Interleukin-17A Serves a Priming Role in Autoimmunity by Recruiting IL-1ß-Producing Myeloid Cells that Promote Pathogenic T Cells.

Interleukin-17A (IL-17A) is a major mediator of tissue inflammation in many autoimmune diseases. Anti-IL-17A is an effective treatment for psoriasis and is showing promise in clinical trials in multiple sclerosis. In this study, we find that IL-17A-defective mice or mice treated with anti-IL-17A at induction of experimental autoimmune encephalomyelitis (EAE) are resistant to disease and have defective priming of IL-17-secreting γδ T (γδT17) cells and Th17 cells. However, T cells from Il17a-/- mice induce EAE in wild-type mice following in vitro culture with autoantigen, IL-1ß, and IL-23. Furthermore, treatment with IL-1ß or IL-17A at induction of EAE restores disease in Il17a-/- mice. Importantly, mobilization of IL-1ß-producing neutrophils and inflammatory monocytes and activation of γδT17 cells is reduced in Il17a-/- mice. Our findings demonstrate that a key function of IL-17A in central nervous system (CNS) autoimmunity is to recruit IL-1ß-secreting myeloid cells that prime pathogenic γδT17 and Th17 cells.

The genomic and enzymatic basis for iridoid biosynthesis in cat thyme (Teucrium marum).

Iridoids are non-canonical monoterpenoids produced by both insects and plants. An example is the cat-attracting and insect-repelling volatile iridoid nepetalactone, produced by Nepeta sp. (catmint) and aphids. Recently, both nepetalactone biosynthetic pathways were elucidated, showing a remarkable convergent evolution. The iridoid, dolichodial, produced by Teucrium marum (cat thyme) and multiple insect species, has highly similar properties to nepetalactone but its biosynthetic origin remains unknown. We set out to determine the genomic, enzymatic, and evolutionary basis of iridoid biosynthesis in T. marum. First, we generated a de novo chromosome-scale genome assembly for T. marum using Oxford Nanopore Technologies long reads and proximity-by-ligation Hi-C reads. The 610.3 Mb assembly spans 15 pseudomolecules with a 32.9 Mb N50 scaffold size. This enabled identification of iridoid biosynthetic genes, whose roles were verified via activity assays. Phylogenomic analysis revealed that the evolutionary history of T. marum iridoid synthase, the iridoid scaffold-forming enzyme, is not orthologous to typical iridoid synthases but is derived from its conserved paralog. We discovered an enzymatic route from nepetalactol to diverse iridoids through the coupled activity of an iridoid oxidase cytochrome P450 and acetyltransferases, via an inferred acylated intermediate. This work provides a genomic resource for specialized metabolite research in mints and demonstration of the role of acetylation in T. marum iridoid diversity. This work will enable future biocatalytic or biosynthetic production of potent insect repellents, as well as comparative studies into iridoid biosynthesis in insects.

Single-cell multi-omics in the medicinal plant Catharanthus roseus.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Targeting GM-CSF in inflammatory and autoimmune disorders.

Granulocyte macrophage-colony stimulating factor (GM-CSF) was originally identified as a growth factor for its ability to promote the proliferation and differentiation in vitro of bone marrow progenitor cells into granulocytes and macrophages. Many preclinical studies, using GM-CSF deletion or depletion approaches, have demonstrated that GM-CSF has a wide range of biological functions, including the mediation of inflammation and pain, indicating that it can be a potential target in many inflammatory and autoimmune conditions. This review provides a brief overview of GM-CSF biology and signaling, and summarizes the findings from preclinical models of a range of inflammatory and autoimmune disorders and the latest clinical trials targeting GM-CSF or its receptor in these disorders.

