Receptor Family Research Articles

Focal adhesions, reticular adhesions, flat clathrin lattices: What divides them, what unites them?

The majority of cells within multicellular organisms requires anchorage to their surroundings in the form of cell-cell or cell-matrix adhesions. In regards to cell-matrix adhesions the transmembrane receptors of the integrin family have long been recognized as the central scaffold around which these adhesion complexes are built. Via their extracellular domains integrins bind extracellular matrix ligands while their intracellular tails interact with a plethora of proteins that link integrin-based adhesions to the cytoskeleton and turn them also into important signaling platforms. Depending on the specific intracellular interactome of the integrins, different types of integrin adhesion complexes have been classified. The best studied ones are the focal adhesions in which integrins become firmly linked to contractile actomyosin fibers allowing for force transduction. But integrins also form an integral part of adhesion structures that lack the strong actomyosin link and are enriched in endocytic proteins. These have been named reticular adhesions, flat clathrin lattices or clathrin plaques. Initially, the different types of integrin adhesion complexes have been viewed as discrete entities with their own separate life cycles. However, in the past years it has become more and more apparent how closely intertwined they are. In fact, it was shown that they can trigger each other´s biogenesis or can even directly convert into each other. Here, we will describe similarities as well as differences between integrin adhesion complexes focusing on the versatile αvß5 integrins and discuss the recently discovered close links and interconversion modes between the different αvß5 integrin adhesion types.

Effect of HNF4 on the Process of Liver Cancer and as a Potential Therapeutic Target

As a ligand-dependent transcription factor of the nuclear receptor family, HNF4 can be regulated by a variety of pathways, and can also be recruited to the DNA sequence through a variety of pathways to regulate downstream target genes by virtue of its three-dimensional structure. HNF4 is usually enriched in organs such as the liver, gallbladder, pancreas, kidneys, etc., and mutations in the HNF4A gene can cause lesions in these organs, including diseases such as liver cancer. However, the pathological role of HNF4 in different diseases is not well understood. Previous studies have shown that 12 variants of HNF4 are jointly regulated by the P1/P2 promoters. HNF4 can be recruited to specific DNA sequences by intermediates (including MLL4, KAT2B, etc.) through DNA-binding domain (DBD) after ligand-binding domain (LBD) has bound ligands. The review goes on to enumerate several pathways, pathways in which HNF4A expression is regulated, and HNF4 acts on promoters or enhancers to regulate the expression of downstream target genes. KDM1A regulates the HNF4A expression pathway, mutations in HNF1 POU domain to downregulate HNF4A promoter activity and NRF2-mediated STAT3 activation to silence HNF4A expression through miR-4, and the functional differences of different subtypes of HNF4 regulated by P1/P2, and the interaction between HNF4 and HIC1 affects the function of HNF4 in regulating FUF/FDF. This review aims to stimulate further research on HNF4, and the specific pathogenesis is clearly elucidated to find suitable targets to treat the corresponding diseases.

The involvement of T1R family receptors expressed outside the oral cavity in the regulation of metabolism

The membrane T1R taste receptor family interacts with sweet substances – carbohydrates, artificial sweeteners and some amino acids. An important result of research in the 21st century was the discovery of abundant expression of these receptors outside of the oral cavity, mainly in cells actively involved in metabolic processes: enteroendocrine cells of the intestine, pancreatic β-cells, adipose and bone tissue, etc. This review integrates and analyzes current data on the role of extraoral T1R receptors in the regulation of metabolism, cell growth and differentiation, which is achieved through modulation of hormone secretion (insulin, GLP-1, GIP), activity of membrane transporters and cell growth and proliferation factors. T1R mediated cellular responses to nutrients, mechanisms of signal transduction, effects on inositol triphosphate, cAMP and intracellular Ca2+ levels, stimulatory effects on glucose transporters SGLT1 and GLUT2, effects on mTOR and hormone secretion are described. The interaction of membrane receptor mechanisms and metabolic detection of glucose by the ATP/ADP ratio in the cell cytoplasm is also discussed. Putative evolutionary adaptation of metabolic processes related to nutrition and manifested in polymorphism of genes encoding T1R proteins is presented. It is suggested that extraoral taste receptors for sweet substances and amino acids may be a target for therapeutic interventions in obesity, hyperglycemia, insulin resistance, and hepatosteatosis.

