Probes for INS

Dorsal root ganglion (DRG) neurons are classified into distinct types to mediate the somatosensation with different modalities. Recently, transcriptional profilings of DRG neurons by single-cell RNA-sequencing have provided new insights into the neuron typing and functional properties. Zinc-finger CCHC domain-containing 12 (Zcchc12) was reported to be the representative marker for a subtype of Gal-positive (Gal+) DRG neurons. However, the characteristics and functions of Zcchc12+ neurons are largely unknown. Here, we genetically labelled Zcchc12+ neurons in Zcchc12-CreERT2::Ai9 mice, and verified that Zcchc12 represented a new subpopulation of DRG neurons in both sexes. Zcchc12+ neurons centrally innervated the superficial laminae in spinal dorsal horn, and peripherally terminated as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. Besides, Zcchc12+ neurons also formed circumferential endings surround the hair follicles in hairy skin. Functionally, in vivo calcium imaging in DRGs revealed that Zcchc12+ neurons were polymodal nociceptors and could be activated by mechanical and noxious thermal stimuli. Behavioral tests showed that selective ablation of Zcchc12+ DRG neurons reduced the sensitivity to noxious heat in mice. Taken together, we identify a new subpopulation of Zcchc12+ nociceptors essential for noxious heat sensation.SIGNIFICANCE STATEMENT:Zcchc12 represents a new subpopulation of DRG neurons. The characteristics and functions of Zcchc12+ neurons are largely unknown. Here we genetically labelled Zcchc12 neurons, and showed that the fibers of Zcchc12+ DRG neurons projected to superficial lamina at spinal dorsal horn, and innervated skin as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. Functionally, Zcchc12+ DRG neurons responded to noxious mechanical and heat stimuli. Ablation of Zcchc12+ DRG neurons impaired the sensation of noxious heat in mice. Therefore, we identify a new subpopulatipn of DRG neurons required for noxious heat sensation.

Stromal-epithelial interactions are critical to the morphogenesis, differentiation, and homeostasis of the prostate, but the molecular identity and anatomy of discrete stromal cell types is poorly understood. Using single-cell RNA sequencing, we identified and validated the in situ localization of three smooth muscle subtypes (prostate smooth muscle, pericytes, and vascular smooth muscle) and two novel fibroblast subtypes in human prostate. Peri-epithelial fibroblasts (APOD+) wrap around epithelial structures, whereas interstitial fibroblasts (C7+) are interspersed in extracellular matrix. In contrast, the mouse displayed three fibroblast subtypes with distinct proximal-distal and lobe-specific distribution patterns. Statistical analysis of mouse and human fibroblasts showed transcriptional correlation between mouse prostate (C3+) and urethral (Lgr5+) fibroblasts and the human interstitial fibroblast subtype. Both urethral fibroblasts (Lgr5+) and ductal fibroblasts (Wnt2+) in the mouse contribute to a proximal Wnt/Tgfb signaling niche that is absent in human prostate. Instead, human peri-epithelial fibroblasts express secreted WNT inhibitors SFRPs and DKK1, which could serve as a buffer against stromal WNT ligands by creating a localized signaling niche around individual prostate glands. We also identified proximal-distal fibroblast density differences in human prostate that could amplify stromal signaling around proximal prostate ducts. In human benign prostatic hyperplasia, fibroblast subtypes upregulate critical immunoregulatory pathways and show distinct distributions in stromal and glandular phenotypes. A detailed taxonomy of leukocytes in benign prostatic hyperplasia reveals an influx of myeloid dendritic cells, T cells and B cells, resembling a mucosal inflammatory disorder. A receptor-ligand interaction analysis of all cell types revealed a central role for fibroblasts in growth factor, morphogen, and chemokine signaling to endothelia, epithelia, and leukocytes. These data are foundational to the development of new therapeutic targets in benign prostatic hyperplasia.

