Naringenin potentiates anti-tumor immunity against oral cancer by inducing lymph node CD169-positive macrophage activation and cytotoxic T cell infiltration

Cancer immunology, immunotherapy : CII

2022 Jan 19

Kawaguchi, S;Kawahara, K;Fujiwara, Y;Ohnishi, K;Pan, C;Yano, H;Hirosue, A;Nagata, M;Hirayama, M;Sakata, J;Nakashima, H;Arita, H;Yamana, K;Gohara, S;Nagao, Y;Maeshiro, M;Iwamoto, A;Hirayama, M;Yoshida, R;Komohara, Y;Nakayama, H;
PMID: 35044489 | DOI: 10.1007/s00262-022-03149-w

The CD169+ macrophages in lymph nodes are implicated in cytotoxic T lymphocyte (CTL) activation and are associated with improved prognosis in several malignancies. Here, we investigated the significance of CD169+ macrophages in oral squamous cell carcinoma (OSCC). Further, we tested the anti-tumor effects of naringenin, which has been previously shown to activate CD169+ macrophages, in a murine OSCC model. Immunohistochemical analysis for CD169 and CD8 was performed on lymph node and primary tumor specimens from 89 patients with OSCC. We also evaluated the effects of naringenin on two murine OSCC models. Increased CD169+ macrophage counts in the regional lymph nodes correlated with favorable prognosis and CD8+ cell counts within tumor sites. Additionally, naringenin suppressed tumor growth in two murine OSCC models. The mRNA levels of CD169, interleukin (IL)-12, and C-X-C motif chemokine ligand 10 (CXCL10) in lymph nodes and CTL infiltration in tumors significantly increased following naringenin administration in tumor-bearing mice. These results suggest that CD169+ macrophages in lymph nodes are involved in T cell-mediated anti-tumor immunity and could be a prognostic marker for patients with OSCC. Moreover, naringenin is a new potential agent for CD169+ macrophage activation in OSCC treatment.

Expression Profile of the GLP-1 Receptor in the Gastrointestinal Tract and Pancreas in Adult Female Mice

Endocrinology

2022 Jan 01

Grunddal, KV;Jensen, EP;Ørskov, C;Andersen, DB;Windeløv, JA;Poulsen, SS;Rosenkilde, MM;Knudsen, LB;Pyke, C;Holst, JJ;
PMID: 34662392 | DOI: 10.1210/endocr/bqab216

Therapies based on glucagon-like peptide-1 receptor (GLP-1R) agonism are highly effective in treating type 2 diabetes and obesity, but the localization of GLP-1Rs mediating the antidiabetic and other possible actions of GLP-1 is still debated. The purpose with this study was to identify sites of GLP-1R mRNA and protein expression in the mouse gastrointestinal system by means of GLP-1R antibody immunohistochemistry, Glp1r mRNA fluorescence in situ hybridization, and 125I-exendin (9-39) autoradiography. As expected, GLP-1R staining was observed in almost all β-cells in the pancreatic islets, but more rarely in α- and δ-cells. In the stomach, GLP-1R staining was found exclusively in the gastric corpus mucous neck cells, known to protect the stomach mucosa. The Brunner glands were strongly stained for GLP-1R, and pretreatment with GLP-1 agonist exendin-4 caused internalization of the receptor and mucin secretion, while pretreatment with phosphate-buffered saline or antagonist exendin (9-39) did not. In the intestinal mucosa, GLP-1R staining was observed in intraepithelial lymphocytes, lamina propria lymphocytes, and enteroendocrine cells containing secretin, peptide YY, and somatostatin, but not cholecystokinin. GLP-1R staining was seen in nerve fibers within the choline acetyl transferase- and nitric oxide-positive myenteric plexuses from the gastric corpus to the distal large intestine being strongest in the mid- and hindgut area. Finally, intraperitoneal administration of radiolabeled exendin (9-39) strongly labeled myenteric fibers. In conclusion, this study expands our knowledge of GLP-1R localization and suggests that GLP-1 may serve an important role in modulating gastrointestinal health and mucosal protection.