Cell-type-aware regulatory landscapes governing monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway of Catharanthus roseus (Madagascar Periwinkle), the source of the anticancer compounds vinblastine and vincristine. In the leaf, the C. roseus MIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell-type specificity of MIA biosynthesis is achieved is poorly understood. We generated single-cell multi-omics data from C. roseus leaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF)-binding site profiles, we constructed a cell-type-aware gene regulatory network for MIA biosynthesis. We showcased cell-type-specific TFs as well as cell-type-specific cis-regulatory elements. Using motif enrichment analysis, co-expression across cell types, and functional validation approaches, we discovered a novel idioblast-specific TF (Idioblast MYB1, CrIDM1) that activates expression of late-stage MIA biosynthetic genes in the idioblast. These analyses not only led to the discovery of the first documented cell-type-specific TF that regulates the expression of two idioblast-specific biosynthetic genes within an idioblast metabolic regulon but also provides insights into cell-type-specific metabolic regulation.

IL-23p19 in osteoarthritic pain and disease.

OBJECTIVE: We have previously reported that the interleukin-23 p19 subunit (IL-23p19) is required for experimental inflammatory arthritic pain-like behavior and disease. Even though inflammation is often a characteristic feature of osteoarthritis (OA), IL-23 is not usually considered as a therapeutic target in OA. We began to explore the role of IL-23p19 in OA pain and disease utilizing mouse models of OA and patient samples. DESIGN: The role of IL-23p19 in two mouse models of OA, namely collagenase-induced OA and monosodium iodoacetate-induced OA, was investigated using gene-deficient male mice. Pain-like behavior and arthritis were assessed by relative static weight distribution and histology, respectively. In knee synovial tissues from a small cohort of human OA patients, a correlation analysis was performed between IL-23A gene expression and Oxford knee score (OKS), a validated Patient Reported Outcome Measure. RESULTS: We present evidence that i) IL-23p19 is required for the development of pain-like behavior and optimal disease, including cartilage damage and osteophyte formation, in two experimental OA models and ii) IL-23A gene expression in OA knee synovial tissues correlates with a lower OKS (r = -0.742, p = 0.0057). CONCLUSIONS: The findings support the possible targeting of IL-23 as a treatment for OA pain and disease progression.

IL-23 regulation of myeloid cell biology during inflammation.

Interleukin (IL)-23 is implicated in the pathogenesis of several inflammatory diseases and is usually linked with helper T cell (Th17) biology. However, there is some data linking IL-23 with innate immune biology in such diseases. We therefore examined the effects of IL-23p19 genetic deletion and/or neutralization on in vitro macrophage activation and in an innate immune-driven peritonitis model. We report that endogenous IL-23 was required for maximal macrophage activation by zymosan as determined by pro-inflammatory cytokine production, including a dramatic upregulation of granulocyte-colony stimulating factor (G-CSF). Furthermore, both IL-23p19 genetic deletion and neutralization in zymosan-induced peritonitis (ZIP) led to a specific reduction in the neutrophil numbers, as well as a reduction in the G-CSF levels in exudate fluids. We conclude that endogenous IL-23 can contribute significantly to macrophage activation during an inflammatory response, mostly likely via an autocrine/paracrine mechanism; of note, endogenous IL-23 can directly up-regulate macrophage G-CSF expression, which in turn is likely to contribute to the regulation of IL-23-dependent neutrophil number and function during an inflammatory response, with potential significance for IL-23 targeting particularly in neutrophil-associated inflammatory diseases.

Eleven biosynthetic genes explain the majority of natural variation in carotenoid levels in maize grain.

Vitamin A deficiency remains prevalent in parts of Asia, Latin America, and sub-Saharan Africa where maize (Zea mays) is a food staple. Extensive natural variation exists for carotenoids in maize grain. Here, to understand its genetic basis, we conducted a joint linkage and genome-wide association study of the US maize nested association mapping panel. Eleven of the 44 detected quantitative trait loci (QTL) were resolved to individual genes. Six of these were correlated expression and effect QTL (ceeQTL), showing strong correlations between RNA-seq expression abundances and QTL allelic effect estimates across six stages of grain development. These six ceeQTL also had the largest percentage of phenotypic variance explained, and in major part comprised the three to five loci capturing the bulk of genetic variation for each trait. Most of these ceeQTL had strongly correlated QTL allelic effect estimates across multiple traits. These findings provide an in-depth genome-level understanding of the genetic and molecular control of carotenoids in plants. In addition, these findings provide a roadmap to accelerate breeding for provitamin A and other priority carotenoid traits in maize grain that should be readily extendable to other cereals.

