Probes for INS

Inflammatory bowel disease (IBD) is a significant global health problem that involves chronic intestinal inflammation and can involve severe comorbidities, including intestinal fibrosis and inflammation-associated colorectal cancer (CRC). Disease-associated alterations to the intestinal microbiota often include fecal enrichment of Enterobacteriaceae, which are strongly implicated in IBD development. This dysbiosis of intestinal flora accompanies changes in microbial metabolites, shaping host:microbe interactions and disease risk. While there have been numerous studies linking specific bacterial taxa with IBD development, our understanding of microbial function in the context of IBD is limited. Several classes of microbial metabolites have been directly implicated in IBD disease progression, including bacterial siderophores and genotoxins. Yet, our microbiota still harbors thousands of uncharacterized microbial products. In-depth discovery and characterization of disease-associated microbial metabolites is necessary to target these products in IBD treatment strategies. Towards improving our understanding of microbiota metabolites in IBD, it is important to recognize how host relevant factors influence microbiota function. For example, changes in host inflammation status, metal availability, interbacterial community structure, and xenobiotics all play an important role in shaping gut microbial ecology. In this minireview, we outline how each of these factors influences gut microbial function, with a specific focus on IBD-associated Enterobacteriaceae metabolites. Importantly, we discuss how altering the intestinal microenvironment could improve the treatment of intestinal inflammation and associated disorders, like intestinal fibrosis and CRC.

The connexin gene family is the most prevalent gene that contributes to hearing loss. Connexins 26 and 30, encoded by GJB2 and GJB6, respectively, are the most abundantly expressed connexins in the inner ear. Connexin 43, which is encoded by GJA1, appears to be widely expressed in various organs, including the heart, skin, the brain, and the inner ear. The mutations that arise in GJB2, GJB6, and GJA1 can all result in comprehensive or non-comprehensive genetic deafness in newborns. As it is predicted that connexins include at least 20 isoforms in humans, the biosynthesis, structural composition, and degradation of connexins must be precisely regulated so that the gap junctions can properly operate. Certain mutations result in connexins possessing a faulty subcellular localization, failing to transport to the cell membrane and preventing gap junction formation, ultimately leading to connexin dysfunction and hearing loss. In this review, we provide a discussion of the transport models for connexin 43, connexins 30 and 26, mutations affecting trafficking pathways of these connexins, the existing controversies in the trafficking pathways of connexins, and the molecules involved in connexin trafficking and their functions. This review can contribute to a new way of understanding the etiological principles of connexin mutations and finding therapeutic strategies for hereditary deafness.

Maturation of the definitive adrenal cortex occurs between 3 and 6 weeks post-partum and involves onset of CYP11B2 expression and establishment of the laminin-encased 3D structure of glomeruli that contain rosettes of 10 to 15 zona glomerulosa (zG) cells that work in coordination to produce optimal amounts of aldosterone. Although this process is dependent on canonical WNT/b-catenin signaling, cellular sources of WNT ligands remain elusive and the mechanisms involved in the extensive extra-cellular matrix remodeling associated with rosette/glomeruli morphogenesis are unknown. Beyond their role in innate immunity, macrophages are involved in extra-cellular matrix remodeling under a wide variety of pathophysiological conditions and have the capacity to produce WNT ligands. This, together with the presence of macrophages within the zG cells, strongly suggest that macrophages may play a role in zG morphogenesis and differentiation. Supporting this idea, a recent publication has shown that intra-tissular aldosterone concentration was reduced in the absence of macrophages under stress conditions. However, whether macrophages play a direct role in controlling aldosterone secretion or an indirect role by remodelling the postnatal zG is unknown. The presence of tissue resident macrophages in a specific zone is dependent on the production of trophic factors such as IL34, CSF1, CSF2 or CX3CL1 by nearby, tissue resident ‘niche’ cells. In return, macrophages are thought to provide positive ‘feedback’ signals to their niche, generating mutually beneficial circuits between the niche and its macrophages. To gain insight into the role of macrophages in zG morphogenesis and homeostasis, we used single cell sequencing and RNAscope analyses to show expression of CX3CL1 in the zG and of CX3CR1 in macrophages. Interestingly, CX3CL1 expression in the zG was downstream of WNT signalling, suggesting existence of a bi-directional interaction between macrophages and zG. To further study the role of macrophages during the maturation of the zG, we pharmacologically depleted macrophages by the small molecule inhibitor Pexidartinib at different time points between 3- and 12-weeks post-partum, when maturation of the zG occurs. Short-term depletion of macrophages resulted in a more disorganized and elongated zG, suggesting a delay in maturation. However, long-term depletion of macrophages resulted in exacerbated maturation of the rosettes, suggesting that the short-term zG defect was followed by establishment of a compensatory mechanism to allow formation of rosettes even in the absence of macrophages. Whether these perturbations of the zG are correlated with an altered production of aldosterone is still under study.

