Galera, P;Alejo, J;Valadez, R;Davies-Hill, T;Menon, M;Hasni, S;Jaffe, E;Pittaluga, S;
| DOI: 10.2139/ssrn.4115599
Kikuchi Fujimoto Disease (KFD) is a rare form of localized lymphadenopathy, commonly affecting young Asian females with a self-limited course. The immunopathogenic mechanisms underlying KFD are still not well understood. KFD and systemic lupus erythematosus (SLE) share several histologic and clinical features, thus posing a diagnostic challenge. The aim of this study was to elucidate the in-situ distribution of immune cells and the cytokine/chemokine milieu of KFD utilizing immunohistochemistry to identify key cellular elements and RNAscope to assess cytokine and chemokine production. This study further compared the clinical, morphologic, and immunologic features of KFD to SLE.18 KFD, 16 SLE and 3 reactive lymph nodes were included. In contrast to KFD and reactive lymph nodes, SLE patients frequently exhibited generalized lymphadenopathy and had significantly higher frequency of systemic manifestations. Both KFD and SLE lymph nodes revealed overlapping morphologic findings with few distinguishing features namely the presence of capsular fibrosis and plasmacytosis in SLE and predominance of CD8-positive T cells in KFD.RNAscope studies in the KFD cohort revealed significantly higher amounts of interferon γ (IFN-γ), CXCL9 and CXCL10 in comparison to the SLE and reactive lymph nodes. These findings suggest a T-helper cell 1 (Th1) response, driven by IFN-γ and IFN-γ induced CXCL9 and CXCL10, is pivotal in the pathogenesis of KFD and is less evident in lymph nodes from SLE patients. Distinguishing histological features between KFD and SLE are subtle. Studying the cytokine/chemokine environment provides valuable insight into the pathophysiology of KFD. In addition, assessing the production of these cytokines/chemokines may provide further diagnostic help in differentiating KFD from SLE.
Namineni S, O'Connor T, Faure-Dupuy S, Johansen P, Riedl T, Liu K, Xu H, Singh I, Shinde P, Li F, Pandyra A, Sharma P, Ringelhan M, Muschaweckh A, Borst K, Blank P, Lampl S, Durantel D, Farhat R, Weber A, Lenggenhager D, K�ndig TM, Staeheli P, Protzer U, Wohlleber D, Holzmann B, Binder M, Breuhahn K, Assmus LM, Nattermann J, Abdullah Z, Rolland M, Dejardin E, Lang PA, Lang KS, Karin M, Lucifora J, Kalinke U, Knolle PA, Heikenwalder M
PMID: 31954207 | DOI: 10.1016/j.jhep.2019.12.019
Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver macrophages. However, also hepatocytes, the parenchymal cells of the liver, possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct anti-viral mechanisms employed by hepatocytes.
METHODS:
Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-?B signaling (IKK??Hep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-?/? signaling-(IFNAR?Hep), or interferon-?/? signaling in myeloid cells-(IFNAR?Myel) were infected.
RESULTS:
Here, we demonstrate that LCMV activates NF-?B signaling in hepatocytes. LCMV-triggered NF-?B activation in hepatocytes did not depend on Kupffer cells or TNFR1- but rather on TLR-signaling. LCMV-infected IKK??Hep livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8+ T-cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKK?, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IKK??Hep mice, enhanced hepatocytic LCMV accumulation was observed in livers of IFNAR?Hep, whereas IFNAR?Myel mice were able to control LCMV-infection. Hepatocytic NF-?B signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-?/?-mediated inhibition of HBV replication in vitro.
CONCLUSIONS:
Together, these data show that hepatocyte-intrinsic NF-?B is a vital amplifier of interferon-?/? signaling pivotal for early, strong ISG responses, influx of immune cells and hepatic viral clearance.
The American Journal of Pathology
Ha Y, Liu H, Zhu S, Yi P, Liu W, Nathanson J, Kayed R, Loucas B, Sun J, Frishman LJ, Motamedi M, Zhang W.