CCL17/TARC in autoimmunity and inflammation-not just a T-cell chemokine.

Chemokine (C-C) ligand 17 (CCL17) was first identified as thymus- and activation-regulated chemokine when it was found to be constitutively expressed in the thymus and identified as a T-cell chemokine. This chemoattractant molecule has subsequently been found at elevated levels in a range of autoimmune and inflammatory diseases, as well as in cancer. CCL17 is a C-C chemokine receptor type 4 (CCR4) ligand, with chemokine (C-C) ligand 22 being the other major ligand and, as CCR4 is highly expressed on helper T cells, CCL17 can play a role in T-cell-driven diseases, usually considered to be via its chemotactic activity on T helper 2 cells; however, given that CCR4 is also expressed by other cell types and there is elevated expression of CCL17 in many diseases, a broader CCL17 biology is suggested. In this review, we summarize the biology of CCL17, its regulation and its potential contribution to the pathogenesis of various preclinical models. Reference is made, for example, to recent literature indicating a role for CCL17 in the control of pain as part of a granulocyte macrophage-colony-stimulating factor/CCL17 pathway in lymphocyte-independent models and thus not as a T-cell chemokine. The review also discusses the potential for CCL17 to be a biomarker and a therapeutic target in human disorders.

The GM-CSF/CCL17 pathway in obesity-associated osteoarthritic pain and disease in mice.

OBJECTIVES: We have previously identified a granulocyte macrophage-colony stimulating factor (GM-CSF)/C-C motif ligand 17 (CCL17) pathway in monocytes/macrophages, in which GM-CSF regulates the formation of CCL17, and it is important for an experimental osteoarthritis (OA) model. We explore here additional OA models, including in the presence of obesity, such as a requirement for this pathway. DESIGN: The roles of GM-CSF, CCL17, CCR4, and CCL22 in various experimental OA models, including those incorporating obesity (eight-week high-fat diet), were investigated using gene-deficient male mice. Pain-like behavior and arthritis were assessed by relative static weight distribution and histology, respectively. Cell populations (flow cytometry) and cytokine messenger RNA (mRNA) expression (qPCR) in knee infrapatellar fat pad were analyzed. Human OA sera were collected for circulating CCL17 levels (ELISA) and OA knee synovial tissue for gene expression (qPCR). RESULTS: We present evidence that: i) GM-CSF, CCL17, and CCR4, but not CCL22, are required for the development of pain-like behavior and optimal disease in three experimental OA models, as well as for exacerbated OA development due to obesity, ii) obesity alone leads to spontaneous knee joint damage in a GM-CSF- and CCL17-dependent manner, and iii) in knee OA patients, early indications are that BMI correlates with a lower Oxford Knee Score (r = -0.458 and p = 0.0096), with elevated circulating CCL17 levels (r = 0.2108 and p = 0.0153) and with elevated GM-CSF and CCL17 gene expression in OA synovial tissue. CONCLUSIONS: The above findings indicate that GM-CSF, CCL17, and CCR4 are involved in obesity-associated OA development, broadening their potential as targets for possible treatments for OA.

Calorie restriction, but not Roux-en-Y gastric bypass surgery, increases [3 H] PK11195 binding in a rat model of obesity.