Membrane progesterone receptor e (paqr9) is necessary for chorion elevation in zebrafish.

Paqr9 is a gene encoding membrane progestin receptor e (mPRe), the fifth subtype of the five mPR subtypes, and is currently classified a member of the progestin and adipoQ receptor (PAQR) family, which consists of 11 genes. To elucidate the physiological functions of the mPR subtypes, we established gene knockout (KO) fish via genome editing of seven paqr genes in zebrafish and analyzed their phenotypes. The null-mutant strain of paqr9 (paqr9-/-) that we established in this study presented reduced chorion elevation and a high percentage of abnormal embryos. Embryos exhibit various kinds of abnormal morphology, which are thought to be caused by insufficient elevation of the chorion. Immunohistochemical staining of ovaries with an anti-Paqr9 antibody revealed that Paqr9 was expressed in the periplasm of oocytes and the surface of chorion in the wild type, whereas signals were absent in paqr9-/- zebrafish. In histological sections, the periplasmic connection between the oocyte plasma membrane and chorion was absent in paqr9-/- oocytes. The number of cortical alveoli (CA) that are responsible for chorion elevation was significantly reduced in paqr9-/- zebrafish. SEM revealed that fiber-supported knob-like structures (KSs) on the chorion were absent in paqr9-/- zebrafish. These results indicate that Paqr9 is required for the preparation of CA during oogenesis. Insufficient formation of the chorion resulted in the abnormal development of embryos.

The Role of Beta-Hydroxybutyrate in Mitigating the Inflammatory and Metabolic Consequences of Uric Acid

Background: Uric acid (UA), a metabolite of purine and fructose metabolism, is linked to inflammation and metabolic disorders, including gout and cardiovascular disease. Its pro-inflammatory effects are largely driven by the activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, leading to increased cytokine production. Beta-hydroxybutyrate (BHB), a ketone produced during fasting or carbohydrate restriction, has been shown to reduce inflammation. This study explores the role of BHB in mitigating the inflammatory and metabolic effects of elevated uric acid levels. Methods: We utilized a murine muscle cell culture treated with UA and BHB. Results: Muscle cells treated with UA had increased production of pro-inflammatory cytokines and reduced cell viability. Co-treatment with BHB reversed these effects, improving cell survival and reducing cytokine levels. Additionally, uric acid impaired mitochondrial function and increased oxidative stress, which were mitigated by BHB. Furthermore, uric acid disrupted insulin signaling, but BHB co-treatment restored insulin sensitivity. Conclusions: These findings suggest that BHB holds therapeutic potential by counteracting the inflammatory and metabolic disruptions caused by elevated uric acid, making it a promising target for conditions such as hyperuricemia and metabolic syndrome.

Purinergic P2X7 receptor mediates hyperoxia-induced injury in pulmonary microvascular endothelial cells via NLRP3-mediated pyroptotic pathway.

Hyperoxia-induced injury is a well-recognized cause of bronchopulmonary dysplasia (BPD). Existing research studies have not well elucidated the exact mechanisms underlying hyperoxia-induced cellular damage. This study examines the involvement of the P2X7 receptor (P2X7R) in hyperoxia-induced damage to human pulmonary microvascular endothelial cells (HPMVECs) via the NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) pathway. HPMVECs developing hyperoxia-induced injury were subjected to the treatment of either selective inhibitors or a P2X7R/NLRP3 agonist. Western blot analysis assisted in the quantification of the levels of P2X7R, NLRP3, caspase-1, and gasdermin D (GSDMD). Additionally, the release of TNF-α, IL-1β, and IL-18 was assessed by ELISA and qRT-PCR. Exposure to hyperoxia diminished cell viability and escalated the levels of P2X7R, caspase-1, NLRP3, GSDMD, and N-terminal-GSDMD. This exposure notably increased the release of TNF-α, IL-1β, and IL-18 in HPMVECs. Notably, the suppression of P2X7R using the inhibitor A438079 decreased pyroptosis and inflammatory responses. Conversely, stimulation of P2X7R by 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) triggered pyroptosis, while inhibition of NLRP3 with glibenclamide ameliorated the damage induced by BzATP. The P2X7R/NLRP3 pathway crucially affects the hyperoxia-induced inflammation and pyroptosis in HPMVECs, hinting the potential of blocking P2X7R/NLRP3-mediated pyroptotic pathway as a valuable therapeutic strategy for BPD.

