Upregulation Of CD36 Research Articles

Inflammatory Dendritic Cells Contribute to Regulate the Immune Response in Sickle Cell Disease.

Sickle cell disease (SCD), one of the most common hemoglobinopathies worldwide, is characterized by a chronic inflammatory component, with systemic release of inflammatory cytokines, due to hemolysis and vaso-occlusive processes. Patients with SCD demonstrate dysfunctional T and B lymphocyte responses, and they are more susceptible to infection. Although dendritic cells (DCs) are the main component responsible for activating and polarizing lymphocytic function, and are able to produce pro-inflammatory cytokines found in the serum of patients with SCD, minimal studies have thus far been devoted to these cells. In the present study, we identified the subpopulations of circulating DCs in patients with SCD, and found that the bloodstream of the patients showed higher numbers and percentages of DCs than that of healthy individuals. Among all the main DCs subsets, inflammatory DCs (CD14+ DCs) were responsible for this rise and correlated with higher reticulocyte count. The patients had more activated monocyte-derived DCs (mo-DCs), which produced MCP-1, IL-6, and IL-8 in culture. We found that a CD14+ mo-DC subset present in culture from some of the patients was the more activated subset and was mainly responsible for cytokine production, and this subset was also responsible for IL-17 production in co-culture with T lymphocytes. Finally, we suggest an involvement of heme oxygenase in the upregulation of CD14 in mo-DCs from the patients, indicating a potential mechanism for inducing inflammatory DC differentiation from circulating monocytes in the patients, which correlated with inflammatory cytokine production, T lymphocyte response skewing, and reticulocyte count.

Foam cell formation but not oxLDL cytotoxicity is inhibited by CD36 down regulation by the macrophage antioxidant 7,8-dihydroneopterin.

Cluster of differentiation 36 (CD36) is a key scavenger receptor in the control of macrophage uptake of oxidised low-density lipoproteins (oxLDL). CD36 expression levels are not down regulated by intracellular cholesterol but are upregulated by oxidised low density lipoprotein (oxLDL) leading to the formation of lipid loaded foam cells, a major constituent of atherosclerotic plaques. We have previous shown that CD36 is down regulated by 7,8-dihydroneopterin, an antioxidant generated by γ-interferon activated macrophages. How CD36 down regulation affects oxLDL induced cytotoxicity, CD36 oxLDL upregulation and foam cell formation is examined using human monocyte like U937 cell line as a model system of human macrophages. Low density lipoprotein (LDL) was prepared by ultracentrifugation from human plasma and oxidised in copper chloride. CD36 levels in U937 cells were measured by western blot analysis. and lipid accumulation was measured by oil red-O staining and 7-ketocholesterol accumulation by high performance liquid chromatography. Cell viability was measured by flow cytometry analysis after propidium iodide staining. 7,8-dihydroneopterin concentrations above 100 μM caused a concentration and time dependent decrease in cellular CD36 levels to 20 % of the untreated cells after 24 h. Upregulation of CD36 by oxLDL was inhibited by 7,8-dihydroneopterin treatment. The CD36 down regulation was associated with decrease in foam cell formation but not a reduction on oxLDL cytotoxicity. 7,8-dihydroneopterin down regulated CD36 in U937 cells, inhibiting foam cell formation but not oxLDL mediated cell death. 7,8-dihydroneopterin may modulate foam cell formation in atherosclerotic plaques.

The cluster of differentiation-36 (CD-36) as a promising marker for many diseases

CD36 is a transmembrane protein involved in fatty acid translocation, scavenging for oxidized fatty acids acting as a receptor for adhesion molecules. It is expressed on macrophages, as well as other types of cells, such as endothelial and adipose cells. CD36 participates in muscle lipid uptake, adipose energy storage, and gut fat absorption. Recently, several preclinical and clinical studies demonstrated that the upregulation of CD36 is a prerequisite for tumor metastasis. Cancer metastasis-related research emerged much later and has been less investigated, though it is equally or even more important. CD36 protein expression can be modified by epigenetic changes and post-transcriptional interference from non-coding RNAs. Some data indicate modulation of CD36 expression in specific cell types by epigenetic changes via DNA methylation patterns or histone tails, or through miRNA interference, but this is largely unexplored. The few papers addressing this topic refer mostly to lipid metabolism-related pathologies, whereas in cancer research, data are even more scarce. The aim of this review was to summarize major epigenetic and post-transcriptional mechanisms that impact CD36 expression in relation to various pathologies while highlighting the areas in need of further exploration.