Transcription start site-level expression of thyroid transcription factor 1 isoforms in lung adenocarcinoma and its clinicopathological significance

The journal of pathology. Clinical research

2021 May 20

Sano, K;Hayashi, T;Suehara, Y;Hosoya, M;Takamochi, K;Kohsaka, S;Kishikawa, S;Kishi, M;Saito, S;Takahashi, F;Kaneko, K;Suzuki, K;Yao, T;Ishijima, M;Saito, T;
PMID: 34014042 | DOI: 10.1002/cjp2.213

There are multiple transcription start sites (TSSs) in agreement with multiple transcript variants encoding different isoforms of NKX2-1/TTF-1 (thyroid transcription factor 1); however, the clinicopathological significance of each transcript isoform of NKX2-1/TTF-1 in lung adenocarcinoma (LAD) is unknown. Herein, TSS-level expression of NKX2-1/TTF-1 isoforms was evaluated in 71 LADs using bioinformatic analysis of cap analysis of gene expression (CAGE)-sequencing data, which provides genome-wide expression levels of the 5'-untranslated regions and the TSSs of different isoforms. Results of CAGE were further validated in 664 LADs using in situ hybridisation. Fourteen of 17 TSSs in NKX2-1/TTF-1 (80% of known TSSs in FANTOM5, an atlas of mammalian promoters) were identified in LADs, including TSSs 1-13 and 15; four isoforms of NKX2-1/TTF-1 transcripts (NKX2-1_001, NKX2-1_002, NKX2-1_004, and NKX2-1_005) were expressed in LADs, although NKX2-1_005 did not contain a homeodomain. Among those, six TSSs regulated NKX2-1_004 and NKX2-1_005, both of which contain exon 1. LADs with low expression of isoforms from TSS region 11 regulating exon 1 were significantly associated with poor prognosis in the CAGE data set. In the validation set, 62 tumours (9.3%) showed no expression of NKX2-1/TTF-1 exon 1; such tumours were significantly associated with older age, EGFR wild-type tumours, and poor prognosis. In contrast, 94 tumours, including 22 of 30 pulmonary invasive mucinous adenocarcinomas (IMAs) exhibited exon 1 expression without immunohistochemical TTF-1 protein expression. Furthermore, IMAs commonly exhibited higher exon 1 expression relative to that of exon 4/5, which contained a homeodomain in comparison with EGFR-mutated LADs. These transcriptome and clinicopathological results reveal that LAD use at least 80% of NKX2-1 TSSs and expression of the NKX2-1/TTF-1 transcript isoform without exon 1 (NKX2-1_004 and NKX2-1_005) defines a distinct subset of LAD characterised by aggressive behaviour in elder patients. Moreover, usage of alternative TSSs regions regulating NKX2-1_005 may occur in subsets of LADs.

Histological Lesions and Replication Sites of PCV3 in Naturally Infected Pigs

Animals : an open access journal from MDPI

2021 May 24

De Conti, ER;Resende, TP;Marshall-Lund, L;Rovira, A;Vannucci, FA;
PMID: 34073660 | DOI: 10.3390/ani11061520

Porcine circovirus type 3 (PCV3) has been recently described as a potential cause of abortions and systemic vasculitis in pigs. Although the virus has been detected by real-time PCR in several porcine tissues from countries worldwide, PCV3-associated diseases have not been satisfactorily clarified. The objective of this study was to investigate the association between the presence of PCV3 mRNA detected by in situ hybridization (ISH) within histological lesions and PCV3 DNA detected by real-time PCR in naturally infected pigs. A total of 25 PCV3 PCR-positive cases were analyzed. Formalin-fixed tissues from these cases were evaluated for histologic lesions and for ISH-RNA positive signals for PCV3. The most frequent tissue type with histopathologic lesions was heart, 76.2%, with lymphoplasmacytic myocarditis and epicarditis as the most frequent lesions observed. Lymphoplasmacytic interstitial pneumonia was also a frequent finding, 47.6%. There were also lesions in kidney, liver, spleen and lymph nodes. PCV3-ISH-RNA positive signals were mostly observed in association with lymphoplasmacytic inflammatory infiltrate in various tissues, including arteries. Based on our results, the minimum set of specimens to be submitted for histopathology and mRNA in situ hybridization to confirm or exclude a diagnosis of PCV3 are heart, lung and lymphoid tissues (i.e., spleen and lymph nodes), especially for differential diagnosis related with PCV2-associated diseases.

