Characterization of Leptin Receptor+ Stromal Cells in Lymph Node

Lymph node (LN)-resident stromal cells play an essential role in the proper functioning of LNs. The stromal compartment of the LN undergoes significant compensatory changes to produce a milieu amenable for regulation of the immune response. We have identified a distinct population of leptin receptor-expressing (LepR+) stromal cells, located in the vicinity of the high endothelial venules (HEVs) and lymphatics. These LepR+ stromal cells expressed markers for fibroblastic reticular cells (FRCs), but they lacked markers for follicular dendritic cells (FDCs) and marginal reticular cells (MRCs). Leptin signaling deficiency led to heightened inflammatory responses within the LNs of db/db mice, leakiness of HEVs, and lymphatic fragmentation. Leptin signaling through the JAK/STAT pathway supported LN stromal cell survival and promoted the anti-inflammatory properties of these cells. Conditional knockout of the LepR+ stromal cells in LNs resulted in HEV and extracellular matrix (ECM) abnormalities. Treatment of ob/ob mice with an agonist leptin fusion protein restored the microarchitecture of LNs, reduced intra-LN inflammatory responses, and corrected metabolic abnormalities. Future studies are needed to study the importance of LN stomal cell dysfunction to the pathogenesis of inflammatory responses in type 2 diabetes (T2D) in humans.

Retinal Development and Pathophysiology in Kcnj13 Knockout Mice

Purpose: We constructed and characterized knockout and conditional knockout mice for KCNJ13, encoding the inwardly rectifying K+ channel of the Kir superfamily Kir7.1, mutations in which cause both Snowflake Vitreoretinal Degeneration (SVD) and Retinitis pigmentosa (RP) to further elucidate the pathology of this disease and to develop a potential model system for gene therapy trials.Methods: A Kcnj13 knockout mouse line was constructed by inserting a gene trap cassette expressing beta-galactosidase flanked by FRT sites in intron 1 with LoxP sites flanking exon two and converted to a conditional knockout by FLP recombination followed by crossing with C57BL/6J mice having Cre driven by the VMD2 promoter. Lentiviral replacement of Kcnj13 was driven by the EF1a or VMD2 promoters.Results: Blue-Gal expression is evident in E12.5 brain ventricular choroid plexus, lens, neural retina layer, and anterior RPE. In the adult eye expression is seen in the ciliary body, RPE and choroid. Adult conditional Kcnj13 ko mice show loss of photoreceptors in the outer nuclear layer, inner nuclear layer thinning with loss of bipolar cells, and thinning and disruption of the outer plexiform layer, correlating with Cre expression in the overlying RPE which, although preserved, shows morphological disruption. Fundoscopy and OCT show signs of retinal degeneration consistent with the histology, and photopic and scotopic ERGs are decreased in amplitude or extinguished. Lentiviral based replacement of Kcnj13 resulted in increased ERG c- but not a- or b- wave amplitudes.Conclusion: Ocular KCNJ13 expression starts in the choroid, lens, ciliary body, and anterior retina, while later expression centers on the RPE with no/lower expression in the neuroretina. Although KCNJ13 expression is not required for survival of the RPE, it is necessary for RPE maintenance of the photoreceptors, and loss of the photoreceptor, outer plexiform, and outer nuclear layers occur in adult KCNJ13 cKO mice, concomitant with decreased amplitude and eventual extinguishing of the ERG and signs of retinitis pigmentosa on fundoscopy and OCT. Kcnj13 replacement resulting in recovery of the ERG c- but not a- and b-waves is consistent with the degree of photoreceptor degeneration seen on histology.

A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration

Emerging evidence suggests that intron-detaining transcripts (IDTs) are a nucleus-detained and polyadenylated mRNA pool for cell to quickly and effectively respond to environmental stimuli and stress. However, the underlying mechanisms of detained intron (DI) splicing are still largely unknown. Here, we suggest that post-transcriptional DI splicing is paused at Bact 29 state, an active spliceosome but not catalytically primed, which depends on SNIP1 (Smad Nuclear Interacting Protein 1) and RNPS1 (a serine-rich RNA binding protein) interaction. RNPS1 and Bact component preferentially dock at DIs and the RNPS1 docking is sufficient to trigger spliceosome pausing. Haploinsufficiency of Snip1 attenuates neurodegeneration and globally rescues IDT accumulation caused by a previously reported mutant U2 snRNA, a basal spliceosomal component. Snip1 conditional knockout in cerebellum decreases DI splicing efficiency and causes neurodegeneration. Therefore, we suggest that SNIP1 and RNPS1 form a molecular brake for the spliceosome pausing, and that its misregulation contributes to neurodegeneration.

