Malinczak CA, Fonseca W, Rasky AJ, Ptaschinski C, Morris S, Ziegler SF, Lukacs NW.
PMID: 31076663 | DOI: 10.1038/s41385-019-0171-3
Many studies have linked severe RSV infection during early-life with an enhanced likelihood of developing childhood asthma, showing a greater susceptibility in boys. Our studies show that early-life RSV infection leads to differential long-term effects based upon the sex of the neonate; leaving male mice prone to exacerbation upon secondary allergen exposure while overall protecting female mice. During initial viral infection, we observed better viral control in the female mice with correlative expression of interferon-β that was not observed in male mice. Additionally, we observed persistent immune alterations in male mice at 4 weeks post infection. These alterations include Th2 and Th17-skewing, innate cytokine expression (Tslp and Il33), and infiltration of innate immune cells (DC and ILC2). Upon exposure to allergen, beginning at 4 weeks following early-life RSV-infection, male mice show severe allergic exacerbation while female mice appear to be protected. Due to persistent expression of TSLP following early-life RSV infection in male mice, genetically modified TSLPR-/- mice were evaluated and demonstrated an abrogation of allergen exacerbation in male mice. These data indicate that TSLP is involved in the altered immune environment following neonatal RSV-infection that leads to more severe responses in males during allergy exposure, later in life. Thus, TSLP may be a clinically relevant therapeutic target early in life.
Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide
Xiao, L;Labaer, J;Guo, J;
PMID: 34063986 | DOI: 10.3390/cells10061277
Understanding the composition, regulation, and function of complex biological systems requires tools that quantify multiple transcripts at their native cellular locations. However, the current multiplexed RNA imaging technologies are limited by their relatively low sensitivity or specificity, which hinders their applications in studying highly autofluorescent tissues, such as formalin-fixed paraffin-embedded (FFPE) tissues. To address this issue, here we develop a multiplexed in situ RNA profiling approach with a high sensitivity and specificity. In this approach, transcripts are first hybridized by target-specific oligonucleotide probes in pairs. Only when these two independent probes hybridize to the target in tandem will the subsequent signal amplification by oligonucleotide hybridization occur. Afterwards, horseradish peroxidase (HRP) is applied to further amplify the signal and stain the target with cleavable fluorescent tyramide (CFT). After imaging, the fluorophores are chemically cleaved and the hybridized probes are stripped by DNase and formamide. Through cycles of RNA staining, fluorescence imaging, signal cleavage, and probe stripping, many different RNA species can be profiled at the optical resolution. In applying this approach, we demonstrated that multiplexed in situ RNA analysis can be successfully achieved in both fixed, frozen, and FFPE tissues.
Characterization of SARS-CoV-2 and host entry factors distribution in a COVID-19 autopsy series
Wang, X;Mannan, R;Xiao, L;Abdulfatah, E;Qiao, Y;Farver, C;Myers, J;Zelenka-Wang, S;McMurry, L;Su, F;Wang, R;Pantanowitz, L;Jentzen, J;Wilson, A;Zhang, Y;Cao, X;Chinnaiyan, A;Mehra, R;
| DOI: 10.1038/s43856-021-00025-z
Background SARS-CoV-2 is a highly contagious virus that causes the disease COVID-19. We have recently reported that androgens regulate the expression of SARS-CoV-2 host entry factors ACE2 and TMPRSS2, and androgen receptor (AR) in lung epithelial cells. We also demonstrated that the transcriptional repression of the AR enhanceosome inhibited SARS-CoV-2 infection in vitro. Methods To better understand the various sites of SARS-CoV-2 infection, and presence of host entry factors, we extensively characterized the tissue distribution and localization of SARS-CoV-2 virus, viral replication, and host entry factors in various anatomical sites sampled via autopsy. We applied RNA in-situ-hybridization (RNA-ISH), immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) approaches. We also assessed histopathological changes in SARS-CoV-2 infected tissues. Results We detect SARS-CoV-2 virus and viral replication in pulmonary tissues by RNA-ISH and IHC and a variety of non-pulmonary tissues including kidney, heart, liver, spleen, thyroid, lymph node, prostate, uterus, and colon by qRT-PCR. We observe heterogeneity in viral load and viral cytopathic effects among various organ systems, between individuals and within the same patient. In a patient with a history of kidney transplant and under immunosuppressant therapy, we observe an unusually high viral load in lung tissue by RNA-ISH, IHC and qRT-PCR. SARS-CoV-2 virus is also detected in this patent’s kidney, liver and uterus. We find ACE2, TMPRSS2 and AR expression to overlap with the infection sites. Conclusions This study portrays the impact of dispersed SARS-CoV-2 infection in diverse organ systems, thereby facilitating avenues for systematic therapeutic approaches.