Characterisation of C101248: A novel selective THIK-1 channel inhibitor for the modulation of microglial NLRP3-inflammasome

Neuropharmacology

2022 Nov 12

Ossola, B;Rifat, A;Rowland, A;Hunter, H;Drinkall, S;Bender, C;Hamlischer, M;Teall, M;Burley, R;Barker, DF;Cadwalladr, D;Dickson, L;Lawrence, JMK;Harvey, JRM;Lizio, M;Xu, X;Kavanagh, E;Cheung, T;Sheardown, S;Lawrence, CB;Harte, M;Brough, D;Madry, C;Matthews, K;Doyle, K;Page, K;Powell, J;Brice, NL;Bürli, RW;Carlton, MB;Dawson, LA;
PMID: 36375694 | DOI: 10.1016/j.neuropharm.2022.109330

Neuroinflammation, specifically the NLRP3 inflammasome cascade, is a common underlying pathological feature of many neurodegenerative diseases. Evidence suggests that NLRP3 activation involves changes in intracellular K+. Nuclear Enriched Transcript Sort Sequencing (NETSseq), which allows for deep sequencing of purified cell types from human post-mortem brain tissue, demonstrated a highly specific expression of the tandem pore domain halothane-inhibited K+ channel 1 (THIK-1) in microglia compared to other glial and neuronal cell types in the human brain. NETSseq also showed a significant increase of THIK-1 in microglia isolated from cortical regions of brains with Alzheimer's disease (AD) relative to control donors. Herein, we report the discovery and pharmacological characterisation of C101248, the first selective small-molecule inhibitor of THIK-1. C101248 showed a concentration-dependent inhibition of both mouse and human THIK-1 (IC50: ∼50 nM) and was inactive against K2P family members TREK-1 and TWIK-2, and Kv2.1. Whole-cell patch-clamp recordings of microglia from mouse hippocampal slices showed that C101248 potently blocked both tonic and ATP-evoked THIK-1 K+ currents. Notably, C101248 had no effect on other constitutively active resting conductance in slices from THIK-1-depleted mice. In isolated microglia, C101248 prevented NLRP3-dependent release of IL-1β, an effect not seen in THIK-1-depleted microglia. In conclusion, we demonstrated that inhibiting THIK-1 (a microglia specific gene that is upregulated in brains from donors with AD) using a novel selective modulator attenuates the NLRP3-dependent release of IL-1β from microglia, which suggests that this channel may be a potential therapeutic target for the modulation of neuroinflammation in AD.

PD-1 and ICOS co-expression identifies tumor-reactive CD4 T cells in human solid tumors

The Journal of clinical investigation

2022 Apr 19

Duhen, R;Fesneau, O;Samson, KA;Frye, AK;Beymer, M;Rajamanickam, V;Ross, D;Tran, E;Bernard, B;Weinberg, AD;Duhen, T;
PMID: 35439168 | DOI: 10.1172/JCI156821

CD4 T helper (Th) cells play a key role in orchestrating immune responses, but the identity of the CD4 Th cells involved in the anti-tumor immune response remains to be defined. We analyzed the immune cell infiltrates of head and neck squamous cell carcinoma and colorectal cancers and identified a subset of CD4 Th cells distinct from FOXP3+ regulatory T cells that co-express PD-1 and ICOS. These tumor-infiltrating CD4 Th cells (CD4 Th TIL) have a tissue-resident memory phenotype, are present in MHC class II-rich areas and proliferate in the tumor suggesting local antigen recognition. The T-cell receptor repertoire of the PD-1+ICOS+ CD4 Th TIL is oligoclonal, with T-cell clones expanded in the tumor, but present at low frequencies in the periphery. Finally, these PD-1+ICOS+ CD4 Th TIL were shown to recognize both tumor-associated antigens and tumor-specific neoantigens. Our findings provide an approach for isolating tumor-reactive CD4 Th TIL directly ex vivo that will help define their role in the anti-tumor immune response and potentially improve future adoptive T-cell therapy approaches.

B4GALNT3 regulates glycosylation of sclerostin and bone mass

EBioMedicine

2023 Apr 04

Movérare-Skrtic, S;Voelkl, J;Nilsson, KH;Nethander, M;Luong, TTD;Alesutan, I;Li, L;Wu, J;Horkeby, K;Lagerquist, MK;Koskela, A;Tuukkanen, J;Tobias, JH;Lerner, UH;Henning, P;Ohlsson, C;
PMID: 37023531 | DOI: 10.1016/j.ebiom.2023.104546

