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.

Sushi, von Willebrand factor type A, EGF pentraxin domain-containing 1 (SVEP1), an extracellular matrix protein, is a human coronary artery disease locus that promotes atherosclerosis. We previously demonstrated that SVEP1 induces vascular smooth muscle cell (VSMC) proliferation and an inflammatory phenotype in the arterial wall to enhance the development of atherosclerotic plaque. The only receptor known to interact with SVEP1 is integrin α9β1, a cell surface receptor that is expressed by VSMCs and myeloid lineage-derived monocytes and macrophages. Our previous in vitro studies suggested that integrin α9β1 was necessary for SVEP1-induced VSMC proliferation and inflammation; however, the underlying mechanisms mediated by integrin α9β1 in these cell types during the development of atherosclerosis remain poorly understood.Here, using cell-specific gene targeting, we investigated the effects of the integrin α9β1 receptor on VSMCs and myeloid cells in mouse models of atherosclerosis. Interestingly, we found that depleting integrin α9β1 in either VSMCs or myeloid cells did not affect the formation or complexity of atherosclerotic plaque in vessels after either 8 or 16 weeks of high fat diet feeding.Our results indicate that integrin α9β1 in these two cell types does not mediate the in vivo effect of SVEP1 in the development of atherosclerosis. Instead, our results suggest either the presence of other potential receptor(s) or alternative integrin α9β1-expressing cell types responsible for SVEP1 induced signaling in the development of atherosclerosis.

Both opioids and nonsteroidal anti-inflammatory drugs (NSAIDS) produce deleterious side effects and fail to provide sustained relief in patients with chronic inflammatory pain. Peripheral neuroinflammation (PN) is critical for initiation and development of inflammatory pain. A better understanding of molecular mechanisms underlying PN would facilitate the discovery of new analgesic targets and the development of new therapeutics. Emerging evidence suggests that peripheral sensory neurons are not only responders to painful stimuli, but are also actively engaged in inflammation and immunity, whereas the intrinsic regulatory mechanism is poorly understood. Here we report the expression of proton-selective ion channel Hv1 in peripheral sensory neurons in rodents and humans, which was previously shown as selectively expressed in microglia in mammalian central nervous system. Neuronal Hv1 was up-regulated by PN or depolarizing stimulation, which in turn aggravates inflammation and nociception. Inhibiting neuronal Hv1 genetically or by a newly discovered selective inhibitor YHV98-4 reduced intracellular alkalization and ROS production in inflammatory pain, mitigated the imbalance in downstream SHP-1-pAKT signaling, and also diminished pro-inflammatory chemokine release to alleviate nociception and morphine-induced hyperalgesia and tolerance. Thus, our data reveal neuronal Hv1 as a novel target in analgesia strategy and managing opioids-related side effects.

Surfactant-induced cumulative irritant contact dermatitis (ICD) is a common and clinically important skin disorder. CCL2 is known to mediate inflammation following tissue damage in various organs. Thus, we investigated whether and how CCL2 contributes to the development of murine cumulative ICD induced by a common surfactant, sodium dodecyl sulfate (SDS). Wild-type mice treated topically with SDS for 6 consecutive days developed skin inflammation that recapitulated the features of human cumulative ICD, including barrier disruption, epidermal thickening, and neutrophil accumulation. CCL2 was upregulated in SDS-treated skin, and local CCL2 blockade attenuated SDS-induced ICD. SDS-induced ICD and neutrophil accumulation were also attenuated in mice deficient in CCR2, the receptor for CCL2. Neutrophil depletion alleviated SDS-induced ICD, suggesting that impaired neutrophil accumulation was responsible for the amelioration of ICD in CCR2-deficient mice. In RNA-seq analyses of SDS-treated skin, the expression levels of Il1b in CCR2-deficient mice were highly downregulated compared with those in wild-type mice. Furthermore, the intradermal administration of IL-1β in the SDS-treated skin of CCR2-deficient mice restored the local accumulation of neutrophils and the development of ICD. Collectively, our results suggest that CCL2-CCR2 signaling in the skin critically promotes the development of SDS-induced ICD by inducing IL-1β expression for neutrophil accumulation.

