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BACKGROUND: Human health is seriously threatened by antibiotic-induced intestinal disorders. Herein, we aimed to determine the effects of Autoinducer-2 (AI-2) combined with Lactobacillus rhamnosus GG (LGG) on the intestinal barrier function of antibiotic-induced intestinal dysbiosis neonatal mice. METHODS: An antibiotic-induced intestinal dysbiosis neonatal mouse model was created using antibiotic cocktails, and the model mice were randomized into the control, AI-2, LGG, and LGG + AI-2 groups. Intestinal short-chain fatty acids and AI-2 concentrations were detected by mass spectrometry and chemiluminescence, respectively. The community composition of the gut microbiota was analyzed using 16S rDNA sequencing, and biofilm thickness and bacterial adhesion in the colon were assessed using scanning electron microscopy. Transcriptome RNA sequencing of intestinal tissues was performed, and the mRNA and protein levels of HCAR2 (hydroxycarboxylic acid receptor 2), claudin3, and claudin4 in intestinal tissues were determined using quantitative real-time reverse transcription PCR and western blotting. The levels of inflammatory factors in intestinal tissues were evaluated using enzyme-linked immunosorbent assays (ELISAs). D-ribose, an inhibitor of AI-2, was used to treat Caco-2 cells in vitro. RESULTS: Compared with the control, AI-2, and LGG groups, the LGG + AI-2 group showed increased levels of intestinal AI-2 and proportions of Firmicutes and Lacticaseibacillus, but a reduced fraction of Proteobacteria. Specifically, the LGG + AI-2 group had considerably more biofilms and LGG on the colon surface than those of other three groups. Meanwhile, the combination of AI-2 and LGG markedly increased the concentration of butyric acid and promoted Hcar2, claudin3 and claudin4 expression levels compared with supplementation with LGG or AI-2 alone. The ELISAs revealed a significantly higher tumor necrosis factor alpha (TNF-α) level in the control group than in the LGG and LGG + AI-2 groups, whereas the interleukin 10 (IL-10) level was significantly higher in the LGG + AI-2 group than in the other three groups. In vitro, D-ribose treatment dramatically suppressed the increased levels of Hcar2, claudin3, and claudin4 in Caco-2 cells induced by AI-2 + LGG. CONCLUSIONS: AI-2 promotes the colonization of LGG and biofilm formation to improve intestinal barrier function in an antibiotic-induced intestinal dysbiosis neonatal mouse model.
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Lacticaseibacillus rhamnosus , Probióticos , Camundongos , Humanos , Animais , Animais Recém-Nascidos , Células CACO-2 , Função da Barreira Intestinal , Disbiose , Antibacterianos/farmacologia , Claudina-4/metabolismo , RiboseRESUMO
Preservation of retinal barrier function is critical to maintenance of retinal health. Therefore, it is not surprising that loss of barrier integrity is a pathologic feature common to degenerative retinal diseases such as diabetic retinopathy. Our prior studies demonstrate the importance of hydroxycarboxylic acid receptor 2/GPR109A (HCAR2/GPR109A) expression in the retinal pigment epithelium (RPE) to outer retinal barrier integrity. However, whether HCAR2/GPR109A is expressed in retinal endothelial cells and has a similar relationship to inner blood retinal barrier regulation is not known. In the current study, we examined relevance of receptor expression to endothelial cell dependent-blood retinal barrier integrity. siRNA technology was used to modulate HCAR2/GPR109A expression in human retinal endothelial cells (HRECs). Cells were cultured in the presence or absence of VEGF, a pro-inflammatory stimulus, and/or various concentrations of the HCAR2/GPR109A-specific agonist beta-hydyroxybutyrate (BHB). HCAR2/GPR109A expression was monitored by qPCR and electrical cell impedance sensing (ECIS) was used to evaluate barrier function. Complementary in vivo studies were conducted in wildtype and HCAR2/GPR109A knockout mice treated intraperitoneally with lipopolysaccharide and/or BHB. Vascular leakage was monitored using fluorescein angiography and Western blot analyses of albumin extravasation. Additionally, retinal function was evaluated by OptoMotry. Decreased (siRNA knockdown) or absent (gene knockout) HCAR2/GPR109A expression was associated with impaired barrier function both in vitro and in vivo. BHB treatment provided some protection, limiting disruptions in retinal barrier integrity and function; an effect that was found to be receptor (HCAR2/GPR109A)-dependent. Collectively, the present studies support a key role for HCAR2/GPR109A in regulating blood-retinal barrier integrity and highlight the therapeutic potential of the receptor toward preventing and treating retinal diseases such as diabetic retinopathy in which compromised barrier function is paramount.