Jerome, K;Sattar, S;Mehedi, M;
PMID: 36779029 | DOI: 10.1016/j.mex.2023.102050
Visualizing and quantifying mRNA and its corresponding protein provides a unique perspective of gene expression at a single-molecule level. Here, we describe a method for differentiating primary cells for making airway epithelium and detecting SARS-CoV-2 Spike (S) mRNA and S protein in the paraformaldehyde-fixed paraffin-embedded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected airway epithelium. For simultaneous detection of mRNA and protein in the same cell, we combined two protocols: 1. RNA fluorescence-based in situ hybridization (RNA-FISH) based mRNA detection and 2. fluorescence-based immunohistochemistry (IHC) based protein detection. The detection of mRNA and proteins in the same cell also allows for quantifying them using the open-source software QuPath, which provides an accurate and more straightforward fluorescent-based quantification of mRNA and protein in the microscopic images of the infected cells. Additionally, we can achieve the subcellular distribution of both S mRNA and S protein. This method identifies SARS-CoV-2 S gene products' (mRNA and protein) degree of expression and their subcellular localization in the infected airway epithelium. Advantages of this method include: •Simultaneous detection and quantification of mRNA and protein in the same cell.•Universal use due to the ability to use mRNA-specific primer-probe and protein-specific antibodies.•An open-source software QuPath provides a straightforward fluorescent-based quantification.
The journal of pathology. Clinical research
Pennel, KA;Quinn, JA;Nixon, C;Inthagard, J;van Wyk, HC;Chang, D;Rebus, S;GPOL Group, ;Hay, J;Maka, NN;Roxburgh, CS;Horgan, PG;McMillan, DC;Park, JH;Roseweir, AK;Steele, CW;Edwards, J;
PMID: 35879507 | DOI: 10.1002/cjp2.290
CXCL8 is an inflammatory chemokine elevated in the colorectal cancer (CRC) tumour microenvironment. CXCR2, the major receptor for CXCL8, is predominantly expressed by neutrophils. In the cancer setting, CXCL8 plays important roles in neutrophil chemotaxis, facilitating angiogenesis, invasion, and metastasis. This study aimed to assess the spatial distribution of CXCL8 mRNA expression in CRC specimens, explore associations with clinical characteristics, and investigate the underlying biology of aberrant CXCL8 levels. CXCR2 expression was also assessed in a second cohort of unique CRC primary tumours and synchronously resected matched liver metastases. A previously constructed tissue microarray consisting of a cohort of stage I-IV CRC patients undergoing surgical resection with curative intent (n = 438) was probed for CXCL8 via RNAscope . Analysis was performed using HALO digital pathology software to quantify expression in the tumour and stromal compartments. Scores were assessed for association with clinical characteristics. Mutational analyses were performed on a subset of these patients to determine genomic differences in patients with high CXCL8 expression. A second cohort of stage IV CRC patients with primary and matched metastatic liver tumours was stained via immunohistochemistry for CXCR2, and scores were assessed for clinical significance. CXCL8 expression within the stromal compartment was associated with reduced cancer-specific survival in the first cohort (p = 0.035), and this relationship was potentiated in right-sided colon cancer cases (p = 0.009). High CXCL8 within the stroma was associated with driving a more stromal-rich phenotype and the presence of metastases. When stromal CXCL8 scores were combined with tumour-infiltrating macrophage counts or systemic neutrophil counts, patients classified as high for both markers had significantly poorer prognosis. CXCR2+ immune cell infiltration was associated with increased stromal invasion in liver metastases (p = 0.037). These data indicate a role for CXCL8 in driving unfavourable tumour histological features and promoting metastases. This study suggests that inhibiting CXCL8/CXCR2 should be investigated in patients with right-sided colonic disease and stroma-rich tumours.
Combined single-molecule fluorescence in situ hybridization and immunohistochemistry analysis in intact murine dorsal root ganglia and sciatic nerve
Li, X;Eadara, S;Jeon, S;Liu, Y;Muwanga, G;Qu, L;Caterina, MJ;Meffert, MK;
PMID: 34142098 | DOI: 10.1016/j.xpro.2021.100555
Single-molecule fluorescence in situ hybridization (smFISH) allows spatial mapping of gene expression. This protocol presents advances in smFISH fidelity and flexibility in intact murine sensory nervous system tissue. An approach using RNAscope probes allows multiplexing, enhanced target specificity, and immunohistochemistry compatibility. Computational strategies increase quantification accuracy of mRNA puncta with a point spread function for clustered transcripts in the dorsal root ganglion and 3D masking for intermingled sciatic nerve cell types. Approaches are validated for mRNAs of modest (Lin28a) and medium (Ppib) steady-state abundance in neurons.