GPR37 is an orphan GPCR and expressed in different brain regions. However, its biological function in pain regulation remains poorly understood. Recently, we identified Neuroprotectin D1 (NPD1) as a novel ligand of GPR37. NPD1 is a specialized pro-resolving mediator (SPM) and bio-synthesized from fish oil DHA (docosahexaenoic acid) . Here we reported a protective role of GRP37/NPD1 signaling in traumatic brain injury (TBI)-induced neuropathic pain. Mild TBI was induced by closed-head impact and the neuropathic pain was assessed by periorbital and cutaneous mechanical allodynia. In contrast to DHA (300 μg) showing no effects, peri-surgical treatment of NPD1 via intravenous injection (300 ng) effectively prevented TBI-induced locomotor deficiency and mechanical hypersensitivity in mice. Intraperitoneal post-treatment of NPD1 also significantly reduced established neuropathic pain in TBI mice. We also found that NPD1 treatment could inhibit TBI-induced neuroinflammation, characterized by microglia and astrocyte activation in the cortex and hippocampus. Furthermore, demyelination occurred after TBI, which was prevented by peri-surgical treatment of NPD1. RNAscope in situ hybridization revealed high Gpr37 mRNA expression in oligodendrocytes of the motor cortex and hippocampus . TBI resulted in a significant decrease in GPR37 expression, which could be restored by NPD1 treatment. GPR37 is protective and mice lacking Gpr37 (Gpr37-/-) exhibited prolonged pain symptoms after TBI. Furthermore, the protective effect of NPD1 was abolished in Gpr37-/- mice. All these findings suggest that activation of NPD1/GPR37 signaling pathway is a promising therapeutic strategy for preventing and treating neuropathic pain and its comorbidities after TBI. Funding: DoD grant W81XWH2110885.

Mammalian genomes encode thousands of long non-coding RNAs (lncRNAs). LncRNAs are extensively expressed in various immune cells. The lncRNAs have been reported to be involved in diverse biological processes, including the regulation of gene expression, dosage compensation, and genomic imprinting. However, very little research has been conducted to explore how they alter innate immune responses during host-pathogen interactions. In this study, we found that a lncRNA, named long non-coding RNA, embryonic stem cells expressed 1 (Lncenc1), was strikingly increased in mouse lungs after gram-negative (G-) bacterial infection or exposure to lipopolysaccharides (LPS). Interestingly, our data indicated that Lncenc1 was upregulated in macrophages but not primary epithelial cells (PEC) or polymorphonuclear leukocytes (PMN). The upregulation was also observed in human THP-1 and U937 macrophages. Besides, Lncenc1 was highly induced during ATP-induced inflammasome activation. Functionally, Lncenc1 showed pro-inflammatory effects in macrophages as demonstrated by increased expressions of cytokine and chemokines, as well as enhanced NF-κB promoter activity. Overexpression of Lncenc1 promoted the releases of IL-1β and IL-18, and Caspase-1 activity in macrophages, suggesting a role in inflammasome activation. Consistently, knockdown of Lncenc1 inhibited inflammasome activation in LPS-treated macrophages. Moreover, knockdown of Lncenc1 using antisense oligo (ASO)-loaded exosomes (EXO) attenuated LPS-induced lung inflammation in mice. Similarly, Lncenc1 deficiency protects mice from bacteria-induced lung injury and inflammasome activation. Taken together, our work identified Lncenc1 as a modulator of inflammasome activation in macrophages during bacterial infection. Our study suggested that Lncenc1 could serve as a therapeutic target for lung inflammation and injury.

SELENON-related congenital myopathy is characterized by proximal weakness starting in infancy, early respiratory insufficiency, and early development of severe scoliosis. While changes in the SELENON gene, which encodes the protein SelN, are known to cause this disease the mechanisms through which loss of SelN lead to myopathy are not well understood. Previous studies suggest that SelN may have multiple roles in muscle, including regulating development of Type II muscle fibers, modulating excitation-contraction coupling through interactions with RYR1 and other muscle calcium channels, and possibly supporting satellite cell activation and proliferation following muscle injury. One particular challenge to understanding the role of SelN in skeletal muscle has been the inability to directly visualize SelN expression within muscle fibers and supporting cells due to a lack of robust antibodies for immunohistochemistry. Studies of mRNA expression and Western blot analysis of protein expression suggest significant post-transcriptional regulation of protein expression with an overall pattern of high expression in developing muscle and other developing tissues and low-level ubitquitous expression in mature tissues but evaluation of SelN expression in more limited sub-populations of cells has not been possible. Experiments in mouse suggest that loss of SelN expression results in decreased satellite cell proliferation following muscle injury. Here, I use a newly developed zebrafish model with mNeonGreen-tagged SelN to directly visualize SelN expression in satellite cells following muscle injury and show that SelN expression increases in activated satellite cells following mechanical muscle injury. This provides support SelN playing a role in satellite cell activation and proliferation during muscle repair following injury.