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Retinopatia Diabética , Receptores Acoplados a Proteínas G , Doenças Retinianas , Animais , Barreira Hematorretiniana/metabolismo , Proteínas de Transporte/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Cetonas/metabolismo , Cetonas/uso terapêutico , Camundongos , RNA Interferente Pequeno/uso terapêutico , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Doenças Retinianas/metabolismoRESUMO
Background: Diabetic retinopathy (DR) is a neurovascular disease, characterized by a deficiency of brain-derived neurotrophic factor (BDNF), a regulator of autophagy. Beta-hydroxybutyrate (BHB), previously reported as a protective agent in DR, has been associated with BDNF promotion. Here, we investigated whether systemic BHB affects the retinal levels of BDNF and local autophagy in diabetic mice with retinopathy; Methods: C57BL/6J mice were administered with intraperitoneal (i.p.) streptozotocin (STZ) (75 mg/kg) injection to develop diabetes. After 2 weeks, they received i.p. injections of BHB (25−50−100 mg/kg) twice a week for 10 weeks. Retinal samples were collected in order to perform immunofluorescence, Western blotting, and ELISA analysis; Results: BHB 50 mg/kg and 100 mg/kg significantly improved retinal BDNF levels (p < 0.01) in diabetic mice. This improvement was negatively associated with autophagosome−lysosome formations (marked by LC3B and ATG14) and to higher levels of connexin 43 (p < 0.01), a marker of cell integrity. Moreover, BHB administration significantly reduced M1 microglial activation and autophagy (p < 0.01); Conclusions: The systemic administration of BHB in mice with DR improves the retinal levels of BDNF, with the consequent reduction of the abnormal microglial autophagy. This leads to retinal cell safety through connexin 43 restoration.
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Diabetes Mellitus Experimental , Retinopatia Diabética , Ácido 3-Hidroxibutírico/farmacologia , Animais , Autofagia , Fator Neurotrófico Derivado do Encéfalo , Conexina 43 , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Retinopatia Diabética/complicações , Retinopatia Diabética/etiologia , Camundongos , Camundongos Endogâmicos C57BL , RetinaRESUMO
Nicotinic acid (NA) activates hydroxycarboxylic acid receptor 2 (HCA2 ), and it is widely used in treating dyslipidaemias. Since its side effects include skin dryness, whereas its deficiency can be accompanied by dyssebacia, characterized by sebaceous gland enlargement, we asked if HCA2 is expressed on human sebocytes, and if NA influences sebocyte functions. By using human immortalized SZ95 sebocytes, we found that non-cytotoxic (≤100 µmol/L; MTT-assay) concentrations of NA had no effect on the homeostatic sebaceous lipogenesis (SLG; Nile Red), but normalized excessive, acne-mimicking SLG induced by several lipogenic agents (arachidonic acid, anandamide, linoleic acid + testosterone; Nile Red; 48-hr treatments). Moreover, it exerted significant anti-proliferative actions (CyQUANT-assay), and increased [Ca2+ ]IC (Fluo-4 AM-based Ca2+ -measurement). Although NA did not prevent the lipopolysaccharide-induced pro-inflammatory response (up-regulation [Q-PCR] and release [ELISA] of several pro-inflammatory cytokines) of the sebocytes, collectively, these data support the concept that NA may be effective in suppressing sebum production in vivo. While exploring the mechanism of the sebostatic actions, we found that sebocytes express HCA2 (Q-PCR, immunofluorescent labelling), siRNA-mediated silencing of which prevented the NA-induced Ca2+ -signal and the lipostatic action. Collectively, our data introduce NA, and HCA2 activators in general, as novel, potent and most likely safe sebostatic agents, with possible anti-acne potential.
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Acne Vulgar/genética , Adenilil Ciclases/genética , Lipogênese/efeitos dos fármacos , Niacina/farmacologia , Glândulas Sebáceas/efeitos dos fármacos , Acne Vulgar/induzido quimicamente , Acne Vulgar/patologia , Ácido Araquidônico/farmacologia , Linhagem Celular , Citocinas/metabolismo , Dislipidemias/tratamento farmacológico , Dislipidemias/patologia , Humanos , Lipogênese/genética , Niacina/efeitos adversos , Niacina/genética , RNA Interferente Pequeno/genética , Glândulas Sebáceas/patologiaRESUMO
Dimethyl fumarate (DMF) was recently approved by the FDA for the treatment of relapsing remitting MS. The pathology of MS is a result of both immune dysregulation and oxidative stress induced damage, and DMF is believed to have therapeutic effects on both of these processes. However, the mechanisms of action of DMF are not fully understood. To determine if DMF is able to activate signaling cascades that affect immune dysregulation, we treated human peripheral blood mononuclear cells with DMF. We discovered that DMF stimulates cyclic adenosine monophosphate (cAMP) production after 1 min treatment in vitro. cAMP is a small molecule second messenger that has been shown to modulate immune response. Using pharmacological inhibitors, we determined that adenylyl cyclase mediates DMF induced cAMP production; DMF activated the prostaglandin EP2 receptor to produce cAMP. This response was not due to increased endogenous production of prostaglandin E2 (PGE2), but was enhanced by addition of exogenous PGE2. Furthermore, we determined that the bioactive metabolite of DMF, monomethyl fumarate (MMF), also stimulates cAMP production. These novel findings suggest that DMF may provide protection against MS by inhibiting immune cell function via the cAMP signaling pathway.