FcRH1: an activation coreceptor on human B cells

AbstractB-cell activation and differentiation is regulated through the coordinated function of a dynamic array of cell surface receptors. At different stages in their differentiation, human B cells may express one or more members of a large family of immunoglobulin Fc receptor homologs (FcRH) with regulatory potential. Among these newly identified transmembrane molecules, FcRH1 is unique in having 2 immunoreceptor tyrosine-based activation motif (ITAM)–like motifs in its intracellular domain. Here we used the Fab fragments of new monoclonal anti-FcRH1 antibodies and mRNA analysis to evaluate FcRH1 expression and function during B-cell differentiation. FcRH1 expression begins in pre-B cells, reaches peak levels on naive B cells, and is down-regulated after B cells are activated to begin to form germinal centers. This FcRH1 down-regulation coincides with dramatic enlargement of the pre-germinal center cells, cell cycle entry, and other overt signs of activation that include CD80 and CD86 up-regulation and immunoglobulin D (IgD) down-regulation. In vitro analysis indicates that ligation of FcRH1 leads to its tyrosine phosphorylation and to modest B-cell activation and proliferation. Concomitant FcRH1 ligation enhances B-cell antigen receptor (BCR)–induced Ca2+ mobilization and proliferation. FcRH1 thus has the potential to serve as an activating coreceptor on B cells.

Pharmacological validation of targets regulating CD14 during macrophage differentiation.

The signalling receptor for LPS, CD14, is a key marker of, and facilitator for, pro-inflammatory macrophage function. Pro-inflammatory macrophage differentiation remains a process facilitating a broad array of disease pathologies, and has recently emerged as a potential target against cytokine storm in COVID19. Here, we perform a whole-genome CRISPR screen to identify essential nodes regulating CD14 expression in myeloid cells, using the differentiation of THP-1 cells as a starting point. This strategy uncovers many known pathways required for CD14 expression and regulating macrophage differentiation while additionally providing a list of novel targets either promoting or limiting this process. To speed translation of these results, we have then taken the approach of independently validating hits from the screen using well-curated small molecules. In this manner, we identify pharmacologically tractable hits that can either increase CD14 expression on non-differentiated monocytes or prevent CD14 upregulation during macrophage differentiation. An inhibitor for one of these targets, MAP2K3, translates through to studies on primary human monocytes, where it prevents upregulation of CD14 following M-CSF induced differentiation, and pro-inflammatory cytokine production in response to LPS. Therefore, this screening cascade has rapidly identified pharmacologically tractable nodes regulating a critical disease-relevant process.

Amorphous silica nanoparticles accelerated atherosclerotic lesion progression in ApoE\u2212/\u2212 mice through endoplasmic reticulum stress-mediated CD36 up-regulation in macrophage

BackgroundThe biosafety concern of silica nanoparticles (SiNPs) is rapidly expanding alongside with its mass production and extensive applications. The cardiovascular effects of SiNPs exposure have been gradually confirmed, however, the interaction between SiNPs exposure and atherosclerosis, and the underlying mechanisms still remain unknown. Thereby, this study aimed to explore the effects of SiNPs on the progression of atherosclerosis, and to investigate related mechanisms.ResultsWe firstly investigated the in vivo effects of SiNPs exposure on atherosclerosis via intratracheal instillation of ApoE−/− mice fed a Western diet. Ultrasound microscopy showed a significant increase of pulse wave velocity (PWV) compared to the control group, and the histopathological investigation reflected a greater plaque burden in the aortic root of SiNPs-exposed ApoE−/− mice. Compared to the control group, the serum levels of total triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) were elevated after SiNPs exposure. Moreover, intensified macrophage infiltration and endoplasmic reticulum (ER) stress was occurred in plaques after SiNPs exposure, as evidenced by the upregulated CD68 and CHOP expressions. Further in vitro, SiNPs was confirmed to activate ER stress and induce lipid accumulation in mouse macrophage, RAW264.7. Mechanistic analyses showed that 4-PBA (a classic ER stress inhibitor) pretreatment greatly alleviated SiNPs-induced macrophage lipid accumulation, and reversed the elevated CD36 expression induced by SiNPs.ConclusionsOur results firstly revealed the acceleratory effect of SiNPs on the progression of atherosclerosis in ApoE−/− mice, which was related to lipid accumulation caused by ER stress-mediated upregulation of CD36 expression in macrophage.Graphical abstract

Immunophenotypic shift in the B-cell precursors from regenerating bone marrow samples: A critical consideration for measurable residual disease assessment in B-lymphoblastic leukemia.