GFRα1 Promotes Axon Regeneration after Peripheral Nerve Injury by Functioning as a Ligand.

The neurotrophic factor, Glial cell line derived neurotrophi factor (GDNF), exerts a variety of biological effects through binding to its receptors, GDNF family receptor alpha-1 (GFRα1), and RET. However, the existence of cells expressing GFRα1 but not RET raises the possibility that GFRα1 can function independently from RET. Here, it is shown that GFRα1 released from repair Schwann cells (RSCs) functions as a ligand in a GDNF-RET-independent manner to promote axon regeneration after peripheral nerve injury (PNI). Local administration of GFRα1 into injured nerve promoted axon regeneration, even more when combined with GDNF blockade. GFRα1 bound to a receptor complex consisting of NCAM and integrin α7β1 of dorsal root ganglion neurons in a GDNF-RET independent manner. This is further confirmed by the Ret Y1062F knock-in mice, which cannot transmit most of GDNF-RET signaling. Finally, local administration of GFRα1 into injured sciatic nerve promoted functional recovery. These findings reveal a novel role of GFRα1 as a ligand, the molecular mechanism supporting axon regeneration by RSCs, and a novel therapy for peripheral nerve repair.

Characterization of a novel short-type peptidoglycan recognition protein from the sea cucumber Apostichopus japonicus.

Peptidoglycan recognition proteins (PGRPs) represent a key component of the family of pattern recognition receptors (PRRs). The functional mechanisms of PGRPs in innate immunity are poorly understood. In this study, we identified a novel short-type PGRP, AjPGRP-S2, from the sea cucumber Apostichopus japonicus. Our data showed that AjPGRP-S2 encoded an extracellular protein that possessed a signal peptide, a complete zinc (Zn2+) binding site, and a disulfide bond. A recombinant AjPGRP-S2 (rAjPGRP-S2) lacking the signal peptide was generated and demonstrated to exhibit amidase activity. Tissue expression analysis revealed that AjPGRP-S2 was highly expressed in coelomocytes and tube feet. Immune-responsive analysis indicated that AjPGRP-S2 was able to bind to various pathogen-associated molecular patterns (PAMPs) from bacteria and fungi, as well as to Gram-positive and -negative bacteria, and was majorly induced by DAP-PGN challenge. Basing on RNA-Seq and Pearson's correlation testing, RNA interference, and pull-down analysis, AjPGRP-S2 was found to be involved in transducing immune signals to the complement system and other PRRs, such as fibrinogen, by protein interactions to further recognize and kill pathogens. To respond comprehensively against pathogenic invasion, AjPGRP-S2 may also have the potential in transducing immune signals to key processes, such as cell adhesion, nerve conduction, apoptosis, and transcription by complex pathways that have yet to be elucidated. Our findings not only promote our understanding of the immune-related function and mechanisms of the PGRP family in A. japonicus, but also provide important data that will facilitate the identification of key evolutionary characteristics associated with invertebrate PGRPs.

Mictlan-D3: A novel medium sized RGD-Disintegrin obtained from Crotalus mictlantecuhtli venom, in vitro tested against human breast Cancer and endothelial cells