PMID: 27960090 | DOI: 10.1016/j.ajpath.2016.10.009
Traumatic optic neuropathy (TON) is an acute injury of the optic nerve secondary to trauma. Loss of retinal ganglion cells (RGCs) is a key pathological process in TON, yet mechanisms responsible for RGC death remain unclear. In a mouse model of TON, real-time noninvasive imaging revealed a dramatic increase in leukocyte rolling and adhesion in veins near the optic nerve (ON) head at 9 hours after ON injury. Although RGC dysfunction and loss were not detected at 24 hours after injury, massive leukocyte infiltration was observed in the superficial retina. These cells were identified as T cells, microglia/monocytes, and neutrophils but not B cells. CXCL10 is a chemokine that recruits leukocytes after binding to its receptor C-X-C chemokine receptor (CXCR) 3. The levels of CXCL10 and CXCR3 were markedly elevated in TON, and up-regulation of CXCL10 was mediated by STAT1/3. Deleting CXCR3 in leukocytes significantly reduced leukocyte recruitment, and prevented RGC death at 7 days after ON injury. Treatment with CXCR3 antagonist attenuated TON-induced RGC dysfunction and cell loss. In vitro co-culture of primary RGCs with leukocytes resulted in increased RGC apoptosis, which was exaggerated in the presence of CXCL10. These results indicate that leukocyte recruitment in retinal vessels near the ON head is an early event in TON and the CXCL10/CXCR3 axis has a critical role in recruiting leukocytes and inducing RGC death.
Taieb, M;Ghannoum, D;Barré, L;Ouzzine, M;
PMID: 37296099 | DOI: 10.1038/s41419-023-05875-0
Genetic mutations in the Xylt1 gene are associated with Desbuquois dysplasia type II syndrome characterized by sever prenatal and postnatal short stature. However, the specific role of XylT-I in the growth plate is not completely understood. Here, we show that XylT-I is expressed and critical for the synthesis of proteoglycans in resting and proliferative but not in hypertrophic chondrocytes in the growth plate. We found that loss of XylT-I induces hypertrophic phenotype-like of chondrocytes associated with reduced interterritorial matrix. Mechanistically, deletion of XylT-I impairs the synthesis of long glycosaminoglycan chains leading to the formation of proteoglycans with shorter glycosaminoglycan chains. Histological and Second Harmonic Generation microscopy analysis revealed that deletion of XylT-I accelerated chondrocyte maturation and prevents chondrocytes columnar organization and arrangement in parallel of collagen fibers in the growth plate, suggesting that XylT-I controls chondrocyte maturation and matrix organization. Intriguingly, loss of XylT-I induced at embryonic stage E18.5 the migration of progenitor cells from the perichondrium next to the groove of Ranvier into the central part of epiphysis of E18.5 embryos. These cells characterized by higher expression of glycosaminoglycans exhibit circular organization then undergo hypertrophy and death creating a circular structure at the secondary ossification center location. Our study revealed an uncovered role of XylT-I in the synthesis of proteoglycans and provides evidence that the structure of glycosaminoglycan chains of proteoglycans controls chondrocyte maturation and matrix organization.
Journal for immunotherapy of cancer
Reschke, R;Yu, J;Flood, B;Higgs, EF;Hatogai, K;Gajewski, TF;
PMID: 34593622 | DOI: 10.1136/jitc-2021-003521
A T cell-inflamed tumor microenvironment is characterized by the accumulation and local activation of CD8+ T cells and Bat3-lineage dendritic cells, which together are associated with clinical response to anti-programmed cell death protein 1 (anti-PD-1)-based immunotherapy. Preclinical models have demonstrated a crucial role for the chemokine CXCL10 in the recruitment of effector CD8+ T cells into the tumor site, and a chemokine gene signature is also seen in T cell-inflamed tumors from patients. However, the cellular source of CXCL10 in human solid tumors is not known. To identify the cellular source of CXCL10 we analyzed 22 pretreatment biopsy samples of melanoma metastases from patients who subsequently underwent checkpoint blockade immunotherapy. We stained for CD45+ and Sox10+ cells with multiparameter immunofluorescence staining, and RNA in situ hybridization technology was used in concert to identify CXCL10 transcripts. The results were correlated with the expression levels of CXCL10 transcripts from bulk RNA sequencing and the best overall response to immune checkpoint inhibition (anti-PD-1 alone or with anti-CTLA-4) in the same patients. We identified CD45+ cells as the major cellular source for CXCL10 in human melanoma metastases, with additional CXCL10 production seen by Sox10+ cells. Up to 90% of CD45+ cells and up to 69% of Sox10+ cells produced CXCL10 transcripts. The CXCL10 staining result was consistent with the level of CXCL10 expression determined by bulk RNA sequencing. The percentages of CD45+ CXCL10+ cells and Sox10+ CXCL10+ cells independently predicted response (p<0.001). The average number of transcripts per cell correlated with the CD45+ cell infiltrate (R=0.37). Immune cells and melanoma cells produce CXCL10 in human melanoma metastases. Intratumoral CXCL10 is a positive prognostic factor for response to immunotherapy, and the RNAscope technique is achievable using paraffin tissue. Strategies that support effector T cell recruitment via induction of CXCL10 should be considered as a mechanism-based intervention to expand immunotherapy efficacy.