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p=0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p=0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

Abstract 2263: Characteristics of the interferon-stimulatory DNA cargo of exosomes produced by irradiated breast cancer cells

Abstract Radiation therapy (RT) used at immunogenic doses (8GyX3) leads to cytosolic accumulation of DNA that binds to the DNA sensor cGAS and activates the cGAMP synthesis, resulting in STING activation and downstream interferon type I (IFN-I) production by cancer cells [1-3]. RT-induced IFN-I is critical for effective anti-tumor immune responses in combination with immune-checkpoint blockade therapy [1]. We have recently demonstrated that tumor-derived exosomes (TEX) secreted by irradiated (8GyX3) TSA murine carcinoma cells (RT-TEX) contain more DNA compared to TEX produced by untreated cells (UT-TEX). Interestingly, only the DNA carried by RT-TEX is capable of STING-dependent activation of recipient dendritic cells (DCs) resulting in CD40, CD80 and CD86 upregulation and IFN-I production. Mice vaccination with RT-TEX, but not UT-TEX, elicits tumor-specific CD8+ T-cell responses that protect mice from tumor development [4]. Here, we tested the hypothesis that not only quantitative but also qualitative differences between the DNA cargo of RT-TEX and UT-TEX may explain its differential ability to activate DCs. To this end, internal DNA purified from TEX and from the cytosolic fraction of the parent TSA cells was characterized for length using Agilent Bioanalyzer. Furthermore, DNA TEX was analyzed by whole-genome sequencing (WGS) and by whole-genome bisulfite sequencing for methylation status. In addition, the percentage of global methylation of DNA of the TSA cells was quantified by 5-methyl cytosine DNA Elisa kit. All experiments were performed in biological triplicates. DNA size analysis revealed an enrichment of DNA fragments with size between 60 and 250 bp in RT-TEX compared to UT-TEX, and in the cytosolic fraction of irradiated compared to untreated TSA cells. The WGS showed that the entire genome was represented in DNA contained within TEX, regardless of the treatment of the parent cells. More than 99% of TEX DNA was of nuclear origin, but mitochondrial DNA was increased in RT-TEX (6.2 fold change, p=0.006). Interestingly, the RT treatment increases the DNA abundance of several regions (median p=0.006). Interestingly, we found reduced levels of methylation in exosomal and total DNA purified from irradiated compared to untreated TSA cells. Our data support the hypothesis that there are potentially important qualitative differences in the DNA cargo of TEX derived from irradiated compared to untreated cancer cells that reflect molecular changes occurring in parent cells. For instance, the enrichment in DNA fragments with size between 60-250 bp in RT-TEX is especially intriguing in light of the recent report that cGAS is optimally activated by this DNA length range [5]. The impact of these qualitative differences in the cargo DNA on activation of the IFN-I pathway in innate immune cells that uptake TEX is under investigation. Identification of DNA signature associated with TEX ability to activate the cGAS/STING pathway could provide a blood-based biomarker for the immunogenic tumor response to radiotherapy. Citation Format: Sheila Spada, Paul Zumbo, Doron Betel, Tuo Zhang, Nils-Petter Rudqvist, Sandra Demaria. Characteristics of the interferon-stimulatory DNA cargo of exosomes produced by irradiated breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2263.

Identification of TGFβ signatures in six murine models mimicking different osteoarthritis clinical phenotypes