Disintegrins are small non-enzymatic proteins present often at low concentration in the venom of viperid snakes. Isolated disintegrins are known for their lack of toxicity as well as their capacity to antagonize integrin receptors. Integrins are a major family of heterodimeric cell surface receptors that mediate cell-cell and cell-extracellular matrix (ECM) interactions. Integrins regulate key functions in cancer pathology and also tumor development. The aim of this study consisted in the isolation and characterization of disintegrins from rattlesnake new species Crotalus mictlantecutli venom. A disintegrin fraction obtained by RP-HPLC and named mictlan-D3, consist in two isoforms of 7439 and 7509 Da with 72 amino acid sequence containing the RGD binding motif. Mictlan-D3 inhibited MDA-MB-231 and HMEC-1 cell adhesion to laminin (LN), fibronectin (FN) and vitronectin (VN), highest inhibition was on MDA-MB-231 cell adhesion to LN by 81 % at 1 μM. The blockade of ⍺Vβ3 integrin was evaluated by wound healing migration assay. Mictlan-D3 inhibited MDA-MB-231 cell migration by 80 % and 38 % after 24 and 72 h of incubation respectively. HMEC-1 cell migration was inhibited by 67.6 % and 27.9 % after 24 and 72 h of incubation. Additionally, mictlan-D3. This work represent the first characterization of disintegrins from the Crotalus mictlantecutli venom.

The CD244 rs3766379 variant showed no association with susceptibility to rheumatoid arthritis while soluble CD244 was associated with disease activity

Cluster of differentiation 244 (CD244), a member of the signaling lymphocytic activation molecule (SLAM) family, is one of the major cell surface receptors of the SLAM family with activating and inhibitory signaling potentials that is involved in the functional regulation of inflammatory reactions. It has recently been suggested that soluble CD244 levels are dysregulated in patients with rheumatoid arthritis (RA), but the evidence is not conclusive. Furthermore, the CD244 rs3766379 variant showed conflicting results regarding association with disease susceptibility. In this cross-sectional case-control study, serum CD244 concentration was determined in 123 RA patients and 60 controls using an enzyme-linked immunosorbent assay kit. The CD244 rs3766379 variant was also genotyped using real-time polymerase chain reaction principles. Results revealed that Log10-transformed serum concentrations (mean ± standard error) of soluble CD244 were significantly elevated in RA patients compared to controls (0.53 ± 0.04 vs. 0.29 ± 0.02 ng/mL; probability = 0.003), especially in patients with high disease activity (0.73 ± 0.12 vs. 0.29 ± 0.02 ng/mL; probability = 0.003), where CD244 showed reliable discrimination between patients and controls (area under the curve = 0.698; probability = 0.023). The rs3766379 allele and genotype frequencies showed no significant differences between patients and controls. Furthermore, this variant did not affect CD244 concentration. In conclusion, CD244 levels were up-regulated in the serum of patients with RA, particularly those with high disease activity. The CD244 rs3766379 variant was not associated with susceptibility to RA and had no effect on serum CD244 concentration.

Evaluation of glycyrrhetinic acid in attenuating adverse effects of a high-fat diet in largemouth bass (Micropterus salmoides)

Glycyrrhetinic acid (GA) has been shown to promote growth characteristics and play a crucial role in anti-inflammatory responses in animals. To investigate the effects of dietary GA supplementation on growth performance, intestinal inflammation, and intestinal barrier protection in largemouth bass (Micropterus salmoides) fed a high-fat diet (HFD), a 77-day feeding experiment was conducted. A total of 750 largemouth bass, initially averaging 17.39 ± 0.09 g in body weight, were randomly allocated to five experimental groups and fed a control diet, a HFD, and the HFD diet supplemented with GA at either 0.5, 1.0, or 1.5 mg/kg, named as control, HDF, HFD+GA 0.5, HFD+GA 1.0, and 1.5 HFD+GA 1.5, respectively. Each group contained three replicates. The study revealed that dietary GA improved final body weight (P < 0.001), percent weight gain (P = 0.041), and feed intake (P < 0.001), all of which had been affected by a HFD in largemouth bass (P < 0.05). Supplementation of HFD with 1.0 mg/kg GA increased the mRNA expressions and protein levels of corresponding tight junctions, occludin, zonula occluden-1 (ZO-1) and claudin-1 in the intestines of largemouth bass. Furthermore, the addition of HFD with both of 0.5 and 1.0 mg/kg GA decreased the mRNA expressions of pro-inflammatory genes such as interleukin-1β (IL-1β), IL-18, and cysteinyl aspartate specific proteinase 1 (caspase-1), as well as proteins associated with pyroptosis-induced inflammation, including NOD-like receptor family and pyrin domain contain 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), gasdermin E (GSDME), and N-terminal domain of GSDME (GSDME-N) (P < 0.05). Finally, dietary GA supplementation alleviated mitochondrial damage and reduced reactive oxygen species (ROS) production induced by the HFD. It is concluded that GA supplementation in HFD enhances growth performance, increases mRNA expression and protein levels of tight junction-related parameters, decreases mRNA expression and protein levels of pyroptosis-related genes, and alleviates intestinal mitochondrial injury and inflammation induced by HFD.