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AMP Cíclico/imunologia , Fumarato de Dimetilo/farmacologia , Imunossupressores/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP2/imunologia , Transdução de Sinais/efeitos dos fármacos , Adenilil Ciclases/imunologia , Dinoprostona/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologiaRESUMO
Osteoarthritis (OA) is widely recognized as the prevailing joint disease associated with aging. The ketogenic diet (KD) has been postulated to impede the advancement of various inflammatory ailments. ß-Hydroxybutyrate (ßOHB), a prominent constituent of ketone bodies, has recently been proposed to possess crucial signaling capabilities. In this study, we propose to explore the role and mechanism of ßOHB in OA. Tissue staining and inflammatory factor assay were employed to evaluate the impacts of KD and ßOHB on OA rats. The oxidative stress conditions in chondrocytes were induced using tert-butyl hydroperoxide (TBHP). The mechanisms were determined using the siRNA of hydroxycarboxylic acid receptor 2 (HCAR2), the antagonist of adenosine monophosphate-activated protein kinase (AMPK), and the inhibitor of mitophagy. The administration of KD demonstrated a reduction in pathological damage to cartilage, as well as a decrease in plasma levels of inflammatory factors. Furthermore, it resulted in an increase in the concentration of ßOHB in the blood and synovial fluid. In vitro experiments showed that ßOHB facilitated mitophagy and adenosine triphosphate production. Besides, ßOHB mitigated chondrocyte senescence, inflammatory factors secretion, extracellular matrix degradation, and apoptosis induced by TBHP. Subsequent investigations indicated that the protective effects of ßOHB were no longer observed following the knockdown of HCAR2, the antagonist of AMPK, or the inhibitor of mitophagy. Moreover, in vivo studies suggested that ßOHB played a protective role by targeting the HCAR2-AMPK-PINK1 axis. In conclusion, ßOHB enhanced chondrocyte mitophagy through the HCAR2/AMPK/PINK1/Parkin pathway, offering a potential therapeutic approach for the treatment of OA.
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Following insults or injury, microglia cells are activated contributing to the cytotoxic response or by promoting an immune-mediated damage resolution. Microglia cells express HCA2R, a hydroxy carboxylic acid (HCA) receptor, which has been shown to mediate neuroprotective and anti-inflammatory effects. In this study we found that HCAR2 expression levels were increased in cultured rat microglia cells after Lipopolysaccharide (LPS) exposure. In a similar fashion, the treatment with MK 1903, a potent full agonist of HCAR2, increased the receptor protein levels. Moreover, HCAR2 stimulation prevented i) cells viability ii) morphological activation iii) pro/anti-inflammatory mediators production in LPS-treated cells. Likewise, HCAR2 stimulation reduced the proinflammatory mediators mRNA expression induced by neuronal chemokine fractalkine (FKN), a neuronal derived chemokine activating its unique receptor, chemokine receptor 1 (CX3CR1) on microglia surface. Interestingly, electrophysiological recordings in vivo revealed that MK1903 was able to prevent the increase of the nociceptive neurons (NS) firing activity mediated by the spinal FKN application in healthy rats. Collectively, our data demonstrate that HCAR2 is functionally expressed in microglia, by showing its capability to shift microglia toward an anti-inflammatory phenotype. Moreover, we indicated the contribute of HCAR2 in the FKN signaling and suggested a possible HCAR2/CX3CR1 functional interaction. This study paves the way for further investigations aimed at understanding the role HCAR2 as potential target in neuroinflammation-based CNS disorders. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".