TGFβ is a key player in cartilage homeostasis and OA pathology. However, few data are available on the role of TGFβ signalling in the different OA phenotypes. Here, we analysed the TGFβ pathway by transcriptomic analysis in six mouse models of OA. We have brought together seven expert laboratories in OA pathophysiology and, used inter-laboratories standard operating procedures and quality controls to increase experimental reproducibility and decrease bias. As none of the available OA models covers the complexity and heterogeneity of the human disease, we used six different murine models of knee OA: from post-traumatic/mechanical models (meniscectomy (MNX), MNX and hypergravity (HG-MNX), MNX and high fat diet (HF-MNX), MNX and seipin knock-out (SP-MNX)) to aging-related OA and inflammatory OA (collagenase-induced OA (CIOA)). Four controls (MNX-sham, young, SP-sham, CIOA-sham) were added. OsteoArthritis Research Society International (OARSI)-based scoring of femoral condyles and ribonucleic acid (RNA) extraction from tibial plateau samples were done by single operators as well as the transcriptomic analysis of the TGFβ family pathway by Custom TaqMan® Array Microfluidic Cards. The transcriptomic analysis revealed specific gene signatures in each of the six models; however, no gene was deregulated in all six OA models. Of interest, we found that the combinatorial Gdf5-Cd36-Ltbp4 signature might discriminate distinct subgroups of OA: Cd36 upregulation is a hallmark of MNX-related OA while Gdf5 and Ltbp4 upregulation is related to MNX-induced OA and CIOA. These findings stress the OA animal model heterogeneity and the need of caution when extrapolating results from one model to another.

The Fungal Iron Chelator Desferricoprogen Inhibits Atherosclerotic Plaque Formation.

Hemoglobin, heme and iron are implicated in the progression of atherosclerosis. Therefore, we investigated whether the hydrophobic fungal iron chelator siderophore, desferricoprogen (DFC) inhibits atherosclerosis. DFC reduced atherosclerotic plaque formation in ApoE−/− mice on an atherogenic diet. It lowered the plasma level of oxidized LDL (oxLDL) and inhibited lipid peroxidation in aortic roots. The elevated collagen/elastin content and enhanced expression of adhesion molecule VCAM-1 were decreased. DFC diminished oxidation of Low-density Lipoprotein (LDL) and plaque lipids catalyzed by heme or hemoglobin. Formation of foam cells, uptake of oxLDL by macrophages, upregulation of CD36 and increased expression of TNF-α were reduced by DFC in macrophages. TNF-triggered endothelial cell activation (vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecules (ICAMs), E-selectin) and increased adhesion of monocytes to endothelium were attenuated. The increased endothelial permeability and intracellular gap formation provoked by TNF-α was also prevented by DFC. DFC acted as a cytoprotectant in endothelial cells and macrophages challenged with a lethal dose of oxLDL and lowered the expression of stress-responsive heme oxygenase-1 as sublethal dose was employed. Saturation of desferrisiderophore with iron led to the loss of the beneficial effects. We demonstrated that DFC accumulated within the atheromas of the aorta in ApoE−/− mice. DFC represents a novel therapeutic approach to control the progression of atherosclerosis.

Nifedipine Exacerbates Lipogenesis in the Kidney via KIM-1, CD36, and SREBP Upregulation: Implications from an Animal Model for Human Study.

Dysregulation of fatty acid oxidation and accumulation of fatty acids can cause kidney injury. Nifedipine modulates lipogenesis-related transcriptional factor SREBP-1/2 in proximal tubular cells by inhibiting the Adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) pathway in vitro. However, the mechanisms by which nifedipine (NF) modulates lipotoxicity in vivo are unclear. Here, we examined the effect of NF in a doxorubicin (DR)-induced kidney injury rat model. Twenty-four Sprague–Dawley rats were divided into control, DR, DR+NF, and high-fat diet (HFD) groups. The DR, DR+NF, and HFD groups showed hypertension and proteinuria. Western blotting and immunohistochemical analysis showed that NF significantly induced TNF-α, CD36, SREBP-1/2, and acetyl-CoA carboxylase expression and renal fibrosis, and reduced fatty acid synthase and AMPK compared to other groups (p < 0.05). Additionally, 18 patients with chronic kidney disease (CKD) who received renal transplants were enrolled to examine their graft fibrosis and lipid contents via transient elastography. Low-density lipoprotein levels in patients with CKD strongly correlated with lipid contents and fibrosis in grafted kidneys (p < 0.05). Thus, NF may initiate lipogenesis through the SREBP-1/2/AMPK pathway and lipid uptake by CD36 upregulation and aggravate renal fibrosis in vivo. Higher low-density lipoprotein levels may correlate with renal fibrosis and lipid accumulation in grafted kidneys of patients with CKD.