Fatty liver is closely associated with elevated concentrations of nonesterified fatty acids (NEFA) and a low level of very low-density lipoproteins (VLDL) in blood of dairy cows. High NEFA inhibit the VLDL synthesis and assembly, and cause hepatic triacylglycerol (TAG) deposition. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, antagonizes NEFA-induced TAG accumulation through modulating expressions of fatty acid synthesis and oxidation genes in cow hepatocytes. However, the role of SIRT3 in the VLDL synthesis and assembly was largely unknown. Here we aimed to test whether SIRT3 would recover the synthesis and assembly of VLDL in cow hepatocytes induced by high NEFA. Primary cow hepatocytes were isolated from 3 Holstein cows. Hepatocytes were infected with SIRT3 overexpression adenovirus (Ad-SIRT3), SIRT3-short interfering (si) RNA, or first infected with Ad-SIRT3 and then incubated with 1.0 mM NEFA (Ad-SIRT3 + NEFA). Expressions of key genes in VLDL synthesis and the VLDL contents in cell culture supernatants were measured. SIRT3 overexpression significantly increased the mRNA abundance of microsomal triglyceride transfer protein (MTP), apolipoprotein B100 (ApoB100) and ApoE (p < 0.01), and raised VLDL contents in the supernatants (p < 0.01). However, SIRT3 silencing displayed a reverse effect in comparison to SIRT3 overexpression. Compared with NEFA treatment alone, the Ad-SIRT3 + NEFA significantly upregulated the mRNA abundance of MTP, ApoB100 and ApoE (p < 0.01), and increased VLDL contents in the supernatants (p < 0.01). Our data demonstrated that SIRT3 restored the synthesis and assembly of VLDL in cow hepatocytes challenged with NEFA, providing an in vitro basis for further investigations testing its feasibility against hepatic TAG accumulation in dairy cows during the perinatal period.

HIV-1 infection within the central nervous system (CNS) includes evolution of the virus, damaging inflammatory cascades, and the involvement of multiple cell types; however, our understanding of how Env tropism and inflammation can influence CNS infectivity is incomplete. In this study, we utilize macrophage-tropic and T cell-tropic HIV-1 Env proteins to establish accurate infection profiles for multiple CNS cells under basal and interferon alpha (IFN-α) or lipopolysaccharide (LPS)-induced inflammatory states. We found that macrophage-tropic viruses confer entry advantages in primary myeloid cells, including monocyte-derived macrophage, microglia, and induced pluripotent stem cell (iPSC)-derived microglia. However, neither macrophage-tropic or T cell-tropic HIV-1 Env proteins could mediate infection of astrocytes or neurons, and infection was not potentiated by induction of an inflammatory state in these cells. Additionally, we found that IFN-α and LPS restricted replication in myeloid cells, and IFN-α treatment prior to infection with vesicular stomatitis virus G protein (VSV G) Envs resulted in a conserved antiviral response across all CNS cell types. Further, using RNA sequencing (RNA-seq), we found that only myeloid cells express HIV-1 entry receptor/coreceptor transcripts at a significant level and that these transcripts in select cell types responded only modestly to inflammatory signals. We profiled the transcriptional response of multiple CNS cells to inflammation and found 57 IFN-induced genes that were differentially expressed across all cell types. Taken together, these data focus attention on the cells in the CNS that are truly permissive to HIV-1, further highlight the role of HIV-1 Env evolution in mediating infection in the CNS, and point to limitations in using model cell types versus primary cells to explore features of virus-host interaction. IMPORTANCE The major feature of HIV-1 pathogenesis is the induction of an immunodeficient state in the face of an enhanced state of inflammation. However, for many of those infected, there can be an impact on the central nervous system (CNS) resulting in a wide range of neurocognitive defects. Here, we use a highly sensitive and quantitative assay for viral infectivity to explore primary and model cell types of the brain for their susceptibility to infection using viral entry proteins derived from the CNS. In addition, we examine the ability of an inflammatory state to alter infectivity of these cells. We find that myeloid cells are the only cell types in the CNS that can be infected and that induction of an inflammatory state negatively impacts viral infection across all cell types.