TRPM7 contributes to pyroptosis and its involvement in status epilepticus

BackgroundPyroptosis, a novel form of programmed cell death, has been implicated in neurodegeneration diseases. However, its role in status epilepticus (SE)—a condition characterized by prolonged or repeated seizures—remains inadequately understood.MethodsSE were induced by intraperitoneal injection of pilocarpine (PILO). Neuronal excitability was assessed through electroencephalogram (EEG) recordings and patch clamp. Chromatin immunoprecipitation (ChIP) assay was applied to verify the interaction of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) protein with the promoters of Nlrp3 (the gene encoding NOD-like receptor family pyrin domain containing 3) and Trpm7 (transient receptor potential melastatin 7). To further investigate the role of TRPM7 in SE, AAV-sh-TRPM7-EGFP transfected mice and TRPM7 conditional knockout (TRPM7-CKO) mice were utilized.ResultsOur findings revealed elevated levels of IL-18 and IL-1β levels in primary epilepsy patients, along with increased expression level of the TRPM7 in SE models. Knockdown of TRPM7 alleviated neuronal damage and pyroptosis, reversing PILO-treated neuronal hyperexcitability. We demonstrated that p-STAT3 binds to the promoters of both Trpm7 and Nlrp3, modulating their transcriptions in SE. Importantly, inhibition of TRPM7 with NS8593, and inflammasome inhibition with MCC950, alleviated neuronal hyperexcitability and pyroptosis in SE. A new compound, SDUY-225, formulated based on the structure of NS8593 mitigated neuronal damage, pyroptosis, and hyperexcitability.ConclusionsTRPM7 contributes to pyroptosis in SE, establishing a positive feedback loop involving the p-STAT3/TRPM7/Zn2+/p-STAT3 signaling pathway. Findings in this study raise the possibility that targeting TRPM7 and NLRP3 represents a promising therapeutic approach for SE.

Structural insights into endogenous ligand selectivity and activation mechanisms of FFAR1 and FFAR2

Free fatty acid receptors (FFARs) play critical roles in metabolic regulation and are potential therapeutic targets for metabolic and inflammatory diseases. A comprehensive understanding of the activation mechanisms and endogenous ligand selectivity of FFARs is essential for drug discovery. Here, we report two cryoelectron microscopy structures of the human FFAR1 bound to the endogenous ligand docosahexaenoic acid (DHA) and Gi1 protein as well as FFAR2 in complex with butyrate and Gi1 at 3.2Å and 3.3Å resolution, respectively. These structures highlight that distinct locations and sizes of the orthosteric ligand binding pockets are crucial determinants of the endogenous ligand selectivity of this receptor subfamily. Additionally, computational analysis reveals a potential allosteric ligand binding pocket in FFAR2. Furthermore, we observe that the upward movement of helix V upon endogenous ligand binding is responsible for receptor activation. These insights will significantly aid in the development of drugs targeting this receptor family.

NLRC3 affects the development of psoriasis by modulating the NF-κB signaling pathway mediated inflammatory response through its interaction with TRAF6