1840-P: GPR40 Deficiency Is Associated with Steatosis, Hepatic Inflammation, and CD36 Upregulation in C57BL/6 Mice

GPR40 is a G-protein coupled receptor for medium and long-chain fatty acids. Since GPR40 is highly expressed in pancreatic islets and GPR40 activation increases glucose-stimulated insulin secretion from pancreatic beta cells, GPR40 is considered as a potential target for treatment of type 2 diabetes mellitus (T2DM). However, although nonalcoholic fatty liver disease (NAFLD) is a common complication of T2DM and GPR40 is also expressed in hepatocytes and macrophages, the role of GPR40 in high-fat diet (HFD)-induced NAFLD in animal models remains unknown. In this study, we fed wild type or GPR40 knockout (KO) C57BL/6 mice low-fat diet (LFD) or HFD for 20 weeks and then analyzed the effect of GPR40 KO on HFD-induced NAFLD. Metabolic assays showed that HFD increased bodyweight, glucose, insulin, insulin resistance, cholesterol and alanine aminotransferase (ALT) as expected, but GPR40 KO did not further change these parameters in HFD-fed mice. Interestingly, in LFD-fed mice, GPR40 KO increased bodyweight, insulin, insulin resistance, cholesterol and ALT. Histological analysis and Oil Red O staining showed that HFD induced steatosis while GPR40 KO only slightly reduced HFD-induced steatosis. Surprisingly, GPR40 KO in LFD-fed mice induced steatosis. Furthermore, study showed that while HFD increased immunostaining of F4/80, a biomarker for macrophages, GPR40 KO also increased F4/80 immunostaining in LFD-fed mice. Finally, we found that while HFD increased mRNA expression of CD36, a scavenger receptor that also imports long-chain fatty acids into cells, by 5-fold in liver, GPR40 KO increased it by 9-fold in LFD-fed mice. Taken together, this study showed that GPR40 deficiency was associated with insulin resistance, dyslipidemia, steatosis and hepatic inflammation in LFD-fed mice. This study also showed that GPR40-deficiency led to a robust upregulation of CD36 expression in liver, which is likely involved in GPR40 deficiency-associated steatosis and hepatic inflammation. Disclosure Z. Lu: None. Y. Li: None. M.F. Lopes-Virella: None. Y. Huang: None. Funding National Institutes of Health (DE027070); U.S. Department of Veterans Affairs (BX000854)

Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function.

BackgroundPreadipocyte is closely related to obesity-induced inflammation. The impairment of autophagic flux by defective lysosomal function has been observed in adipose tissue from obese mice. While the fatty acid translocase CD36 is an important immuno-metabolic receptor, it remains unclear whether preadipocyte CD36 is involved in adipose tissue inflammation and whether CD36 regulates lysosomal function.MethodsUsing visceral adipose tissue from obese patients, a high-fat diet (HFD)-induced obese mice model, primary mouse preadipocytes and 3T3L1 cells we analyzed whether and how preadipocyte CD36 modulates lysosomal function and adipose tissue inflammation.FindingsCD36 expression in preadipocytes is induced in obese patients and HFD-fed mice, accompanied with the disruption of lysosome function. CD36 knockout protects primary preadipocytes of HFD-fed mice from lysosomal impairment. In vitro, CD36 interacts with Fyn to phosphorylate and activate Inositol (1,4,5)-trisphosphate receptor 1 (IP3R1), causing excess calcium transport from endoplasmic reticulum (ER) to lysosome, which results in lysosomal impairment and inflammation. Moreover, IP3R inhibitor 2-aminoethoxydiphenyl borate (2APB) attenuates lysosomal impairment, inflammation and lipid accumulation in CD36-overexpressing preadipocytes.InterpretationOur data support that the abnormal upregulation of CD36 in preadipocytes may contribute to the development of adipose tissue inflammation. CD36/Fyn/IP3R1-mediated lysosomal calcium overload leads to lysosomal impairment and inflammation in preadipocyte. Thus targeting improving lysosomal calcium homeostasis may represent a novel strategy for treating obesity-induced inflammation.