Roux-en-Y gastric bypass surgery (RYGB) remains an effective weight-loss method used to treat obesity. While it is successful in combating obesity, there are many lingering questions related to the changes in the brain following RYGB surgery, one of them being its effects on neuroinflammation. While it is known that chronic high-fat diet (HFD) contributes to obesity and neuroinflammation, it remains to be understood whether bariatric surgery can ameliorate diet-induced inflammatory responses. To examine this, rats were assigned to either a normal diet (ND) or a HFD for 8 weeks. Rats fed a HFD were split into the following groups: sham surgery with ad libitum access to HFD (sham-HF); sham surgery with calorie-restricted HFD (sham-FR); RYGB surgery with ad libitum access to HFD (RYGB). Following sham or RYGB surgeries, rats were maintained on their diets for 9 weeks before being euthanized. [3 H] PK11195 autoradiography was then performed on fresh-frozen brain tissue in order to measure activated microglia. Sham-FR rats showed increased [3 H] PK11195 binding in the amygdala (63%), perirhinal (60%), and ectorhinal cortex (53%) compared with the ND rats. Obese rats who had the RYGB surgery did not show this increased inflammatory effect. Since the sham-FR and RYGB rats were fed the same amount of HFD, the surgery itself seems responsible for this attenuation in [3 H] PK11195 binding. We speculate that calorie restriction following obese conditions may be seen as a stressor and contribute to inflammation in the brain. Further research is needed to verify this mechanism.

Macrophage activation and polarization: nomenclature and experimental guidelines.

Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles-the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation-with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature.

Extracellular ATP targets Arabidopsis RIBONUCLEASE 1 to suppress mycotoxin stress-induced cell death.

Extracellular ATP is a purinergic signal with important functions in regulating plant growth and stress-adaptive responses, including programmed cell death. While signalling events proximate to receptor activation at the plasma membrane have been characterised, downstream protein targets and the mechanism of cell death activation/regulation are unknown. We designed a proteomic screen to identify ATP-responsive proteins in Arabidopsis cell cultures exposed to mycotoxin stress via fumonisin B1 (FB1) application. Arabidopsis RIBONUCLEASE 1 (RNS1) was identified by the screen, and transgenic plants overexpressing native RNS1 showed greater susceptibility to FB1, while a gene knockout rns1 mutant and antisense RNS1 transgenic plants were resistant to FB1-induced cell death. Native RNS1 complemented rns1 mutants and restored the cell death response to FB1, while a catalytically inactive version of the ribonuclease could not. The FB1 resistance of salicylic acid (SA)-depleted nahG-expressing plants was abolished by transformation with native RNS1, but not the catalytically dead version. The mechanism of FB1-induced cell death is activation of RNS1-dependent RNA cleavage, which is blocked by ATP via RNS1 suppression, or enhanced by SA through induction of RNS1 expression. Our study reveals RNS1 as a previously unknown convergence point of ATP and SA signalling in the regulation of stress-induced cell death.

Internal vascular channel architecture in human auditory ossicles.

The vascular supply of the human auditory ossicles has long been of anatomical and clinical interest. While the external blood supply has been well-described, there is only limited information available regarding the internal vascular architecture of the ossicles, and there has been little comparison of this between individuals. Based on high-resolution micro-CT scans, we made reconstructions of the internal vascular channels and cavities in 12 sets of ossicles from elderly donors. Despite considerable individual variation, a common basic pattern was identified. The presence of channels within the stapes footplate was confirmed. The long process of the incus and neck of the stapes showed signs of bony erosion in all specimens examined. More severe erosion was associated with interruption of some or all of the main internal vascular channels which normally pass down the incudal long process; internal excavation of the proximal process could interrupt vascular channels in ossicles which did not appear to be badly damaged from exterior inspection. An awareness of this possibility may be helpful for surgical procedures that compromise the mucosal blood supply. We also calculated ossicular densities, finding that the malleus tends to be denser than the incus. This is mainly due to a lower proportion of vascular channels and cavities within the malleus.

Abstinence following intermittent methylphenidate exposure dose-dependently modifies brain glucose metabolism in the rat brain.