ObjectiveThe function and mechanism of NOD-like receptor family CARD-containing 3 (NLRC3) in psoriasis are not yet reported, even though it plays a crucial role in innate and adaptive immunity by inhibiting inflammation. Therefore, this research aims to investigate the role and mechanism of NLRC3 in psoriasis. MethodsHaCaT cells were induced to form a psoriasis cell model using 20 ng/mL IL-1β, 20 ng/mL IL-17A, 20 ng/mL IL-23, 50 ng/mL TNF-α, and 20 ng/mL oncostatin M. Cell Counting Kit-8 (CCK-8) assay and flow cytometry were assessed to determine the proliferation, cell cycle, and apoptosis of HaCaT cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was utilized to measure the knockdown efficiency of NLRC3 and TRAF6 interfering RNA in HaCaT cells. Western blot analysis was performed to determine the expression levels of NLRC3, TRAF6, and proteins associated with the NF-κB signaling pathway. A mouse model of psoriasis-like dermatitis was established by evenly applying miquimod cream (62.5 mg/day) to both ears. Hematoxylin-eosin staining was used to measure ear thickness and inflammatory infiltrates in mice. Histological analysis, immunohistochemistry, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL were performed to detect cell proliferation and apoptosis. Interactions between NLRC3 and TRAF6 were predicted using the STRING database (https://cn.string-db.org/). Co-Immunoprecipitation was used to confirm interactions between NLRC3 and TRAF6. Ubiquitination of TRAF6 was assessed by Western blot. ResultsKnockdown of NLRC3 expression promoted cell proliferation and inhibited cell apoptosis in HaCaT cells. In vivo, knockdown of NLRC3 expression significantly increased the infiltration of inflammatory cells and the proliferation of Ki-67 positive cells within mouse ear epidermis, while decreasing the number of apoptotic cells. NLRC3 interacted with TRAF6 and influenced its K63 ubiquitination level. Knockdown of TRAF6 expression resulted in increased cell proliferation and decreased cell apoptosis in HaCaT cells. In vivo, knockdown of TRAF6 expression led to a significant increase in inflammatory cell infiltration and Ki-67 positive cells in mouse ear epidermis, and a decrease in apoptotic cells. Inhibiting the NF-κB signaling pathway alleviated the progression of psoriasis, and interfering with TRAF6 activated the NF-κB signaling axis, contributing to the onset and advancement of psoriasis. ConclusionNLRC3 affects the occurrence of psoriasis by regulating TRAF6 and influencing the NF-κB signaling axis-mediated inflammatory response. This finding offers a theoretical foundation for the treatment of psoriasis.

Acid-sensing ion channel 1a promotes alcohol-associated liver disease in mice via regulating endoplasmic reticulum autophagy.

Alcohol-associated liver disease (ALD) is a hepatocyte dysfunction disease caused by chronic or excessive alcohol consumption, which can lead to extensive hepatocyte necrosis and even liver failure. Currently, the pathogenesis of ALD and the anti-ALD mechanisms have not been fully elucidated yet. In this study, we investigated the effects of endoplasmic reticulum autophagy (ER-phagy) in ALD and the role of acid-sensing ion channel 1a (ASIC1a) in ER stress-mediated ER-phagy. A mouse model of ALD was established using the Gao-Binge method and the AML12 cell line treated with alcohol was used as an in vitro model. We showed that ASIC1a expression was significantly increased and ER-phagy was activated in both the in vivo and in vitro models. In alcohol-treated AML12 cells, we showed that blockade of ASIC1a with PcTx-1 or knockdown of ASIC1a reduced alcohol-induced intracellular Ca2+ accumulation and ER stress. In addition, inhibition of ER stress with 4-PBA reduced the level of ER-phagy. Furthermore, knockdown of the ER-phagy receptor family with sequence similarity 134 member B (FAM134B) alleviated alcohol-triggered hepatocyte injury and apoptosis. In conclusion, this study demonstrates that alcohol activates ER stress-induced ER-phagy and liver injury by increasing ASIC1a expression and ASIC1a-mediated Ca2+ influx, providing a novel strategy for the treatment of ALD.

Immuno-oncologic profiling by stage-dependent transcriptome and proteome analyses of spontaneously regressing canine cutaneous histiocytoma.