Garcia-Alonso, L;Lorenzi, V;Mazzeo, CI;Alves-Lopes, JP;Roberts, K;Sancho-Serra, C;Engelbert, J;Marečková, M;Gruhn, WH;Botting, RA;Li, T;Crespo, B;van Dongen, S;Kiselev, VY;Prigmore, E;Herbert, M;Moffett, A;Chédotal, A;Bayraktar, OA;Surani, A;Haniffa, M;Vento-Tormo, R;
PMID: 35794482 | DOI: 10.1038/s41586-022-04918-4
Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.
PLoS One. 2014 Nov 3;9(11):e111658.
Choi J, Lee HE, Kim MA, Jang BG, Lee HS, Kim WH.
PMID: 25364819 | DOI: 10.1371/journal.pone.0111658
We investigated MET mRNA expression status using RNA in situ hybridization (ISH) technique in primary and metastatic lesions of 535 surgically resected gastric carcinoma (GC) cases. We compared the results with those of immunohistochemistry and silver in situ hybridization, and examined the association with clinicopathologic characteristics and prognosis. Among 535 primary GCs, 391 (73.1%) were scored 0, 87 (16.3%) were scored 1, 38 (7.1%) were scored 2, 12 (2.2%) were scored 3 and 7 (1.3%) were scored 4 by RNA ISH. High MET mRNA expression (score ≥3) was associated with lymph node metastasis (P = .014), distant metastasis (P = .001), and higher TNM stage (P<.001). MET mRNA expression was correlated with protein expression (r = 0.398; P<.001) and gene copy number (r = 0.345; P<.001). The patients showing high-MET mRNA in primary or metastatic lesions had shorter overall survival than those showing low-MET mRNA (primary tumors, P = .002; metastatic lymph nodes, P<.001). The patients showing positive conversion of MET mRNA status in metastatic lymph node had shorter overall survival than those with no conversion (P = .011). Multivariate analysis demonstrated that high MET mRNA expression in metastatic lymph node was an independent prognostic factor for overall survival (P = .007). Therefore, this study suggests that MET mRNA expression assessed by RNA ISH could be useful as a potential marker to identify MET oncogene-addicted GC.
Ouwendijk WJ, Getu S, Mahalingam R, Gilden D, Osterhaus AD, Verjans GM.
PMID: 26676825 | DOI: -
Primary simian varicella virus (SVV) infection in non-human primates causes varicella, after which the virus becomes latent in ganglionic neurons and reactivates to cause zoster. The host response in ganglia during establishment of latency is ill-defined. Ganglia from five African green monkeys (AGMs) obtained at 9, 13, and 20 days post-intratracheal SVV inoculation (dpi) were analyzed by ex vivo flow cytometry, immunohistochemistry, and in situ hybridization. Ganglia at 13 and 20 dpi exhibited mild inflammation. Immune infiltrates consisted mostly of CD8dim and CD8bright memory T cells, some of which expressed granzyme B, and fewer CD11c+ and CD68+ cells. Chemoattractant CXCL10 transcripts were expressed in neurons and infiltrating inflammatory cells but did not co-localize with SVV open reading frame 63 (ORF63) RNA expression. Satellite glial cells expressed increased levels of activation markers CD68 and MHC class II at 13 and 20 dpi compared to those at 9 dpi. Overall, local immune responses emerged as viral DNA load in ganglia declined, suggesting that intra-ganglionic immunity contributes to restricting SVV replication.
Ma F, Ye H, He HH, Gerrin SJ, Chen S, Tanenbaum BA, Cai C, Sowalsky AG, He L, Wang H, Balk SP, Yuan X.
PMID: 27043282 | DOI: 10.1172/JCI78815.