Methylphenidate (MP) is a psychostimulant chronically prescribed for the treatment of attention deficit hyperactivity disorder (ADHD). Additionally, MP users may take breaks from using the medication during "drug holidays," which may include short-term or long-term breaks from medication. The present study utilized fluorodeoxyglucose (FDG) positron emission tomography (PET) to analyze the effects of chronic oral MP use and abstinence on brain glucose metabolism (BGluM) in rats at two different doses: high dose (HD) and low dose (LD). The schedule of treatment was 3 weeks on-treatment and 1 week off-treatment for a period of 13 weeks, followed by an abstinence period of 4 total weeks. Results showed that chronic MP treatment using this schedule did not lead to significant changes in BGluM when comparing the control to HD MP groups. However, significant activation in BGluM was observed after periods of abstinence between control and HD MP rats in the following brain regions: the trigeminal nucleus, reticular nucleus, inferior olive, lemniscus, mesencephalic reticular formation, inferior colliculus, and several areas of the cerebellum. These brain regions and functional brain circuit play a role in facial sensory function, the auditory pathway, organizing connections between the thalamus and cortex, motor learning, auditory function, control over eye movement, auditory information integration, and both motor and cognitive functions. These results, when considered with previous studies, indicate that MP schedule of use may have differing effects on BGluM. BGluM following long-term MP use was dependent on MP dose and schedule of use in rats. This study was conducted in non-ADHD model rats with the aim to establish an understanding of the effects of MP itself, especially given the growing chronic off-label and prescribed use of MP. Further studies are needed for analysis of the drug's effects on an ADHD model.

High Fat Diet Increases [3H] Flunitrazepam Binding in the Mouse Brain that is Dependent on the Expression of the Dopamine D2 Gene.

Dopamine is an important neuromodulator in the brain that binds to dopamine D1-like receptors (D1, D5) as well as dopamine D2-like receptors (D2, D3, D4). The D2 receptor is known to play an integral role in a variety of physiological processes including addictive behaviors, locomotion, motivation, feeding behavior, and more. It was recently reported that dopamine is a direct-acting modulator of mammalian GABA(A) receptors. To this end, we wanted to examine how the expression of the dopamine D2 gene impacts the expression of GABA(A) receptors in the brain under different dietary conditions. Adult female Drd2 wild-type (WT), heterozygous (HT), and knockout (KO) mice were given either normal or high-fat diet for a period of 30 weeks. Following this, their brains were collected for [3H] Flunitrazepam binding in order to assess GABA(A) receptor expression. A high fat diet significantly increased [3H] Flunitrazepam binding in the regions of the somatosensory cortex, striatum, and various other cortical areas within WT mice. In contrast, no effect of diet was observed in HT or KO mice. As such, HT and KO mice displayed reduced [3H] Flunitrazepam binding in these areas relative to WT mice under high-fat dietary conditions. The effect of a high-fat diet on [3H] Flunitrazepam binding is consistent with recent evidence showing increases in GABA neurotransmitter levels following a high-fat diet. We demonstrate for the first time that the expression of the D2 gene plays a prominent role in the ability of a high-fat diet to impact GABA(A) receptors in the mouse brain.

CCL17 in Inflammation and Pain.

It has been reported that a GM-CSF→CCL17 pathway, originally identified in vitro in macrophage lineage populations, is implicated in the control of inflammatory pain, as well as arthritic pain and disease. We explore, in this study and in various inflammation models, the cellular CCL17 expression and its GM-CSF dependence as well as the function of CCL17 in inflammation and pain. This study used models allowing the convenient cell isolation from Ccl17E/+ reporter mice; it also exploited both CCL17-dependent and unique CCL17-driven inflammatory pain and arthritis models, the latter permitting a radiation chimera approach to help identify the CCL17 responding cell type(s) and the mediators downstream of CCL17 in the control of inflammation and pain. We present evidence that 1) in the particular inflammation models studied, CCL17 expression is predominantly in macrophage lineage populations and is GM-CSF dependent, 2) for its action in arthritic pain and disease development, CCL17 acts on CCR4+ non-bone marrow-derived cells, and 3) for inflammatory pain development in which a GM-CSF→CCL17 pathway appears critical, nerve growth factor, CGRP, and substance P all appear to be required.