Canine cutaneous histiocytoma (CCH) is a tumor that originates from dermal Langerhans cells and affects particularly young dogs. The common spontaneous regression of CCH makes it an interesting model in comparative oncology research. Previous studies have indicated that anti-tumor immune responses may be involved, but details remain speculative to date. Here, we asked which specific immuno-oncological dynamics underlie spontaneous regression of CCH on mRNA and protein levels. QuantSeq 3' mRNA sequencing with functional over-representation analysis and an nCounter RNA hybridization assay were employed on 21 formalin-fixed, paraffin-embedded CCH samples representing three different tumor stages (dataset information: GSE261387-Immuno-Oncologic Profiling by Stage-Dependent Transcriptome and Proteome Analyses of Spontaneously Regressing Canine Cutaneous Histiocytoma-OmicsDI). Nine additional samples were subjected to matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Surprisingly, only minor stage-specific differences were found. When we investigated expression of B7 family ligands and CD28 family receptors holding co-stimulatory and -inhibitory functions, respectively, we found a higher abundance of CD80, CD86, CTLA4 and CD28, which may trigger a balanced activation of lymphocyte-mediated immune responses. CD80 and CD86 expressing cells were further quantified by in situ hybridization and compared with data from three cases of canine histiocytic sarcoma (HS), a malignant tumor variant originating from antigen-presenting interstitial dendritic cells. A stage-specific increase of CD80 expressing cells was recorded in CCH from the tumor bottom to the top, while CD86 was continuously and homogenously expressed at high levels. Overall expression of CD80 in CCH was similar to that in HS (73.3 ± 37.4% vs 62.1 ± 46.4%), while significantly more CD86 expressing tumor cells were found in CCH (94.7 ± 10.3%) when compared to HS (57.6 ± 11.0%). Our data suggest that major immuno-oncological pathways are not regulated during regression of CCH on the mRNA or protein levels as detectable by the methods used. Instead, our data provide further evidence supporting previous hypotheses towards a role of immune stimulatory B7 family ligands and CD28 family receptors in the regression of CCH.

GDNF family receptor alpha-like (GFRAL) expression is restricted to the caudal brainstem

ObjectiveGrowth differentiation factor 15 (GDF15) acts on the receptor dimer of GDNF family receptor alpha-like (GFRAL) and Rearranged during transfection (RET). While Gfral-expressing cells are known to be present in the area postrema and nucleus of the solitary tract (AP/NTS) located in the brainstem, the presence of Gfral-expressing cells in other sites within the central nervous system and peripheral tissues is not been fully addressed. Our objective was to thoroughly investigate whether GFRAL is expressed in peripheral tissues and in brain sites different from the brainstem. MethodsFrom Gfral:eGFP mice we collected tissue from 12 different tissues, including brain, and used single molecule in-situ hybridizations to identify cells within those tissues expressing Gfral. We then contrasted the results with human Gfral-expression by analyzing publicly available single-cell RNA sequencing data. ResultsIn mice we found readably detectable Gfral mRNA within the AP/NTS but not within other brain sites. Within peripheral tissues, we failed to detect any Gfral-labelled cells in the vast majority of examined tissues and when present, were extremely rare. Single cell sequencing of human tissues confirmed GFRAL-expressing cells are detectable in some sites outside the AP/NTS in an extremely sparse manner. Importantly, across the utilized methodologies, smFISH, genetic Gfral reporter mice and scRNA-Seq, we failed to detect Gfral-labelled cells with all three. ConclusionsThrough highly sensitive and selective technologies we show Gfral expression is overwhelmingly restricted to the brainstem and expect that GDF15 and GFRAL-based therapies in development for cancer cachexia will specifically target AP/NTS cells.

Sex-specific NLRP3 activation in neutrophils promotes neutrophil recruitment and NETosis in the murine model of diffuse alveolar hemorrhage.

Diffuse alveolar hemorrhage (DAH) is a life-threatening complication of systemic lupus erythematosus and small vessel vasculitis. We previously showed that neutrophil extracellular traps (NETs) were associated with the pathogenesis of pristane-induced DAH and demonstrated that neutrophil NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome assembly participated in NET generation under sterile stimulation. We investigated whether NLRP3 inflammasome assembly in neutrophils may drive pulmonary NETosis in a mouse model of pristane-induced DAH. C57BL/6J mice received a single intraperitoneal injection of 0.5mL of pristane. Neutrophil NLRP3 inflammasome assembly and NETs were characterized by immunofluorescence staining of apoptosis-associated speck-like protein a CARD (ASC), co-staining of DNA, and citrullinated histones, respectively. Clinical status of mice was assessed 11 days after pristane injection by measurement of arterial oxygen saturation and of weight loss; severity of lung injury was determined using a quantification score from hematoxylin-eosin-stained slides. Pristane induced ASC speck formation in neutrophils and we confirmed that NLRP3 inflammasome was involved in NET generation after pristane stimulation in vitro. NLRP3 deficiency reduced the severity of pristane-induced DAH in female, but not male mice. Interestingly, NLRP3 deficiency reduced the number of neutrophils and NETs in the lungs of females compared to males. Our results suggest a link between female sex-specific NLRP3 inflammasome activation and subsequent pulmonary NETosis in the development of pristane-induced DAH. Therefore, we identified NLRP3 inflammasome as a potential new therapeutic target in this severe complication of pro-female autoimmune disease for which specific inhibitors of NLRP3 are currently developed.