Differential immune pathways in classic and mixed variants of anaplastic thyroid cancer.

e18579 Background: Patients with anaplastic thyroid cancer (ATC) have poor outcomes due to treatment resistance with an estimated 5-year survival rate of 7%. Although “classic” ATC can arise de novo from follicular thyroid cells, it can also arise in association with papillary thyroid carcinoma (PTC), as a “mixed” histology. The immunologic and molecular differences between these histological subtypes have not been well-characterized. We aimed to investigate the differences in immune signatures in these histological subtypes to assess immune pathway differences in the classic ATC and mixed ATC/PTC tumor microenvironment. Methods: Classic ATC and mixed ATC/PTC cases (N = 12) from 1998 to 2018 with viable formalin-fixed paraffin-embedded (FFPE) tissue were identified and selected by a board-certified pathologist. 4 samples exhibited both PTC and ATC components while 8 were classic ATC cases, as confirmed by a pathologist. In the 4 mixed ATC/PTC histology specimens, 3 were identified to contain greater than 80% ATC involvement while the fourth was characterized as 40% ATC and 60% PTC. The NanoString Immune Profiling panel contains more than 770 genes. It was performed with extracted RNA to evaluate and compare the gene expression of classic ATC versus mixed ATC/PTC. Results: Majority of patients (N = 11) developed metastatic disease, a common feature of ATC, and are currently deceased (N = 10). The NanoString panel identified several differentially expressed immune pathways in the mixed ATC/PTC in comparison to the classic ATC group. There is upregulation of CD3 (p &lt; 0.01) and CD8 (p &lt; 0.01) transcripts in the mixed ATC/PTC group, but not CD4 transcripts (p &gt; 0.05). Additionally, we found that genes associated with B cell and T cell function were significantly upregulated in the mixed ATC group, including CD27 (p &lt; 0.01), CD28 (p &lt; 0.05), BTLA (p &lt; 0.01), CTLA4 (p &lt; 0.01), LAG3 (p &lt; 0.05), and TIGIT(p &lt; 0.01). We did not observe any significant differences in the expression of PD1 (p &gt; 0.05) or MR1 (p &gt; 0.05) between classic ATC and mixed ATC variants. Conclusions: Our study demonstrates a significant difference in the immune landscape between classic and mixed variants of ATC. Our results indicate there may be an increase in immune cell infiltration and tumor inflammation in the mixed ATC variant, which suggests patients with the mixed ATC variant may show a positive response to immunotherapy.

Role of miR-30a-5p and miR-26a-5p in induction of terminal differentiation in human K562 erythroleukemia cells

Background & Aim Background Plasmodium vivax (Pv) infections affect ∼300 million people every year. However, studies into this parasite species are hampered by the inability to set up a continuous, long-term in vitro blood stage culture for Pv. Establishing an in vitro “red blood cell matrix” that would allow continuous access to reticulocytes would facilitate the establishment of continuous, long-term in vitro Pv blood stage cultures and ultimately the study of parasite vulnerabilities that can be exploited in therapeutic approaches. Our study aims to establish culture conditions that allow for the differentiation of K562 cells into enucleated erythroid cells, serving as a constant source of reticulocytes for continuous, long-term Pv blood stage cultures. Methods, Results & Conclusion Materials and Methods Since the K562 cell line (K562) does not express the Duffy blood group antigen receptor (Fy), essential for Pv invasion, the Duffy FyB receptor was stably introduced into the K562 by lentiviral gene transfer. In addition, we combined different factors, which were known to enhance the terminal erythroid differentiation of K562 (knockdown of miR-30a-5p and -26a-5p, co-cultivation with macrophages, EPO, transferrin, insulin and mithramycin A). Effects were analysed by quantification of total cell numbers, surface markers and Giemsa staining. Hb expression was detected by Benzidin staining and PCR. Results K562 cells transduced with anti-miR-26a-5p and -30a-5p (C26) and stimulated with the induction cocktail showed a significantly increase in the amount of enucleated reticulocytes as compared to cells transduced with the empty vector. This is reflected in a stronger down-regulation of CD45 and up-regulation of CD71 as well as in the number of enucleated reticulocytes and the increase in hemoglobin production. CD235a expression was already higher in clone C26 prior to stimulation with the induction cocktail as compared to unstimulated K562 empty cells. The Duffy receptor expression remained stable in stimulated cells of the clone C26, while it was downregulated in K562 empty vector cells over the stimulation period. Conclusions The data show that knockdown of miR-26a-5p and -30a-5p are necessary to induce terminal differentiation of K562 cells. Further optimisation of culture conditions to enhance the yield of RBC blasts from K562 is necessary, as is the confirmation of functionality and susceptibility to Pv infection of these cells.

Graft-versus-host disease of the CNS is mediated by TNF upregulation in microglia.