B cell–derived IL-27 promotes control of persistent LCMV infection

Recent studies have identified a critical role for B cell–produced cytokines in regulating both humoral and cellular immunity. Here, we show that B cells are an essential source of interleukin-27 (IL-27) during persistent lymphocytic choriomeningitis virus (LCMV) clone 13 (Cl-13) infection. By using conditional knockout mouse models with specific IL-27p28 deletion in B cells, we observed that B cell–derived IL-27 promotes survival of virus-specific CD4 T cells and supports functions of T follicular helper (Tfh) cells. Mechanistically, B cell–derived IL-27 promotes CD4 T cell function, antibody class switch, and the ability to control persistent LCMV infection. Deletion of IL-27ra in T cells demonstrated that T cell–intrinsic IL-27R signaling is essential for viral control, optimal CD4 T cell responses, and antibody class switch during persistent LCMV infection. Collectively, our findings identify a cellular mechanism whereby B cell–derived IL-27 drives antiviral immunity and antibody responses through IL-27 signaling on T cells to promote control of LCMV Cl-13 infection.

NLK is required for Ras/ERK/SRF/ELK signaling to tune skeletal muscle development by phosphorylating SRF and antagonizing the SRF/MKL pathway

Serum response factor (SRF) regulates differentiation and proliferation by binding to RhoA-actin-activated MKL or Ras-MAPK-activated ELK transcriptional coactivators, but the molecular mechanisms responsible for SRF regulation remain unclear. Here, we show that Nemo-like kinase (NLK) is required for the promotion of SRF/ELK signaling in human and mouse cells. NLK was found to interact with and phosphorylate SRF at serine residues 101/103, which in turn enhanced the association between SRF and ELK. The enhanced affinity of SRF/ELK antagonized the SRF/MKL pathway and inhibited mouse myoblast differentiation in vitro. In a skeletal muscle-specific Nlk conditional knockout mouse model, forming muscle myofibers underwent hypertrophic growth, resulting in an increased muscle and body mass phenotype. We propose that both phosphorylation of SRF by NLK and phosphorylation of ELKs by MAPK are required for RAS/ELK signaling, confirming the importance of this ancient pathway and identifying an important role for NLK in modulating muscle development in vivo.

Adipocyte-specific deletion of PIP5K1c reduces diet-induced obesity and insulin resistance by increasing energy expenditure

Background Phosphatidylinositol 4-phosphate 5-kinase type I c (PIP5K1c) catalyses the synthesis of phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphorylating phosphatidylinositol 4 phosphate, which plays multiple roles in regulating focal adhesion formation, invasion, and cell migration signal transduction cascades. Here, a new physiological mechanism of PIP5K1c in adipocytes and systemic metabolism is reported. Methods Adipose-specific conditional knockout mice were generated to delete the PIP5K1c gene in adipocytes. In addition, in vitro research investigated the effect of PIP5K1c deletion on adipogenesis. Results Deletion of PIP5K1c in adipocytes significantly alleviated high fat diet (HFD)-induced obesity, hyperlipidaemia, hepatic steatosis, and insulin resistance. PIP5K1c deficiency in adipocytes also decreased adipocyte volume in HFD-induced obese mice, whereas no significant differences were observed in body weight and adipose tissue weight under normal chow diet conditions. PIP5K1c knockout in adipocytes significantly enhanced energy expenditure, which protected mice from HFD-induced weight gain. In addition, adipogenesis was markedly impaired in mouse stromal vascular fraction (SVF) from PIP5K1c-deleted mice. Conclusion Under HFD conditions, PIP5K1c regulates adipogenesis and adipose tissue homeostasis. Together, these data indicate that PIP5K1c could be a novel potential target for regulating fat accumulation, which could provide novel insight into the treatment of obesity.

Evaluation of animal model congruence to human depression based on large-scale gene expression patterns of the CNS

Depression is a complex mental health disorder that is difficult to study. A wide range of animal models exist and for many of these data on large-scale gene expression patterns in the CNS are available. The goal of this study was to evaluate how well animal models match human depression by evaluating congruence and discordance of large-scale gene expression patterns in the CNS between almost 300 animal models and a portrait of human depression created from male and female datasets. Multiple approaches were used, including a hypergeometric based scoring system that rewards common gene expression patterns (e.g., up-up or down-down in both model and human depression), but penalizes opposing gene expression patterns. RRHO heat maps, Uniform Manifold Approximation Plot (UMAP), and machine learning were used to evaluate matching of models to depression. The top ranked model was a histone deacetylase (HDAC2) conditional knockout in forebrain neurons. Also highly ranked were various models for Alzheimer’s, including APPsa knock-in (2nd overall), APP knockout, and an APP/PS1 humanized double mutant. Other top models were the mitochondrial gene HTRA2 knockout (that is lethal in adulthood), a modified acetylcholinesterase, a Huntington’s disease model, and the CRTC1 knockout. Over 30 stress related models were evaluated and while some matched highly with depression, others did not. In most of the top models, a consistent dysregulation of MAP kinase pathway was identified and the genes NR4A1, BDNF, ARC, EGR2, and PDE7B were consistently downregulated as in humans with depression. Separate male and female portraits of depression were also evaluated to identify potential sex specific depression matches with models. Individual human depression datasets were also evaluated to allow for comparisons across the same brain regions. Heatmap, UMAP, and machine learning results supported the hypergeometric ranking findings. Together, this study provides new insights into how large-scale gene expression patterns may be similarly dysregulated in some animals models and humans with depression that may provide new avenues for understanding and treating depression.