Alcohol binge drinking decreases brain glucose metabolism and functional connectivity in adolescent rats.

Alcohol misuse represents a serious health concern, especially during adolescence, with approximately 18% of high school students engaging in binge drinking. Despite widespread misuse of alcohol, its effects on how the brain functions is not fully understood. This study utilized a binge drinking model in adolescent rats to examine effects on brain function as measured by brain glucose metabolism (BGluM). Following an injection of [18 FDG] fluro-2-deoxy-D-glucose, rats had voluntary access to either water or various concentrations of ethanol to obtain the following targeted doses: water (no ethanol), low dose ethanol (0.29 ± 0.03 g/kg), moderate dose ethanol (0.98 ± 0.05), and high dose ethanol (2.19 ± 0.23 g/kg). Rats were subsequently scanned using positron emission tomography. All three doses of ethanol were found to decrease BGluM in the restrosplenial cortex, visual cortex, jaw region of the somatosensory cortex, and cerebellum. For both the LD and MD ethanol dose, decreased BGluM was seen in the superior colliculi. The MD ethanol dose also decreased BGluM in the subiculum, frontal association area, as well as the primary motor cortex. Lastly, the HD ethanol dose decreased BGluM in the hippocampus, thalamus, raphe nucleus, inferior colliculus, and the primary motor cortex. Similar decreases in the hippocampus were also seen in the LD group. Taken together, these results highlight the negative consequences of acute binge drinking on BGluM in many regions of the brain involved in sensory, motor, and cognitive processes. Future studies are needed to assess the long-term effects of alcohol binge drinking on brain function as well as its cessation.

Granulocyte-Macrophage Colony Stimulating Factor As an Indirect Mediator of Nociceptor Activation and Pain.

The interaction between the immune system and the nervous system has been at the center of multiple research studies in recent years. Whereas the role played by cytokines as neuronal mediators is no longer contested, the mechanisms by which cytokines modulate pain processing remain to be elucidated. In this study, we have analyzed the involvement of granulocyte-macrophage colony stimulating factor (GM-CSF) in nociceptor activation in male and female mice. Previous studies have suggested GM-CSF might directly activate neurons. However, here we established the absence of a functional GM-CSF receptor in murine nociceptors, and suggest an indirect mechanism of action, via immune cells. We report that GM-CSF applied directly to magnetically purified nociceptors does not induce any transcriptional changes in nociceptive genes. In contrast, conditioned medium from GM-CSF-treated murine macrophages was able to drive nociceptor transcription. We also found that conditioned medium from nociceptors treated with the well established pain mediator, nerve growth factor, could also modify macrophage gene transcription, providing further evidence for a bidirectional crosstalk.SIGNIFICANCE STATEMENT The interaction of the immune system and the nervous system is known to play an important role in the development and maintenance of chronic pain disorders. Elucidating the mechanisms of these interactions is an important step toward understanding, and therefore treating, chronic pain disorders. This study provides evidence for a two-way crosstalk between macrophages and nociceptors in the peripheral nervous system, which may contribute to the sensitization of nociceptors by cytokines in pain development.

Immune Cytokines and Their Receptors in Inflammatory Pain.

There is burgeoning interest in the interaction between the immune and nervous systems. Pain is mediated by primary sensory neurons (nociceptors) that can respond to a variety of thermal, mechanical and chemical signals. Cytokines are now recognized as important mediators of inflammatory pain. They can induce nociceptor sensitization indirectly via mediators, wherein neurons become primed and thus become more responsive to stimulation; alternatively, there is also evidence that cytokines can directly activate neurons via their specific receptors present on the neuronal cells. We review here the evidence for and against these respective mechanisms, focusing on arthritis and inflammatory skin models. A number of striking inconsistencies amongst the conclusions made in the literature are highlighted and discussed.