BACKGROUND The long saphenous vein (LSV) is commonly utilised in CABG surgery to facilitate revascularisation. However, over time these grafts develop intimal hyperplasia (IH) and accelerated atherosclerosis, leading to stenosis and occlusion. A common feature of IH is vascular calcification (VC) within the affected vessel. Recently, the matricellular protein osteopontin (OPN) has been implicated in this process at endothelial injury sites in porcine models, but this has not been expanded to humans. Consecutively, studies have implicated the arterial haemodynamic environment as a major driver of the pro-inflammatory conditions facilitating VC and IH. As such, treatment with a synthetic glucocorticoid, dexamethasone, which has proven beneficial in inhibiting IH in murine models, may beneficially modulate this process in humans. This work aims to assess the role of OPN on VC and IH in an ex vivo model, whether dexamethasone can modulate this process, and whether detection of VC in situ can act as a novel clinical monitoring approach to graft patency.

The olfactory epithelium undergoes constant neurogenesis throughout life in mammals. Several factors including key signaling pathways and inflammatory microenvironment regulate the maintenance and regeneration of the olfactory epithelium. In this study, we identify TMEM59 (also known as DCF1) as a critical regulator to the epithelial maintenance and regeneration. Single-cell RNA-Seq data show downregulation of TMEM59 in multiple epithelial cell lineages with aging. Ablation of TMEM59 leads to apparent alteration at the transcriptional level, including genes associated with olfactory transduction and inflammatory/immune response. These differentially expressed genes are key components belonging to several signaling pathways, such as NF-κB, chemokine, etc. TMEM59 deletion impairs olfactory functions, attenuates proliferation, causes loss of both mature and immature olfactory sensory neurons, and promotes infiltration of inflammatory cells, macrophages, microglia cells and neutrophils into the olfactory epithelium and lamina propria. TMEM59 deletion deteriorates regeneration of the olfactory epithelium after injury, with significant reduction in the number of proliferative cells, immature and mature sensory neurons, accompanied by the increasing number of inflammatory cells and macrophages. Anti-inflammation by dexamethasone recovers neuronal generation and olfactory functions in the TMEM59-KO animals, suggesting the correlation between TMEM59 and inflammation in regulating the epithelial maintenance. Collectively, TMEM59 regulates olfactory functions, as well as neuronal generation in the olfactory epithelium via interaction with inflammation, suggesting a potential role in therapy against olfactory dysfunction associated with inflamm-aging.

Chronic pain, which affects around 1/3 of the world population and is often comorbid with memory deficit and mood depression, is a leading source of suffering and disability. Studies in past decades have shown that hyperexcitability of primary sensory neurons resulting from abnormal expression of ion channels and central sensitization mediated pathological synaptic plasticity, such as long-term potentiation in spinal dorsal horn, underlie the persistent pain. The memory/emotional deficits are associated with impaired synaptic connectivity in hippocampus. Dysregulation of numerous endogenous proteins including receptors and intracellular signaling molecules is involved in the pathological processes. However, increasing knowledge contributes little to clinical treatment. Emerging evidence has demonstrated that the neuroinflammation, characterized by overproduction of pro-inflammatory cytokines and glial activation, is reliably detected in humans and animals with chronic pain, and is sufficient to induce persistent pain and memory/emotional deficits. The abnormal expression of ion channels and pathological synaptic plasticity in spinal dorsal horn and in hippocampus are resulting from neuroinflammation. The neuroinflammation is initiated and maintained by the interactions of circulating monocytes, glial cells and neurons. Obviously, unlike infectious diseases and cancer, which are caused by pathogens or malignant cells, chronic pain is resulting from alterations of cells and molecules which have numerous physiological functions. Therefore, normalization (counterbalance) but not simple inhibition of the neuroinflammation is the right strategy for treating neuronal disorders. Currently, no such agent is available in clinic. While experimental studies have demonstrated that intracellular Mg2+ deficiency is a common feature of chronic pain in animal models and supplement Mg2+ are capable of normalizing the neuroinflammation, activation of upregulated proteins that promote recovery, such as translocator protein (18k Da) or liver X receptors, has a similar effect. In this article, relevant experimental and clinical evidence is reviewed and discussed.