The pathogenesis of gastroesophageal reflux disease (GERD) is not fully understood. It involves the activation of mucosal immune-mediated and inflammatory responses. Toll-like receptors (TLR) 2 and TLR4 are pattern-recognition receptors of the innate immune system; they recognize microbial and endogenous ligands. Farnesoid X receptor (FXR) is a bile acid receptor that regulates the inflammatory response. We aimed to evaluate TLR2, TLR4 and FXR expression patterns in GERD. We re-evaluated 84 oesophageal biopsy samples according to the global severity (GS) score, including 26 cases with histologically normal oesophagus, 28 with histologically mild oesophagitis and 30 with severe oesophagitis. We used immunohistochemistry and in situ hybridization to assess the expression patterns of TLR2, TLR4 and FXR in oesophageal squamous cells. Immunohistochemistry showed that nuclear and cytoplasmic TLR2 was expressed predominantly in the basal layer of normal oesophageal epithelium. In oesophagitis, TLR2 expression increased throughout the epithelium, and the superficial expression was significantly more intensive compared to normal epithelium, p <0.01. Nuclear and cytoplasmic TLR4 was expressed throughout the thickness of squamous epithelium, with no change in oesophagitis. FXR was expressed in the nuclei of squamous cells, and the intensity of the expression increased significantly in oesophagitis (p <0.05). FXR expression correlated with basal TLR2. In situ hybridization confirmed the immunohistochemical expression patterns of TLR2 and TLR4. In GERD, TLR2, but not TLR4, expression was upregulated which indicates that innate immunity is activated according to a specific pattern in GERD. FXR expression was increased in GERD and might have a regulatory connection to TLR2.

Digital spatial profiling (DSP) is an emerging powerful technology for proteomics and transcriptomics analyses in a spatially resolved manner for formalin-fixed paraffin-embedded (FFPE) samples developed by nanoString Technologies. DSP applies several advanced technologies, including high-throughput readout technologies (digital optical barcodes by nCounter instruments or next generation sequencing (NGS)), programmable digital micromirror device (DMD) technology, and microfluidic sampling technologies into traditional immunohistochemistry (IHC) and RNA in situ hybridization (ISH) approaches, creating an innovative tool for discovery, translational research, and clinical uses. Since its launch in 2019, DSP has been rapidly adopted, especially in immuno-oncology and tumor microenvironment research areas, and has revealed valuable information that was inaccessible before. In this article, we report the successful setup and validation of the first DSP technology platform in China. Both DSP spatial protein and RNA profiling approaches were validated using FFPE colorectal cancer tissues. Regions of interest (ROIs) were selected in the areas enriched with tumor cells, stroma/immune cells, or normal epithelial cells, and multiplex spatial profiling of both proteins and RNAs were performed. DSP spatial profiling data were processed and normalized accordingly, validating the high quality and consistency of the data. Unsupervised hierarchical clustering as well as principal component analysis (PCA) grouped tumor, stroma/immune cells, and normal epithelial cells into distinct clusters, indicating that the DSP approach effectively captured the spatially resolved proteomics and transcriptomics profiles of different compartments within the tumor microenvironment. In summary, the results confirmed the expected sensitivity and robustness of the DSP approach in profiling both proteins and RNAs in a spatially resolved manner. As a novel technology in highly complex spatial analyses, DSP endows refined analytical power from the tumor microenvironment perspective with the potential of scaling up to more analyzable targets at relatively low cell input levels. We expect that the DSP technology will greatly advance a wide range of biomedical research, especially in immuno-oncology and tumor microenvironment research areas.