The transcription factor SOX9 is critical for prostate development, and dysregulation of SOX9 is implicated in prostate cancer (PCa). However, the SOX9-dependent genes and pathways involved in both normal and neoplastic prostate epithelium are largely unknown. Here, we performed SOX9 ChIP sequencing analysis and transcriptome profiling of PCa cells and determined that SOX9 positively regulates multiple WNT pathway genes, including those encoding WNT receptors (frizzled [FZD] and lipoprotein receptor-related protein [LRP] family members) and the downstream β-catenin effector TCF4. Analyses of PCa xenografts and clinical samples both revealed an association between the expression of SOX9 and WNT pathway components in PCa. Finally, treatment of SOX9-expressing PCa cells with a WNT synthesis inhibitor (LGK974) reduced WNT pathway signaling in vitro and tumor growth in murine xenograft models. Together, our data indicate that SOX9 expression drives PCa by reactivating the WNT/β-catenin signaling that mediates ductal morphogenesis in fetal prostate and define a subgroup of patients who would benefit from WNT-targeted therapy.
Bradley CA, Dunne PD, Bingham V, McQuaid S, Khawaja H, Craig S, James J, Moore WL, McArt DG, Lawler M, Dasgupta S, Johnston PG, Van Schaeybroeck S.
PMID: 27793046 | DOI: 10.18632/oncotarget.12933
c-MET and its ligand HGF are frequently overexpressed in colorectal cancer (CRC) and increased c-MET levels are found in CRC liver metastases. This study investigated the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Pre-clinically, we found marked upregulation of c-MET at both protein and mRNA levels in several invasive CRC cells. Down-regulation of c-MET using RNAi suppressed migration/invasion of parental and invasive CRC cells. Stimulation of CRC cells with rh-HGF or co-culture with HGF-expressing colonic myofibroblasts, resulted in significant increases in their migratory/invasive capacity. Importantly, HGF-induced c-MET activation promoted rapid downregulation of c-MET protein levels, while the MET transcript remained unaltered. Using RNA in situ hybridization (RNA ISH), we further showed that MET mRNA, but not protein levels, were significantly upregulated in tumor budding foci at the invasive front of a cohort of stage III CRC tumors (p < 0.001). Taken together, we show for the first time that transcriptional upregulation of MET is a key molecular event associated with CRC invasion and tumor budding. This data also indicates that RNA ISH, but not immunohistochemistry, provides a robust methodology to assess MET levels as a potential driving force of CRC tumor invasion and metastasis.
Hoch, T;Schulz, D;Eling, N;Gómez, JM;Levesque, MP;Bodenmiller, B;
PMID: 35363540 | DOI: 10.1126/sciimmunol.abk1692
Intratumoral immune cells are crucial for tumor control and antitumor responses during immunotherapy. Immune cell trafficking into tumors is mediated by binding of specific immune cell receptors to chemokines, a class of secreted chemotactic cytokines. To broadly characterize chemokine expression and function in melanoma, we used multiplexed mass cytometry-based imaging of protein markers and RNA transcripts to analyze the chemokine landscape and immune infiltration in metastatic melanoma samples. Tumors that lacked immune infiltration were devoid of most of the profiled chemokines and exhibited low levels of antigen presentation and markers of inflammation. Infiltrated tumors were characterized by expression of multiple chemokines. CXCL9 and CXCL10 were often localized in patches associated with dysfunctional T cells expressing the B lymphocyte chemoattractant CXCL13. In tumors with B cells but no B cell follicles, T cells were the sole source of CXCL13, suggesting that T cells play a role in B cell recruitment and potentially in B cell follicle formation. B cell patches and follicles were also enriched with TCF7+ naïve-like T cells, a cell type that is predictive of response to immune checkpoint blockade. Our data highlight the strength of targeted RNA and protein codetection to analyze tumor immune microenvironments based on chemokine expression and suggest that the formation of tertiary lymphoid structures may be accompanied by naïve and naïve-like T cell recruitment, which may contribute to antitumor activity.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Schaaf, CR;Polkoff, KM;Carter, A;Stewart, AS;Sheahan, B;Freund, J;Ginzel, J;Snyder, JC;Roper, J;Piedrahita, JA;Gonzalez, LM;
PMID: 37159340 | DOI: 10.1096/fj.202300223R
Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/β-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.