Acute graft-versus-host disease (GVHD) can affect the central nervous system (CNS). The role of microglia in CNS-GVHD remains undefined. In agreement with microglia activation, we found that profound morphological changes and MHC-II and CD80 upregulation occurred upon GVHD induction. RNA sequencing-based analysis of purified microglia obtained from mice with CNS-GVHD revealed TNF upregulation. Selective TNF gene deletion in microglia of Cx3cr1creER Tnffl/- mice reduced MHC-II expression and decreased CNS T cell infiltrates and VCAM-1+ endothelial cells. GVHD increased microglia TGF-β-activated kinase-1 (TAK1) activation and NF-κB/p38 MAPK signaling. Selective Tak1 deletion in microglia using Cx3cr1creER Tak1fl/fl mice resulted in reduced TNF production and microglial MHC-II and improved neurocognitive activity. Pharmacological TAK1 inhibition reduced TNF production and MHC-II expression by microglia, Th1 and Th17 T cell infiltrates, and VCAM-1+ endothelial cells and improved neurocognitive activity, without blocking graft-versus-leukemia effects. Consistent with these findings in mice, we observed increased activation and TNF production of microglia in the CNS of GVHD patients. In summary, we prove a role for microglia in CNS-GVHD, identify the TAK1/TNF/MHC-II axis as a mediator of CNS-GVHD, and provide a TAK1 inhibitor-based approach against GVHD-induced neurotoxicity.

PDK4-Deficiency Reprograms Intrahepatic Glucose and Lipid Metabolism to Facilitate Liver Regeneration in Mice.

Liver regeneration requires intrahepatic and extrahepatic metabolic reprogramming to meet the high hepatic bioenergy demand for liver cell repopulation. This study aims to elucidate how pyruvate dehydrogenase kinase 4 (PDK4), a critical regulator of glucose and lipid metabolism, coordinates metabolic regulation with efficient liver growth. We found that hepatic Pdk4 expression was elevated after two‐thirds partial hepatectomy (PHx). In Pdk4 −/− PHx mice, the liver/body weight ratio was more rapidly restored, accompanied by more aggressive hepatic DNA replication; however, Pdk4 −/− mice developed more severe hypoglycemia. In Pdk4 −/− PHx livers, the pro‐regenerative insulin signaling was potentiated, as demonstrated by early peaking of the phosphorylation of insulin receptor, more remarkable induction of the insulin receptor substrate proteins, IRS1 and IRS2, and more striking activation of Akt. The hepatic up‐regulation of CD36 contributed to the enhanced transient regeneration‐associated steatosis in Pdk4 −/− PHx mice. Notably, CD36 overexpression in mice promoted the recovery of liver/body weight ratio and elevated intrahepatic adenosine triphosphate after PHx. CD36 expression was transcriptionally suppressed by FOXO1 (forkhead box protein O1), which was stabilized and translocated to the nucleus following AMPK (adenosine monophosphate–activated protein kinase) activation. PHx remarkably induced AMPK activation, which became incompetent to respond in Pdk4 −/− livers. Moreover, we defined that PDK4‐regulated AMPK activation directly depended on intracellular adenosine monophosphate in vitro and in regenerative livers. Conclusion: PDK4 inhibition reprograms glucose and lipid metabolism to promote liver regeneration by enhancing hepatic insulin/Akt signaling and activating an AMPK/FOXO1/CD36 regulatory axis of lipid. These findings may lead to potential therapeutic strategies to prevent hepatic insufficiency and liver failure.

Decreased PEDF Promotes Hepatic Fatty Acid Uptake and Lipid Droplet Formation in the Pathogenesis of NAFLD.

Non-alcoholic fatty liver disease (NAFLD), the leading cause of chronic liver diseases worldwide, ranges from simple steatosis to steatohepatitis, with the risk for progressive fibrosis or even cirrhosis. While simple steatosis is a relatively benign condition, the buildup of toxic lipid metabolites can induce chronic inflammation, ultimately triggering disease progression. Pigment epithelium-derived factor (PEDF) is a secreted, multifunctional glycoprotein with lipid metabolic activities. PEDF promotes lipolysis through binding to adipose triglyceride lipase (ATGL), a key enzyme for triglyceride breakdown. In the current study, we aimed to delineate how changes in PEDF expression affect hepatic lipid accumulation. Our data revealed that hepatic PEDF was downregulated in a mouse NAFLD model. We further showed that decreased PEDF levels in hepatocytes in vitro resulted in elevated fatty acid uptake and lipid droplet formation, with concomitant upregulation of fatty acid transport proteins CD36 and fatty acid binding protein 1 (FABP1). RNA sequencing analysis of PEDF knocked down hepatocytes revealed an alteration in gene expression profile toward lipid accumulation. Additionally, decreased PEDF promotes mobilization of fatty acids, an observation distinct from blocking ATGL activity. Taken together, our data suggest that hepatic PEDF downregulation causes molecular changes that favor triglyceride accumulation, which may further lead to NAFLD progression.