Background: Orai1 is a plasma membrane Ca2+ channel that is involved in store-operated calcium entry (SOCE). In pulmonary cells, SOCE regulates gene expression and stimulates cytokine, mucin, and protease secretion. Activation of Orai1/SOCE results in recruitment of neutrophils to the lungs. Orai1 activation is also upstream of transcription factors such as nuclear factor of activated T cells, which facilitate onset of inflammation. In cystic fibrosis (CF), the immune response is dysregulated, and the lung is chronically inflamed, but Orai1 expression in the CF lung is poorly understood. Orai1 is a promising target for drug development, so we tested the hypothesis that Orai1 was upregulated in CF lungs. Methods: We used LungMAP to analyze single-cell ribonucleic acid (RNA) sequencing data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, CF, and asthma (disease control) donors (4 male/4 female per group). We imaged sections by confocal and super resolution microscopy and analyzed Orai1 and STIM1 expression, colocalization, and particle size in different pulmonary cell types. Results: Orai1 was broadly expressed throughout the lung, but expression was greatest in immune cells. At messenger RNA and protein levels, there were no consistent trends in expression levels between the three groups. Orai1 must interact with STIM1 to activate SOCE, so we used STIM1/Orai1 colocalization as a marker of Orai1 activity. Using this approach, we found significantly greater colocalization between these proteins in CF and asthma lung epithelia (CF 50%, asthma 54%, normal 15%), interstitia (CF 57%, asthma 49%, normal 16%), and luminal immune cells (CF 66%, asthma 70%, normal 38%). Orai1 also aggregates as part of its interaction process. Using super-resolution microscopy, we found significantly more Orai1 and STIM1 aggregation in immune cells from CF and asthmatic lungs (average Orai1 particle size: CF 52 nm, asthma 63 nm, normal 28 nm; average STIM1 particle size: CF 77 nm, asthma 59 nm, normal 14 nm). We also looked at Orai1 in peripheral blood neutrophils from normal and CF donors (5 per group). All CF subjects took elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA), but under baseline conditions, there were significantly bigger puncta in CF neutrophils (CF 10 nm, normal 6 nm), suggesting that these patients continued to have significant inflammation despite taking ELX/TEZ/IVA, and mean percentage predicted forced expiratory volume in 1 second in our CF cohort was 55 ± 22%, indicating that these patients had persistent lung disease. Conclusions: This is the first comprehensive analysis of Orai1 and STIM1 expression in lungs from normal and CF donors. We found evidence that Orai1 was more active in CF than normal lungs. This novel application of super-resolution microscopy has the potential to be used in clinical settings for analysis of ex vivo patient samples and to evaluate inflammation in people with CF. Although traditional biomarkers of inflammation such as serum cytokine levels are useful for rapid detection of systemic inflammation, our technique allows for precise localization of upstream inflammatory signaling biomarkers at the cellular level and in fixed samples. Therefore, these data suggest that Orai1 has a key role in CF lung inflammation and attest to the potential of anti-inflammatory therapeutics that target Orai1.We used LungMAP to analyze single-cell ribonucleic acid (RNA) sequencing data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, CF, and asthma (disease control) donors (4 male/4 female per group). W

Immune cell metabolism, or immunometabolism, has recently become of interest for its role in inflammation and disease. A growing field of research has identified that metabolic rewiring and immune cell activation are intimately connected, however the mechanisms driving these connections have remained poorly understood. The tricarboxylic acid cycle and its intermediates have become recognized as major players in disease and inflammation. The immunometabolite itaconate has been identified as a potent immunomodulator produced in high quantities in activated macrophages. Itaconate is produced by the enzyme aconitate decarboxylase 1 (Acod1), which is highly upregulated in proinflammatory macrophages. Although itaconate and Acod1 have been found to be upregulated in macrophages under stimulated conditions, the potential role of itaconate production in other non-immune cells remains poorly understood. Itaconate and its exogenous derivative forms have been found to be potent mediators of inflammation, and specifically have been found to decrease proinflammatory cytokine production in cultured macrophages. In this dissertation, we sought to identify the role of itaconate in three separate murine models of disease: cerebral ischemia/reperfusion injury, diet-induced obesity, and ulcerative colitis. We hypothesized that deletion of Acod1 would lead to greater disease severity in these models and that macrophages would be the primary cell type responsible. To understand the role of endogenously produced itaconate, mice lacking Acod1 (Acod1-/- ) were used. We demonstrate that global Acod1 deletion leads to significantly worsened disease severity in all three models studied. Specifically, Acod1 deletion leads to increased lesion volume size compared to wild type (WT) mice in a model of ischemia/reperfusion stroke. The observed increased lesion volume did not appear to be caused by increased inflammation, indicating a separate potential mechanism driving these changes. In a model of diet-induced obesity, mice lacking Acod1 showed similar weight gain compared to WT mice, however, Acod1-/- mice had elevated blood glucose levels after 12 weeks of high fat diet. Acod1-/- mice also had elevated inflammatory gene expression. Furthermore, naïve Acod1-/- mice had significant increases in fat deposition when on chow diet at 3 and 6 months of age. Lastly, Acod1-/- mice exposed to an acute ulcerative colitis model induced by dextran sulfate xi sodium (DSS) treatment showed increased disease severity with more severe and sustained body weight loss and increased inflammatory gene expression. Importantly, cell specific knockout of Acod1 in myeloid cells (MyAcod1-/- ) with LysM-Cre did not phenocopy disease severity in any of the three in vivo models. This suggests that myeloid cells, specifically macrophages, are not the primary cell type responsible for the observed phenotypes seen in the global Acod1-/- studies. These data define a novel role for Acod1 in transient ischemia/reperfusion occlusion stroke, diet-induced obesity, and ulcerative colitis. Furthermore, these differences do not appear to be regulated by Acod1 and itaconate in macrophage/myeloid cells. These findings suggest that Acod1 and itaconate are likely working through other cell types. To further explore potential mechanisms driving the observed Acod1-/- phenotypes, we sought to identify if Acod1 and endogenous itaconate were capable of modulating ferroptosis induced cell death. Using bone marrow derived macrophages as a model cell type capable of expressing Acod1, we found that macrophages lacking Acod1 had significantly increased susceptibility to RSL3 induced ferroptosis death compared to WT cells. Further analysis found that Acod1-/- macrophages also showed decreased glutathione levels compared to their WT counterparts. Lastly, we found that supplementing Acod1-/- cells with exogenous itaconate restored protection from RSL3 induced cell death and increased glutathione level to what is observed in WT macrophages. These findings would suggest that Acod1 and endogenous itaconate play a role in ferroptosis protection through sustaining intracellular glutathione levels, and could be a relevant mechanism regarding the protective role of Acod1 from ischemia/reperfusion injury.