Heterozygous Predicted Loss-of-function Variants of TRAF3 in Patients with Common Variable Immunodeficiency.

TRAF3, a versatile adaptor protein within the TRAF family, participates in various signaling pathways involving the tumor necrosis factor receptor, toll-like receptor, and retinoic acid-inducible gene I-like receptor families. In 2010, autosomal dominant TRAF3 deficiency was reported in a patient with herpes simplex virus-1 encephalitis, consistent with the role of TRAF3 in type I interferon production. Recently, a novel, completely different clinical phenotype was described in patients with TRAF3 haploinsufficiency (TRAF3Hl), characterized by recurrent bacterial infections, autoimmune features, systemic inflammation, and hypergammaglobulinemia. In this study, we conducted a TRAF3-targeted reanalysis of next-generation sequencing data from 800 patients with inborn errors of immunity. Through this reassessment and additional familial investigations, we identified three previously unidentified cases of TRAF3Hl within two different families. These individuals harbored stop-gain variants (p.Arg163* and p.Gln407*) and experienced recurrent bacterial infections with hypogammaglobulinemia. Previously, the patients had been diagnosed with common variable immunodeficiency (CVID) and were receiving immunoglobulin replacement therapy. In addition, a TRAF3 start-loss variant (c.3G > A) was identified in a fourth patient, but after familial and molecular studies, it was not considered disease-causing, excluding TRAF3Hl in this patient. This study illustrates the usefulness of targeted reanalysis of genes with reported novel phenotypes. We rescued three patients with TRAF3Hl, presenting similarities and differences with the previously reported patients. The most significant differences were hypogammaglobulinemia and a CVID-like presentation. These data expand the clinical phenotype of TRAF3Hl and pave the way for further investigation into loss-of-function variants in patients with CVID.

Lipophorin receptor knockdown reduces hatchability of kissing bug Rhodnius prolixus eggs

Lipophorin is the primary lipoprotein present in the hemolymph of insects, responsible for the lipids' transport between organs. It interacts with specific sites on cell membranes in an essential process for transferring lipids. The lipophorin receptor is the protein responsible for the interaction between lipophorin and cell membranes. In the kissing bug Rhodnius prolixus, much information on the interaction of lipophorin with organs is available. However, molecular data on the lipophorin receptor and its functions is still needed. Here, we explored lipophorin receptor gene expression and functions using a functional genomics approach. The R. prolixus genome encodes seven genes from the low-density lipoprotein receptor family, including a single ortholog of the lipophorin receptor. All organs analyzed (anterior and posterior midguts, fat body, ovaries, and flight muscle) expressed this gene. In the fat body, blood-feeding strongly reduced lipophorin receptor gene expression. Lipophorin receptor knockdown by RNA interference delayed egg laying and reduced the triacylglycerol in laid eggs without altering lipid stores in the fat body or lipid levels in the hemolymph. In the ovaries, lipophorin receptor knockdown reduces the expression of acetyl-CoA carboxylase and a fatty acid synthase while altered the gene expression profile in the fat body, causing an increase in the expression of carnitine palmitoyltransferase 1 and a reduction in Brummer lipase and vitellogenin 2. RNA interference treatment reduced the hatching of the eggs, causing the collapse and darkening of the laid eggs, in addition to the hatching of deformed first-stage nymphs. Furthermore, the structure of the chorion showed distortions in patterns and cracks and reduced hydrocarbon levels. These results show that the lipophorin receptor alone is not essential for lipid physiology in R. prolixus. However, this protein plays a fundamental role in the viability of eggs and, consequently, in insect reproduction.