Anderson CM, Zhang B, Miller M, Butko E, Wu X, Laver T, Kernag C, Kim J, Luo Y, Lamparski H, Park E, Su N, Ma XJ.
PMID: 27191821 | DOI: 10.1002/jcb.25606.
Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal-to-noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA-box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development.
Ferretti E, Hohaus S, Di Napoli A, Belmonte B, Cuccaro A, Cupelli E, Galli E, Rufini V, Tripodi G, Fraternali-Orcioni G, Pistoia V, Corcione A.
PMID: - | DOI: 10.18632/oncotarget.19665
ABSTRACT
Hodgkin Lymphoma (HL) is a tumor of B-cell origin characterized by Hodgkin and Reed-Stenberg (H/RS) cells embedded in an inflammatory tissue where numerous cytokines/chemokines contribute to shape the microenvironment, leading to the typical clinical symptoms.
We investigated: i) the expression of Interleukin-IL-31 (IL-31) and Thymic Stromal Lymphopoietin (TSLP), two Th2-related cytokines with tumor-promoting and pruritogenic functions, and of the respective receptors in HL invaded lymph nodes by flow cytometry, and ii) the potential association of IL-31/TSLP plasma concentrations with clinical characteristics by ELISA.
H/RS cells and the major immune cell types infiltrating HL lymph nodes expressed intracytoplasmic and surface IL-31/TSLP, and their receptors. A subgroup of patients showing at diagnosis elevated IL-31 and TSLP plasma levels had an International Prognostic Score>2, indicative of high risk of relapse, and a subsequent positive interim PET-scan, indicative of insufficient response to chemotherapy. No correlation was found between IL-31/TSLP plasma levels and overall or event-free survival.
In conclusion, IL-31/TSLP and their receptors are expressed in HL cells and in immune cells infiltrating affected lymph nodes, where both cytokines may contribute to local immune suppression. The clinical impact of IL-31 and TSLP plasma levels has to be further defined in larger patient cohorts.
Expression of immunoglobulin constant domain genes in neurons of the mouse central nervous system
Scheurer, L;Das Gupta, RR;Saebisch, A;Grampp, T;Benke, D;Zeilhofer, HU;Wildner, H;
PMID: 34433614 | DOI: 10.26508/lsa.202101154
General consensus states that immunoglobulins are exclusively expressed by B lymphocytes to form the first line of defense against common pathogens. Here, we provide compelling evidence for the expression of two heavy chain immunoglobulin genes in subpopulations of neurons in the mouse brain and spinal cord. RNA isolated from excitatory and inhibitory neurons through ribosome affinity purification revealed Ighg3 and Ighm transcripts encoding for the constant (Fc), but not the variable regions of IgG3 and IgM. Because, in the absence of the variable immunoglobulin regions, these transcripts lack the canonical transcription initiation site used in lymphocytes, we screened for alternative 5' transcription start sites and identified a novel 5' exon adjacent to a proposed promoter element. Immunohistochemical, Western blot, and in silico analyses strongly support that these neuronal transcripts are translated into proteins containing four Immunoglobulin domains. Our data thus demonstrate the expression of two Fc-encoding genes Ighg3 and Ighm in spinal and supraspinal neurons of the murine CNS and suggest a hitherto unknown function of the encoded proteins.
Opposing effects of Wnt/β-catenin signaling on epithelial and mesenchymal cell fate in the developing cochlea
Development (Cambridge, England)
Billings, SE;Myers, NM;Quiruz, L;Cheng, AG;
PMID: 34061174 | DOI: 10.1242/dev.199091
During embryonic development, the otic epithelium and surrounding periotic mesenchymal cells originate from distinct lineages and coordinate to form the mammalian cochlea. Epithelial sensory precursors within the cochlear duct first undergo terminal mitosis before differentiating into sensory and non-sensory cells. In parallel, periotic mesenchymal cells differentiate to shape the lateral wall, modiolus and pericochlear spaces. Previously, Wnt activation was shown to promote proliferation and differentiation of both otic epithelial and mesenchymal cells. Here, we fate-mapped Wnt-responsive epithelial and mesenchymal cells in mice and found that Wnt activation resulted in opposing cell fates. In the post-mitotic cochlear epithelium, Wnt activation via β-catenin stabilization induced clusters of proliferative cells that dedifferentiated and lost epithelial characteristics. In contrast, Wnt-activated periotic mesenchyme formed ectopic pericochlear spaces and cell clusters showing a loss of mesenchymal and gain of epithelial features. Finally, clonal analyses via multi-colored fate-mapping showed that Wnt-activated epithelial cells proliferated and formed clonal colonies, whereas Wnt-activated mesenchymal cells assembled as aggregates of mitotically quiescent cells. Together, we show that Wnt activation drives transition between epithelial and mesenchymal states in a cell type-dependent manner.
