastatic HCC.

Nonerythropoietic Erythropoietin Mimetic Peptide ARA290 Ameliorates Chronic Stress-Induced Depression-Like Behavior and Inflammation in Mice.

Major depressive disorder (MDD) is a highly prevalent psychiatric disorder. But the treatment of depression remains challenging. Anti-inflammatory treatments frequently produce antidepressant effects. EPO-derived helix-B peptide ARA290 has been reported to retain the anti-inflammatory and tissue-protective functions of EPO without erythropoiesis-stimulating effects. The effects of ARA290 on MDD remain elusive. This study established chronic unpredictable mild stress and chronic social defeat stress mouse models. Daily administration of ARA290 during chronic stress induction in two mouse models ameliorated depression-like behavior, similar to fluoxetine. With marginal effects on peripheral blood hemoglobin and red cells, ARA290 and fluoxetine reversed chronic stress-induced increased frequencies and/or numbers of CD11b+Ly6Ghi neutrophils and CD11b+Ly6Chi monocytes in the bone marrow and meninges. Furthermore, both drugs reversed chronic stress-induced microglia activation. Thus, ARA290 ameliorated chronic stress-induced depression-like behavior in mice through, at least partially, its anti-inflammatory effects.

Hepatic RACK1 deficiency protects against fulminant hepatitis through myeloid-derived suppressor cells.

Fulminant hepatitis (FH) is a life-threatening disease with partially understood pathogenesis. It has been demonstrated that myeloid-derived suppressor cells (MDSCs) are recruited into the liver during this process, and their augmented accumulation by various strategies protects against liver injury. However, the underlying mechanism(s) remain elusive. Receptor for activated C kinase 1 (RACK1), a multi-functional scaffold protein, is highly expressed in normal liver and has been implicated in liver physiology and diseases, but the in vivo role of hepatic RACK1 in FH remains unknown. Methods: Survival curves and liver damage were monitored to investigate the in vivo role of hepatic RACK1 in FH. The liver microenvironment was explored by microarray-based transcriptome analysis, flow cytometry, immunoblotting, and immunohistochemistry. MDSCs were identified with phenotypic and functional characteristics. Functional antibodies were used to target MDSCs. Co-culture techniques were used to study the underlying mechanism(s) of protection. The interaction of RACK1 with histone deacetylase 1 (HDAC1) and the consequent effects on HDAC1 ubiquitination were analyzed. Ectopic expression of HDAC1 with recombinant adeno-associated virus serotype 8 was conducted to confirm the role of HDAC1 in the protective effects of hepatic RACK1 deficiency against FH. Post-translational modifications of RACK1 were also investigated during the induction of FH. Results: Liver-specific RACK1 deficiency rendered mice resistant to FH. RACK1-deficient livers exhibited high basal levels of chemokine (C-X-C motif) ligand 1 (CXCL1) and S100 calcium-binding protein A9 (S100A9), associated with MDSC accumulation under steady-state conditions. Targeting MDSCs with an antibody against either Gr1 or DR5 abrogated the protective effects of liver-specific RACK1 deficiency. Accumulated MDSCs inhibited inflammatory cytokine production from macrophages and enhanced IκB kinase (IKK)/NF-κB pathway activation in hepatocytes. Further investigation revealed that RACK1 maintained HDAC1 protein level in hepatocytes by direct binding, thereby controlling histone H3K9 and H3K27 acetylation at the Cxcl1 and S100a9 promoters. Ectopic expression of HDAC1 in livers with RACK1 deficiency partially reversed the augmented Cxcl1/S100a9 → MDSCs → IKK/NF-κB axis. During FH induction, RACK1 was phosphorylated at serine 110, enhancing its binding to ubiquitin-conjugating enzyme E2T and promoting its ubiquitination and degradation. Conclusion: Liver-specific RACK1 deficiency protects against FH through accelerated HDAC1 degradation and the consequent CXCL1/S100A9 upregulation and MDSC accumulation.

Identification of hematopoietic stem cells residing in the meninges of adult mice at steady state.

Steady-state extramedullary hematopoiesis during adulthood is an emerging field of great interest. The meninges contain both innate and adaptive immune cells, which provide immunosurveillance of the central nervous system (CNS). Hematopoietic progenitors that give rise to meningeal immune cells remain elusive. Here, we report that steady-state meninges of adult mice host hematopoietic stem cells (HSCs), as defined by long-term, efficient, multi-lineage reconstitution and self-renewal capacity in the meninges, blood, spleen, and bone marrow of sublethally irradiated adult recipients. HSCs lodge in the meninges after birth with local expression of pro-hematopoietic niche factors. Meningeal HSCs are locally maintained in homeostasis and get replenished from the blood only when the resident pool is reduced. With a tissue-specific expression profile, meningeal HSCs can provide the CNS with a constant supply of leukocytes more adapted to local microenvironment.