Low-grade inflammation is the hallmark of non-alcoholic fatty liver diseases (NAFLD) and non-alcoholic steatohepatitis (NASH). The leakage of microbiota-derived products can contribute to liver inflammation during NAFLD/NASH development. Here, we assessed the roles of gut microbial DNA-containing extracellular vesicles (mEVs) in regulating liver cellular abnormalities in the course of NAFLD/NASH.We performed studies with Vsig4-/- , C3-/- , cGAS-/- , and their wild-type littermate mice. Vsig4+ macrophage population and bacterial DNA abundance were examined in both mouse and human liver by either flow cytometric or immunohistochemistry analysis. Gut mEVs were adoptively transferred into Vsig4-/- , C3-/- , cGAS-/- , or littermate WT mice, and hepatocyte inflammation and HSC fibrogenic activation were measured in these mice.Non-alcoholic fatty liver diseases and non-alcoholic steatohepatitis development was concomitant with a diminished liver Vsig4+ macrophage population and a marked bacterial DNA enrichment in both hepatocytes and HSCs. In the absence of Vsig4+ macrophages, gut mEVs translocation led to microbial DNA accumulation in hepatocytes and HSCs, resulting elevated hepatocyte inflammation and HSC fibrogenic activation. In contrast, in lean WT mice, Vsig4+ macrophages remove gut mEVs from bloodstream through a C3-dependent opsonization mechanism and prevent the infiltration of gut mEVs into hepatic cells. Additionally, Vsig4-/- mice more quickly developed significant liver steatosis and fibrosis than WT mice after Western diet feeding. In vitro treatment with NASH mEVs triggered hepatocyte inflammation and HSC fibrogenic activation. Microbial DNAs are key cargo for the effects of gut mEVs by activating cGAS/STING.Accumulation of microbial DNAs fuels the development of NAFLD/NASH-associated liver abnormalities.

Sensory neurons derived from human induced pluripotent stem cells (hiPSCs) are a promising model. One limitation posed by the use of monocultures is the loss of cellular heterogeneity found in tissues. Here we make use of high-throughput RNA sequencing to quantify gene expression in hiPSC-derived mono-cultured and co-cultured sensory neurons. The following groups were compared: human induced pluripotent stem cells (hiPSCs) prior to differentiation, mature hiPSC-derived sensory neurons, mature co-cultures containing hiPSC-derived astrocytes and sensory neurons, mouse dorsal root ganglion (DRG) tissues, and mouse DRG cultures. We find that co-culture of sensory neurons and astrocytes enhances expression of transcripts enriched in native DRG tissues. Numerous well-established genes linked to pain and most translation factors are consistently expressed in both hiPSC and mouse samples. Marker genes for various neuronal subtypes are also present in the hiPSC cultures. As a proof of concept for the potential use of the dataset for hypothesis generation, we validated the expression of eukaryotic initiation factor 5A (eIF5A) in DRG tissue and hiPSC samples. eIF5A is a unique translation factor in that it requires a post-translational hypusine modification to be active. We show that inhibition of hypusine synthesis prevents hyperalgesic priming by inflammatory mediators in vivo and diminishes hiPSC neuronal firing in vitro. In total, we present the transcriptomes of hiPSC sensory neuron models and evaluate the requirement of a functional translation factor. This work was supported by NIH grants R01NS100788 (ZTC), R01NS114018 (ZTC), and 1UG3TR003149 (BJB).

The chronic infection established by the human immunodeficiency virus 1 (HIV-1) produces serious CD4+ T cell immunodeficiency despite the decrease in HIV-1 ribonucleic acid (RNA) levels and the raised life expectancy of people living with HIV-1 (PLWH) through treatment with combined antiretroviral therapies (cART). HIV-1 enters the central nervous system (CNS), where perivascular macrophages and microglia are infected. Serious neurodegenerative symptoms related to HIV-associated neurocognitive disorders (HAND) are produced by infection of the CNS. Despite advances in the treatment of this infection, HAND significantly contribute to morbidity and mortality globally. The pathogenesis and the role of inflammation in HAND are still incompletely understood. Principally, growing evidence shows that the CNS is an anatomical reservoir for viral infection and replication, and that its compartmentalization can trigger the evolution of neurological damage and thus make virus eradication more difficult. In this review, important concepts for understanding HAND and neuropathogenesis as well as the viral proteins involved in the CNS as an anatomical reservoir for HIV infection are discussed. In addition, an overview of the recent advancements towards therapeutic strategies for the treatment of HAND is presented. Further neurological research is needed to address neurodegenerative difficulties in people living with HIV, specifically regarding CNS viral reservoirs and their effects on eradication.