The interest and utility of high-plex spatial profiling of RNA and protein biomarkers has increased over the last few years. The implementation of high-plex analyte spatial platforms, such as GeoMx^® Digital Spatial Profiler (DSP), is increasing within discovery and development of biomarkers associated with clinical outcome. The surge in spatial platforms comes with an increased adoption of digital pathology in translational and clinical research studies. The integration of these two workflows has the potential to benefit diagnostic and therapeutic development. This study aims to facilitate the implementation of DSP in tissue analysis workflows helping researchers involved in drug discovery and development efforts to (1) assess platform feasibility for their research, (2) design effective DSP experiments, and (3) enable generation of high-quality, analyzable spatial data from large cohortstudies. The GeoMx DSP Biopharma and CRO Consortium has developed consensus-based best practices incorporating expertise of members from biopharma and contract research organizations (CROs). Best practices guidelines for spatial profiling of tissue biopsies in drug discovery and development using GeoMx stands to advance current standard practices in tissue analysis. These recommendations encompass every step in the implementation of DSP for standard tissue analysis workflows, emphasizing the importance of multidisciplinary stakeholder involvement, defining experimental conditions and testing these prior to execution of large-scale studies, and considerations in assessing assay performance. This document offers a practical reference for optimal implementation of GeoMx DSP in exploratory sample analysis for use in research supporting drug discovery and development. Here we present a practical reference for the optimal implementation of GeoMx DSP in exploratory analysis for drug discovery and development studies. Best practices insights for the application of this technology to breast cancer research have been previously published (1) and should also be taken in consideration when designing relevantspatial studies.

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.

We developed an animal model of activity-induced muscle pain that combines muscle fatigue with a non-painful, low-dose muscle insult. We previously showed that pharmacological blockade of acid-sensing ion channel 3 (ASIC3) in muscle prevents the activity-induced muscle pain, however, genetic deletion of ASIC3 in primary afferents innervating muscle has no effect on pain. We hypothesized that genetic deletion of ASIC3 on macrophages would prevent activity-induced muscle pain. ASIC3fl/fl mice were crossed with Cx3cr1-Cre mice to generate macrophage/monocyte-specific deletion of ASIC3. To confirm the genetic deletion of ASIC3, expression of ASIC3 was assessed from the peritoneal macrophages using quantitative PCR. Muscle pain was induced by 2, 20μl pH 5.0 injections, 5 days apart, in the gastrocnemius muscle combined with fatiguing muscle contractions in male and female mice. To assess hyperalgesia, muscle withdrawal thresholds (MWT) of the gastrocnemius muscle were measured before and after induction of the model. There was a decrease in ASIC3 mRNA expression in peritoneal macrophages from Cx3cr1CreASIC3fl/fl mice. The decrease in MWT was prevented in Cx3cr1CreASIC3fl/fl mice in both male and female mice, when compared to genetic controls (Cx3cr1CreASIC3+/+)(p

Organ development involves the sustained production of diverse cell types with spatiotemporal precision. In the vertebrate jaw, neural-crest-derived progenitors produce not only skeletal tissues but also later-forming tendons and salivary glands. Here we identify the pluripotency factor Nr5a2 as essential for cell-fate decisions in the jaw. In zebrafish and mice, we observe transient expression of Nr5a2 in a subset of mandibular postmigratory neural-crest-derived cells. In zebrafish nr5a2 mutants, nr5a2-expressing cells that would normally form tendons generate excess jaw cartilage. In mice, neural-crest-specific Nr5a2 loss results in analogous skeletal and tendon defects in the jaw and middle ear, as well as salivary gland loss. Single-cell profiling shows that Nr5a2, distinct from its roles in pluripotency, promotes jaw-specific chromatin accessibility and gene expression that is essential for tendon and gland fates. Thus, repurposing of Nr5a2 promotes connective tissue fates to generate the full repertoire of derivatives required for jaw and middle ear function.