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Quimiocinas CXC , Microglia , Ratos , Animais , Quimiocinas CXC/metabolismo , Quimiocinas CXC/farmacologia , Lipopolissacarídeos/farmacologia , Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CX3CL1/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismoRESUMO
Monomethyl fumarate (MMF), metabolite of dimethyl fumarate (DMF), an immunosuppressive drug approved for the treatment of multiple sclerosis (MS), is a potent agonist for hydroxycarboxylic acid receptor 2 (HCAR2), eliciting signals that dampen cell activation or lead to inflammation such as the skin flushing reaction that is one of the main side effects of the treatment, together with gastrointestinal inflammation. Our aim is to further understand the molecular basis underlying these differential effects of the drug. We have used wild-type and HCAR2 knock-out mice to investigate, in vitro and ex vivo under steady-state and pathological conditions, the HCAR2-mediated signaling pathways activated by MMF in dendritic cells (DC), which promote differentiation of T cells, and in intestinal epithelial cells (IEC) where activation of a pro-inflammatory pathway, such as the cyclooxygenase-2 pathway involved in skin flushing, could underlie gastrointestinal side effects of the drug. To understand how DMF treatment might impact on gut inflammation induced by experimental autoimmune encephalomyelitis (EAE), the animal model for MS, we have used 3D X-ray phase contrast tomography and flow cytometry to monitor possible intestinal alterations at morphological and immunological levels, respectively. We show that HCAR2 is a pleiotropically linked receptor for MMF, mediating activation of different pathways leading to different outcomes in different cell types, depending on experimental in-vitro and in-vivo conditions. In the small intestine of EAE-affected mice, DMF treatment affected migration of tolerogenic DC from lamina propria to mesenteric lymph nodes, and/or reverted their profile to pro-inflammatory, probably as a result of reduced expression of aldehyde dehydrogenase and transforming growth factor beta as well as the inflammatory environment. Nevertheless, DMF treatment did not amplify the morphological alterations induced by EAE. On the basis of our further understanding of MMF signaling through HCAR2, we suggest that the pleiotropic signaling of fumarate via HCAR2 should be addressed for its pharmaceutical relevance in devising new lead compounds with reduced inflammatory side effects.
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Fumaratos/farmacologia , Imunossupressores/farmacologia , Esclerose Múltipla/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Biomarcadores , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental , Imuno-Histoquímica , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Pattern recognition receptors (PRRs) can recognize pathogen-associated molecular patterns and activate downstream signalling pathways, resulting in modulation of host immunity against pathogens. Here, we investigated whether PRR-mediated recognition is involved in host immune responses to the blood-feeding nematode Haemonchus contortus. METHODS: During blood-feeding, H. contortus secretes immune-modulating antigens into host blood. Therefore, we stimulated sheep peripheral blood mononuclear cells (PBMCs) with H. contortus soluble extract (HcAg) and performed transcriptional profiling. RESULTS: HcAg upregulated two genetically linked CLRs (CLEC2L and KLRG2), two NLRs attenuating inflammation (NLRP12 and NLRC3) and one G protein-coupled receptor with potent anti-inflammatory effects (HCAR2). Furthermore, several Th2-related transcription factors (ATF3, IRF4, BCL3 and NFATC) were also upregulated, which may confer anti-inflammatory type 2 immune responses to HcAg. CONCLUSIONS: Together, our preliminary studies provide new insights into how the host innate immune system controls type 2 immunity to H. contortus. Further work will be needed to identify H. contortus products recognized by the host innate immune system and determine the Th2 polarization ability of these putative PRR ligands.
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Hemoncose/veterinária , Haemonchus/química , Proteínas de Helminto/farmacologia , Imunidade Inata , Leucócitos Mononucleares/imunologia , Extratos de Tecidos/farmacologia , Animais , Antígenos de Helmintos/imunologia , Perfilação da Expressão Gênica , Hemoncose/sangue , Proteínas de Helminto/imunologia , Interações Hospedeiro-Patógeno , Leucócitos Mononucleares/parasitologia , Proteínas NLR/genética , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/parasitologia , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: The mechanisms of action of dimethyl fumarate (DMF), and its metabolite, monomethyl fumarate (MMF), for the treatment of multiple sclerosis are not completely elucidated. OBJECTIVES: To discuss the role of DMF/MMF-induced hydroxycarboxylic acid receptor 2 (HCA2/GPR109A) pathway activation in the immune response and treatment of MS. METHODS: A narrative (traditional) review of the current literature. RESULTS: Studies have shown that binding of DMF/MMF to HCA2 on dendritic cells inhibits the production of pro-inflammatory cytokines in vitro and in MS murine models. Evidence suggests that activation of HCA2 expressed in immune cells and gut epithelial cells by DMF/MMF, may induce anti-inflammatory responses in the intestinal mucosa. CONCLUSION: Although the DMF/MMF mechanism of action remains unclear, evidence suggests that the activation of HCA2/GPR109A pathway downregulates the immune response and may activate anti-inflammatory response in the intestinal mucosa, possibly leading to reduction in CNS tissue damage in MS patients.