Background & Aim: One of the major bottlenecks of cancer cellular therapy development is off-target toxicity. It is caused by activated T-cells that unexpectedly recognize epitopes presented on healthy tissues instead of interacting only with the intended target on cancer cells. This mechanism can lead to severe immuno-toxicity resulting in organ dysfunction or even death. Unfortunately, experimental identification of epitopes that may trigger off-target toxicity is both costly and time consuming. In an attempt to accelerate this process, we created ArdImmune Tox, a computational tool assessing epitopes for their potential toxicity. Methods, Results & Conclusion: ArdImmune Tox builds up on the recent advances in computational immunology and Artificial Intelligence (AI). In the first step ArdImmune Tox mimics an experimental approach (X-scan) by simultaneously modifying multiple amino acids in the target peptide creating many possible off-target epitopes (OTE). Next, the peptide collection is mapped against a curated reference dataset encompassing proteomes from more than 1000 patients. In this step we retain peptides found in proteins expressed in healthy human tissues and keep track of their frequency. In order to establish which peptides are presented by the HLAs on the cell surface we use a dedicated machine-learning model developed in-house, which was trained on mass spectroscopy data of peptide-HLA (pHLA) presentation. These peptides are then scored for similarity to the target peptide based on selected physico-chemical properties. Importantly, only amino acids in positions identified by our approach as interacting with TCRs are considered. We present results of ArdImmune Tox on three cancer immunotherapy cases, where the OTEs were identified experimentally. In all of them ArdImmune Tox correctly identified the OTEs, ranking them among the 3 highest scoring potential off-target peptides, whereas BLAST algorithm-based approaches either positioned OTEs much lower in the ranking or even failed to identify them altogether. In conclusion, we introduce ArdImmuneTox - a novel, effective, in silico approach for the identification of peptide- based off-target toxicity and T-cell cross-reactivity in immunotherapies. Our approach shows superior performance to currently used sequence comparison-based methods. Our results indicate that ArdImmune-Tox can aid in the rapid and cost-effective selection of safe epitope targets for cancer immunotherapies.