Post mortem evaluation of inflammation, oxidative stress, and PPARγ activation in a nonhuman primate model of cardiac sympathetic neurodegeneration.

Cardiac dysautonomia is a common nonmotor symptom of Parkinson's disease (PD) associated with loss of sympathetic innervation to the heart and decreased plasma catecholamines. Disease-modifying strategies for PD cardiac neurodegeneration are not available, and biomarkers of target engagement are lacking. Systemic administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) recapitulates PD cardiac dysautonomia pathology. We recently used positron emission tomography (PET) to visualize and quantify cardiac sympathetic innervation, oxidative stress, and inflammation in adult male rhesus macaques (Macaca mulatta; n = 10) challenged with 6-OHDA (50mg/kg; i.v.). Twenty-four hours post-intoxication, the animals were blindly and randomly assigned to receive daily doses of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone (n = 5; 5mg/kg p.o.) or placebo (n = 5). Quantification of PET radioligand uptake showed increased oxidative stress and inflammation one week after 6-OHDA which resolved to baseline levels by twelve weeks, at which time pioglitazone-treated animals showed regionally preserved sympathetic innervation. Here we report post mortem characterization of heart and adrenal tissue in these animals compared to age and sex matched normal controls (n = 5). In the heart, 6-OHDA-treated animals showed a significant loss of sympathetic nerve fibers density (tyrosine hydroxylase (TH)-positive fibers). The anatomical distribution of markers of sympathetic innervation (TH) and inflammation (HLA-DR) significantly correlated with respective in vivo PET findings across left ventricle levels and regions. No changes were found in alpha-synuclein immunoreactivity. Additionally, CD36 protein expression was increased at the cardiomyocyte intercalated discs following PPARγ-activation compared to placebo and control groups. Systemic 6-OHDA decreased adrenal medulla expression of catecholamine producing enzymes (TH and aromatic L-amino acid decarboxylase) and circulating levels of norepinephrine, which were attenuated by PPARγ-activation. Overall, these results validate in vivo PET findings of cardiac sympathetic innervation, oxidative stress, and inflammation and illustrate cardiomyocyte CD36 upregulation as a marker of PPARγ target engagement.

Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells.

Vitiligo is an acquired pigmentary disorder resulting from selective destruction of melanocytes. Emerging studies have suggested that T helper cell 17 (Th17) is potentially implicated in vitiligo development and progression. It was recently discovered that metabotropic glutamate receptor 4 (mGluR4) can modulate Th17-mediated adaptive immunity. However, the influence of mGluR4 on melanogenesis of melanocytes has yet to be elucidated. In the present study, we primarily cultured mouse bone marrow-derived dendritic cells (BMDC) and then knocked down and over-expressed mGluR4 using transfection. Transduced BMDC were co-cultured with CD4+ T cells and the expression of Th17-related cytokines were measured. The morphology and melanogenesis of B16 cells were observed after being treated with co-culture medium of CD4+ T cells and transduced BMDC. We found that mGluR4 knockdown did not affect the co-stimulatory CD80 and CD86 upregulation after lipopolysaccharide stimulation but did increase the expression of Th17-related cytokines, and further down-regulated the expression of microphthalmia-associated transcription factor (MITF) and the downstream genes, decreased melanin production, and destroyed the morphology of B16 cells. Conversely, over-expression of mGluR4 reduced the expression of CD80 and CD86, suppressed the production of Th17-related cytokines, increased the expression of MITF, and did not destroy the morphology of B16 cells. Our study confirmed that mGluR4 modulated the Th17 cell polarization and resulted in the alteration of melanogenesis and morphology of B16 cells. Collectively, these findings suggest mGluR4 might be a potent target involved in the immune pathogenesis of vitiligo.