Global sclerostin inhibition reduces fracture risk efficiently but has been associated with cardiovascular side effects. The strongest genetic signal for circulating sclerostin is in the B4GALNT3 gene region, but the causal gene is unknown. B4GALNT3 expresses the enzyme beta-1,4-N-acetylgalactosaminyltransferase 3 that transfers N-acetylgalactosamine onto N-acetylglucosaminebeta-benzyl on protein epitopes (LDN-glycosylation).To determine if B4GALNT3 is the causal gene, B4galnt3-/- mice were developed and serum levels of total sclerostin and LDN-glycosylated sclerostin were analysed and mechanistic studies were performed in osteoblast-like cells. Mendelian randomization was used to determine causal associations.B4galnt3-/- mice had higher circulating sclerostin levels, establishing B4GALNT3 as a causal gene for circulating sclerostin levels, and lower bone mass. However, serum levels of LDN-glycosylated sclerostin were lower in B4galnt3-/- mice. B4galnt3 and Sost were co-expressed in osteoblast-lineage cells. Overexpression of B4GALNT3 increased while silencing of B4GALNT3 decreased the levels of LDN-glycosylated sclerostin in osteoblast-like cells. Mendelian randomization demonstrated that higher circulating sclerostin levels, genetically predicted by variants in the B4GALNT3 gene, were causally associated with lower BMD and higher risk of fractures but not with higher risk of myocardial infarction or stroke. Glucocorticoid treatment reduced B4galnt3 expression in bone and increased circulating sclerostin levels and this may contribute to the observed glucocorticoid-induced bone loss.B4GALNT3 is a key factor for bone physiology via regulation of LDN-glycosylation of sclerostin. We propose that B4GALNT3-mediated LDN-glycosylation of sclerostin may be a bone-specific osteoporosis target, separating the anti-fracture effect of global sclerostin inhibition, from indicated cardiovascular side effects.Found in acknowledgements.

Cerebellar spreading depolarization mediates paroxysmal movement disorder

Cell reports

2021 Sep 21

Lu, B;Lou, SS;Xu, RS;Kong, DL;Wu, RJ;Zhang, J;Zhuang, L;Wu, XM;He, JY;Wu, ZY;Xiong, ZQ;
PMID: 34551285 | DOI: 10.1016/j.celrep.2021.109743

Paroxysmal kinesigenic dyskinesia (PKD) is the most common paroxysmal dyskinesia, characterized by recurrent episodes of involuntary movements provoked by sudden changes in movement. Proline-rich transmembrane protein 2 (PRRT2) has been identified as the major causative gene for PKD. Here, we report that PRRT2 deficiency facilitates the induction of cerebellar spreading depolarization (SD) and inhibition of cerebellar SD prevents the occurrence of dyskinetic movements. Using Ca2+ imaging, we show that cerebellar SD depolarizes a large population of cerebellar granule cells and Purkinje cells in Prrt2-deficient mice. Electrophysiological recordings further reveal that cerebellar SD blocks Purkinje cell spiking and disturbs neuronal firing of the deep cerebellar nuclei (DCN). The resultant aberrant firing patterns in DCN are tightly, temporally coupled to dyskinetic episodes in Prrt2-deficient mice. Cumulatively, our findings uncover a pivotal role of cerebellar SD in paroxysmal dyskinesia, providing a potent target for treating PRRT2-related paroxysmal disorders.

Mitochondrial glutamine transporter SLC1A5_var, a potential target to suppress astrocyte reactivity in Parkinson's Disease

Cell death & disease

2022 Nov 09

Liu, Y;Cao, L;Song, Y;Kang, Z;Liu, T;Ding, J;Hu, G;Lu, M;
PMID: 36351889 | DOI: 10.1038/s41419-022-05399-z

SLC1A5 variant (SLC1A5_var) is identified as a mitochondrial glutamine transporter in cancer cells recently. However, the role of SLC1A5_var in Parkinson's disease (PD) is completely unknown. Here, we found the significant downregulation of SLC1A5_var in astrocytes and midbrain of mice treated with MPTP/MPP+ and LPS. Importantly, overexpression of SLC1A5_var ameliorated but knockdown of SLC1A5_var exacerbated MPTP/MPP+- and LPS-induced mitochondrial dysfunction. Consequently, SLC1A5_var provided beneficial effects on PD pathology including improvement of PD-like motor symptoms and rescue of dopaminergic (DA) neuron degeneration through maintaining mitochondrial energy metabolism. Moreover, SLC1A5_var reduced astrocyte reactivity via inhibition of A1 astrocyte conversion. Further investigation demonstrated that SLC1A5_var restrained the secretion of astrocytic pro-inflammatory cytokines by blunting TLR4-mediated downstream pathways. This is the first study to prove that astrocytic SLC1A5_var inhibits neuroinflammation, and rescues the loss of DA neurons and motor symptoms involved in PD progression, which provides a novel target for PD treatment.