Yao, W;German, B;Chraa, D;Braud, A;Hugel, C;Meyer, P;Davidson, G;Laurette, P;Mengus, G;Flatter, E;Marschall, P;Segaud, J;Guivarch, M;Hener, P;Birling, MC;Lipsker, D;Davidson, I;Li, M;
PMID: 36107619 | DOI: 10.1172/jci.insight.161438
Malignant melanoma is a major public health issue displaying frequent resistance to targeted therapy and immunotherapy. A major challenge is to better understand how melanoma cells evade immune elimination and how tumor growth and metastasis is facilitated by tumor microenvironment. Here, we show that expression of the cytokine TSLP by epidermal keratinocytes is induced by cutaneous melanoma in both mice and humans. Using genetically engineered models of melanoma and tumor cell grafting combined with TSLP knockout or overexpression, we defined a crosstalk between melanoma cells, keratinocytes and immune cells in establishing a tumor promoting microenvironment. Keratinocyte-derived TSLP is induced by signal(s) derived from melanoma cells and subsequently acts via immune cells to promote melanoma progression and metastasis. Furthermore, we show that TSLP signals through TSLPR-expressing dendritic cells to play an unrecognized role in promoting GATA3+ Tregs expressing a gene signature including ST2, CCR8, ICOS, PD-1, CTLA-4 and OX40 and exhibiting a potent suppressive activity on CD8+ T cell proliferation and IFNγ production. An analogous population of GATA3-expressing Tregs was also identified in human melanoma tumors. Together, our study provides novel insights into the role of TSLP in programming a pro-tumoral immune microenvironment in cutaneous melanoma.
Bresciani, N;Demagny, H;Lemos, V;Pontanari, F;Li, X;Sun, Y;Li, H;Perino, A;Auwerx, J;Schoonjans, K;
PMID: 35714811 | DOI: 10.1016/j.jhep.2022.05.040
Transporters of the SLC25 mitochondrial carrier superfamily bridge cytoplasmic and mitochondrial metabolism by channeling metabolites across mitochondrial membranes and are pivotal for metabolic homeostasis. Despite their physiological relevance as gatekeepers of cellular metabolism, most of the SLC25 family members remain uncharacterized. We undertook a comprehensive tissue distribution analysis of all Slc25 family members across metabolic organs and identified SLC25A47 as a liver-specific mitochondrial carrier.We used a murine loss-of-function model to unravel the role of this transporter in mitochondrial and hepatic homeostasis. We performed extensive metabolic phenotyping and molecular characterization of newly generated Slc25a47hep-/- and Slc25a47-Fgf21hep-/- mice.Slc25a47hep-/- mice displayed a wide variety of metabolic abnormalities, as a result of sustained energy deficiency in the liver originating from impaired mitochondrial respiration in this organ. This mitochondrial phenotype was associated with an activation of the mitochondrial stress response (MSR) in the liver, and the development of fibrosis, which was exacerbated upon feeding a high-fat high-sucrose diet. The MSR induced the secretion of several mitokines, amongst which FGF21 played a preponderant role on systemic physiology. To dissect the FGF21-dependent and -independent physiological changes induced in Slc25a47hep-/- mice, we generated a double Slc25a47-Fgf21hep-/- mouse model and demonstrated that several aspects of the hypermetabolic state were driven by hepatic secretion of FGF21. On the other hand, the metabolic fuel inflexibility observed in Slc25a47hep-/- mice could not be rescued with the genetic removal of Fgf21.Collectively, our data place SLC25A47 at the center of mitochondrial homeostasis, which upon dysfunction triggers robust liver-specific and systemic adaptive stress responses. The prominent role of SLC25A47 in hepatic fibrosis identifies this carrier, or its transported metabolite, as a potential target for therapeutic intervention.SLC25A47 is a liver-specific mitochondrial carrier. Slc25a47hep-/- mice are unable to maintain mitochondrial homeostasis in hepatocytes and show impaired mitochondrial respiration resulting in chronic energy deficiency, mitochondrial stress, and fibrosis in hepatocytes. Hepatic mitochondrial stress is characterized by the secretion of the mitokine FGF21 which drives a strong and systemic hypermetabolic state impacting whole-body physiology.