BackgroundAryl Hydrocarbon Receptor (AHR) is a ligand-activated transcription factor that regulates the activities of multiple innate and adaptive immune cell types. Multiple ligands such as kynurenine bind to AHR driving its nuclear translocation and transcriptional activation, leading to an immunosuppressive tumor microenvironment.1 2 AHR activation is implicated in tumor development in multiple cancer types. In addition, high levels of serum kynurenine are associated with resistance to checkpoint inhibitors.3 To overcome AHR-mediated immunosuppression in cancers, we developed a selective oral AHR inhibitor IK-175 and took a combined computational and tissue-based approach to select cancer indications for its clinical development.MethodsThe aim of this work is to identify tumor indications dependent on AHR signaling and design patient selection strategies based on a proprietary transcriptional signature, mRNA and protein detection assays to evaluate AHR pathway activation in tumors.ResultsGenomic profiling of solid and hematological cancers from TCGA and Project GENIE databases identified bladder and esophageal tumors among others, as frequently harboring AHR gene amplifications.A proprietary gene signature of AHR activation was developed integrating literature, pathway analysis, RNAseq and nanostring data from PBMC, T-cells and cell lines upon AHR inhibition. Transcriptional analysis of the TCGA data using this signature demonstrated bladder cancer has the highest expressions of AHR and AHR signature genes, suggesting increased pathway activity in bladder cancer relative to other cancer types. Increased AHR signature gene expression was associated with worse overall survival in the TCGA bladder cancer cohort. Furthermore, RNAscope analysis of a tissue microarray containing 10 different tumor types revealed bladder cancer had one of the highest AHR transcript expression in the tumor compartment.Finally, nuclear localization of AHR protein was assessed as an indicator of pathway activation through the development of a novel IHC method. Extensive TMA screening of AHR protein in 15 different indications demonstrated bladder cancer as the tumor type with the highest prevalence of AHR nuclear expression.ConclusionsIn summary, we demonstrated high prevalence of nuclear AHR protein expression, AHR gene amplification and target gene expression in bladder cancer, suggesting aberrant AHR activation may play an important role in the progression of this tumor type. This study provides rationale for therapeutic targeting of AHR in bladder cancer patients. Ikena is currently evaluating the anti-tumor activity of IK-175 as a single agent and in combination with nivolumab in bladder cancer in a Phase 1a/1b clinical study (NCT04200963).ReferencesQuintana FJ, Sherr DH. Aryl hydrocarbon receptor control of adaptive immunity. Pharmacol Rev 2013 Aug 1;65(4):1148-61.Murray IA, Patterson AD, Perdew GH. Aryl hydrocarbon receptor ligands in cancer: friend and foe. Nat Rev Cancer 2014 Dec;14(12):801-14.Li, Haoxin et al. ‘Metabolomic adaptations and correlates of survival to immune checkpoint blockade.’ Nature Communications 2019 Sep 25;10:1-4346.

The emergence of single-cell RNA sequencing enables simultaneous sequencing of thousands of cells, making the analysis of cell population heterogeneity more efficient. In recent years, single-cell RNA sequencing has been used in the investigation of heterogeneous cell populations, cellular developmental trajectories, stochastic gene transcriptional kinetics, and gene regulatory networks, providing strong support in life science research. However, the application of single-cell RNA sequencing in the field of oral science has not been reviewed comprehensively yet. Therefore, this paper reviews the development and application of single-cell RNA sequencing in oral science, including fields of tissue development, teeth and jaws diseases, maxillofacial tumors, infections, etc., providing reference and prospects for using single-cell RNA sequencing in studying the oral diseases, tissue development, and regeneration.

Fluorescence in situ hybridization (FISH) has become an important tool in laboratory experimentation by providing a qualitative or semi-quantitative technique to detect nucleic acids across different sample types and species. It also serves as a promising platform for the discovery of novel RNA biomarkers and the development of molecular diagnostic assays. While technologies to detect hundreds or thousands of gene transcripts in situ with single-cell resolution are rapidly coming online, smaller scale FISH analysis continues to be highly useful in neuroscience research. In this chapter, we describe a robust, relatively fast and low cost, turnkey in situ hybridization technology (ISH) to identify one or more RNA targets together with immunohistochemical analyses. Specifically, we present a customized version of the protocol that works particularly well for spinal cord and primary sensory ganglia tissues.

A major social challenge of modern societies is to prevent and treat neurodegenerative diseases suffered by the constantly increasing elderly population. We are investigating the changes in gene expression in single neurons in response to nerve terminal dysfunction in the brain of genetically modified mice. Machine Learning approaches imported from other disciplines are key for the bioinformatic analysis of data sets comprising thousands of cells expressing thousands of genes each. Expression changes in specific genes might be essential to maintain neuronal homeostasis and, therefore, potentially useful to be targeted in the context of therapeutic strategies.

Single-cell genomics, particularly single-cell transcriptome profiling by RNA sequencing have transformed the possibilities to relate genes to functions, structures, and eventually phenotypes. We can now observe changes in each cell's transcriptome and among its neighborhoods, interrogate the sequence of transcriptional events, and assess their influence on subsequent events. This paradigm shift in biology enables us to infer causal relationships in these events with high accuracy. Here we review the latest single-cell studies in plants that uncover how cellular phenotypes emerge as a result of the transcriptome process such as waves of expression, trajectories of development and responses to the environment, and spatial information. With an eye on the advances made in animal and human studies, we further highlight some of the needed areas for future research and development, including computational methods.