Delta opioid receptors on nociceptive sensory neurons mediate peripheral endogenous analgesia in colitis

Background Inflammatory visceral pain is endogenously controlled by enkephalins locally released by mucosal CD4+ T lymphocytes in mice. The present study aimed at identifying opioid receptor(s) expressed on nociceptive sensory nerves involved in this peripheral opioid-mediated analgesia. Methods The peripheral analgesia associated with the accumulation of CD4+ T lymphocytes within the inflamed colonic mucosa was assessed in conditional knockout mice specifically deleted for either of the two opioid receptors for enkephalins (i.e., µ (MOR) and δ (DOR) receptors) in Nav1.8-expressing sensory neurons in the dextran sulfate sodium (DSS)-induced colitis model. Results Endogenous analgesia is lost in conditional knockout mice for DOR, but not MOR at the later phase of the DSS-induced colitis. The absence of either of the opioid receptors on sensory nerves had no impact on both the colitis severity and the rate of T lymphocytes infiltrating the inflamed colonic mucosa. Conclusion The key role of DOR on primary afferents in relieving intestinal inflammatory pain opens new therapeutic opportunities for peripherally restricted DOR analgesics to avoid most of the side effects associated with MOR-targeting drugs used in intestinal disorders.

Stress-induced antinociception to noxious heat requires α1A-adrenaline receptors of spinal inhibitory neurons in mice

It is well known that acute exposure to physical stress produces a transient antinociceptive effect (called stress-induced analgesia [SIA]). One proposed mechanism for SIA involves noradrenaline (NA) in the central nervous system. NA has been reported to activate inhibitory neurons in the spinal dorsal horn (SDH), but its in vivo role in SIA remains unknown. In this study, we found that an antinociceptive effect on noxious heat after acute exposure to restraint stress was impaired in mice with a conditional knockout of α1A-adrenaline receptors (α1A-ARs) in inhibitory neurons (Vgat-Cre;Adra1aflox/flox mice). A similar reduction was also observed in mice treated with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, a selective neurotoxin for NAergic neurons in the locus coeruleus (LC). Furthermore, whole-cell patch-clamp recordings using spinal cord slices revealed that NA-induced increase in the frequency of spontaneous inhibitory postsynaptic currents in the substantia gelatinosa neurons was suppressed by silodosin, an α1A-AR antagonist, and by conditional knockout of α1A-ARs in inhibitory neurons. Moreover, under unstressed conditions, the antinociceptive effects of intrathecal NA and phenylephrine on noxious heat were lost in Vgat-Cre;Adra1aflox/flox mice. Our findings suggest that activation of α1A-ARs in SDH inhibitory neurons, presumably via LC-NAergic neurons, is necessary for SIA to noxious heat.

Ketogenic diet ameliorates lipid dysregulation in type 2 diabetic mice by downregulating hepatic pescadillo 1

Background Previous reports implied a possible link between PES1 and lipid metabolism. However, the role of PES1 in regulating T2DM related lipid metabolism and the effect of ketogenic diet (KD) on PES1 have not been reported. The aim of present study is to explore the role of PES1 in effects of KD on diabetic mice and its mediated mechanism. Methods Male C57BL/6J and KKAy mice were fed with standard diet (SD) and KD, respectively. Simultaneously, McArdle 7777 cells were treated by β-hydroxybutyric acid (β-HB), Pes1 siRNA or Pes1 overexpression plasmid, respectively. Additionally, liver-conditional knockout (CKO) of Pes1 in vivo was applied. Results Hepatic PES1 expression in diabetic mice was markedly increased, which was suppressed by KD feeding with an accompanying reduction of hepatic and plasma triglycerides (TG). In mice with CKO of Pes1, the protein levels of p300, SREBP1c, FASN, SCD1, Caspase1, NLRP3 and GSDMD were dramatically downregulated in livers, and the plasma and hepatic TG, IL-1β and IL-18 were decreased as well. The similar outcomes were also observed in β-HB and Pes1 knockdown treated hepatocytes. By contrast, Pes1 overexpression in cultured hepatocytes showed that these levels were significantly enhanced, which were, however reduced under β-HB treatment. Mechanistically, we discovered that β-HB decreased CHOP binding to the Pes1 promoters, resulting in the downregulation of PES1, thereby reducing PES1 binding to p300 and Caspase1 promoters. The inhibition of p300 and Caspase1 expression elicited the dramatic suppression of acetylation of SREBP1c via its interaction with p300, and the decreased GSDMD levels. Besides, knockdown of Caspase1 also alleviated the TG levels in cultured hepatocytes. Conclusion KD may improve lipid dysregulation in type 2 diabetic mice by downregulating hepatic PES1 expression.