Epigenomic landscape of human colorectal cancer unveils an aberrant core of pan-cancer enhancers orchestrated by YAP/TAZ

Nature communications

2021 Apr 20

Della Chiara, G;Gervasoni, F;Fakiola, M;Godano, C;D'Oria, C;Azzolin, L;Bonnal, RJP;Moreni, G;Drufuca, L;Rossetti, G;Ranzani, V;Bason, R;De Simone, M;Panariello, F;Ferrari, I;Fabbris, T;Zanconato, F;Forcato, M;Romano, O;Caroli, J;Gruarin, P;Sarnicola, ML;Cordenonsi, M;Bardelli, A;Zucchini, N;Ceretti, AP;Mariani, NM;Cassingena, A;Sartore-Bianchi, A;Testa, G;Gianotti, L;Opocher, E;Pisati, F;Tripodo, C;Macino, G;Siena, S;Bicciato, S;Piccolo, S;Pagani, M;
PMID: 33879786 | DOI: 10.1038/s41467-021-22544-y

Cancer is characterized by pervasive epigenetic alterations with enhancer dysfunction orchestrating the aberrant cancer transcriptional programs and transcriptional dependencies. Here, we epigenetically characterize human colorectal cancer (CRC) using de novo chromatin state discovery on a library of different patient-derived organoids. By exploring this resource, we unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic and independent of interpatient heterogeneity. We show that the transcriptional coactivators YAP/TAZ act as key regulators of the conserved CRC gained enhancers. The same YAP/TAZ-bound enhancers display active chromatin profiles across diverse human tumors, highlighting a pan-cancer epigenetic rewiring which at single-cell level distinguishes malignant from normal cell populations. YAP/TAZ inhibition in established tumor organoids causes extensive cell death unveiling their essential role in tumor maintenance. This work indicates a common layer of YAP/TAZ-fueled enhancer reprogramming that is key for the cancer cell state and can be exploited for the development of improved therapeutic avenues.

Functional antagonism between ΔNp63α and GCM1 regulates human trophoblast stemness and differentiation

Nature communications

2022 Mar 25

Wang, LJ;Chen, CP;Lee, YS;Ng, PS;Chang, GD;Pao, YH;Lo, HF;Peng, CH;Cheong, ML;Chen, H;
PMID: 35338152 | DOI: 10.1038/s41467-022-29312-6

The combination of EGF, CHIR99021, A83-01, SB431542, VPA, and Y27632 (EGF/CASVY) facilitates the derivation of trophoblast stem (TS) cells from human blastocysts and first-trimester, but not term, cytotrophoblasts. The mechanism underlying this chemical induction of TS cells remains elusive. Here we demonstrate that the induction efficiency of cytotrophoblast is determined by functional antagonism of the placental transcription factor GCM1 and the stemness regulator ΔNp63α. ΔNp63α reduces GCM1 transcriptional activity, whereas GCM1 inhibits ΔNp63α oligomerization and autoregulation. EGF/CASVY cocktail activates ΔNp63α, thereby partially inhibiting GCM1 activity and reverting term cytotrophoblasts into stem cells. By applying hypoxia condition, we can further reduce GCM1 activity and successfully induce term cytotrophoblasts into TS cells. Consequently, we identify mitochondrial creatine kinase 1 (CKMT1) as a key GCM1 target crucial for syncytiotrophoblast differentiation and reveal decreased CKMT1 expression in preeclampsia. Our study delineates the molecular underpinnings of trophoblast stemness and differentiation and an efficient method to establish TS cells from term placentas.

Glucokinase neurons of the paraventricular nucleus of the thalamus sense glucose and decrease food consumption

iScience

2021 Oct 01

Kessler, S;Labouèbe, G;Croizier, S;Gaspari, S;Tarussio, D;Thorens, B;
| DOI: 10.1016/j.isci.2021.103122

The paraventricular nucleus of the thalamus (PVT) controls goal-oriented behavior through its connections to the nucleus accumbens (NAc). We previously characterized Glut2aPVT neurons that are activated by hypoglycemia, and which increase sucrose seeking behavior through their glutamatergic projections to the NAc. Here, we identified glucokinase (Gck)-expressing neurons of the PVT (GckaPVT) and generated a mouse line expressing the Cre recombinase from the glucokinase locus (GckCre/+ mice). Ex vivo calcium imaging and whole-cell patch clamp recordings revealed that GckaPVT neurons that project to the NAc were mostly activated by hyperglycemia. Their chemogenetic inhibition or optogenetic stimulation, respectively, enhanced food intake or decreased sucrose-seeking behavior. Collectively, our results describe a neuronal population of Gck-expressing neurons in the PVT, which has opposite glucose sensing properties and control over feeding behavior than the previously characterized Glut2aPVT neurons. This study allows a better understanding of the complex regulation of feeding behavior by the PVT.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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