Search Results (42)

Porcine circovirus type 2 (PCV2) is a globally prevalent swine pathogen that induces immunosuppression, predisposing pigs to subclinical infections. In intensive farming systems, PCV2 persistently impairs growth performance and vaccine efficacy, leading to substantial economic losses in the swine industry. Emerging evidence suggests that certain viruses exploit Suppressor of Cytokine Signaling 3 (SOCS3), a key immune checkpoint protein, to subvert host innate immunity by suppressing cytokine signaling. While SOCS3 has been implicated in various viral infections, its regulatory role in PCV2 replication remains undefined. This study aims to elucidate the mechanisms underlying the interplay between SOCS3 and PCV2 during viral pathogenesis. Porcine SOCS3 was amplified using RT-PCR and stably overexpressed in PK-15 cells through lentiviral delivery. Bioinformatics analysis facilitated the design of three siRNA candidates targeting SOCS3. We systematically investigated the effects of SOCS3 overexpression and knockdown on PCV2 replication kinetics and host antiviral responses by quantifying the viral DNA load and the mRNA levels of cytokines. PCV2 infection upregulated SOCS3 expression at both transcriptional and translational levels in PK-15 cells. Functional studies revealed that SOCS3 overexpression markedly enhanced viral replication, whereas its knockdown suppressed viral proliferation. Intriguingly, SOCS3-mediated immune modulation exhibited a divergent regulation of antiviral cytokines: PCV2-infected SOCS3-overexpressing cells showed elevated IFN-β but suppressed TNF-α expressions, whereas SOCS3 silencing conversely downregulated IFN-β while amplifying TNF-α responses. This study unveils a dual role of SOCS3 during subclinical porcine circovirus type 2 (PCV2) infection: it functions as a host-derived pro-viral factor that facilitates viral replication while simultaneously reshaping the cytokine milieu to suppress overt inflammatory responses. These findings provide novel insights into the mechanisms underlying PCV2 immune evasion and persistence and establish a theoretical framework for the development of host-targeted control strategies. Although our results identify SOCS3 as a key host determinant of PCV2 persistence, the precise molecular pathways involved require rigorous experimental validation. Full article

Acute pancreatitis (AP) is an inflammatory disease initiated by the death of exocrine acinar cells, but its pathogenesis remains unclear. Signal transducer and activator of transcription 3 (STAT3) is a multifunctional factor that regulates immunity and the inflammatory response. The protective role of STAT3 is reported in Coxsackievirus B3 (CVB3)-induced cardiac fibrosis, yet the exact role of STAT3 in modulating viral-induced STAT1 activation and type I interferon (IFN)-stimulated gene (ISG) transcription in the pancreas remains unclarified. In this study, we tested whether STAT3 regulated viral-induced STAT1 translocation. We found that CVB3, particularly capsid VP1 protein, markedly upregulated the phosphorylation and nuclear import of STAT3 (p-STAT3) while it significantly impeded the nuclear translocation of p-STAT1 in the pancreases and hearts of mice on day 3 postinfection (p.i.). Immunoblotting and an immunofluorescent assay demonstrated the increased expression and nuclear translocation of p-STAT3 but a blunted p-STAT1 nuclear translocation in CVB3-infected acinar 266-6 cells. STAT3 shRNA knockdown or STAT3 inhibitors reduced viral replication via the rescue of STAT1 nuclear translocation and increasing the ISRE activity and ISG transcription in vitro. The knockdown of STAT1 blocked the antiviral effect of the STAT3 inhibitor. STAT3 inhibits STAT1 activation by virally inducing a potent inhibitor of IFN signaling, the suppressor of cytokine signaling-3 ((SOCS)-3). Sustained pSTAT1 and the elevated expression of ISGs were induced in SOCS3 knockdown cells. The in vivo administration of HJC0152, a pharmaceutical STAT3 inhibitor, mitigated the viral-induced AP and myocarditis pathology via increasing the IFNβ as well as ISG expression on day 3 p.i. and reducing the viral load in multi-organs. These findings define STAT3 as a negative regulator of the type I IFN response via impeding the nuclear STAT1 translocation that otherwise triggers ISG induction in infected pancreases and hearts. Our findings identify STAT3 as an antagonizing factor of the IFN-STAT1 signaling pathway and provide a potential therapeutic target for viral-induced AP and myocarditis. Full article

Obesity and metabolic syndrome alter serum lipid profiles. They also increase vulnerability to viral infections and worsen the survival rate and symptoms after infection. How serum lipids affect influenza virus proliferation is unclear. Here, we investigated the effects of lysophosphatidylcholines on influenza A virus (IAV) proliferation. IAV particles in the culture medium were titrated using extraction-free quantitative PCR, and viral RNA and protein levels were assessed using real-time PCR and Western blot, respectively. RNA sequencing data were analyzed using PCA and heatmap analysis, and pathway analysis was performed using the KEGG mapper and PathIN tools. Statistical analysis was conducted using SPSS21.0. LPC treatment of THP-1 cells significantly increased IAV proliferation and IAV RNA and protein levels, and saturated LPC was more active in IAV RNA expression than unsaturated LPC was. The functional analysis of genes affected by LPCs showed that the expression of genes involved in IAV signaling, such as suppressor of cytokine signaling 3 (SOCS3), phosphoinositide-3-kinase regulatory subunit 3 (PI3K) and AKT serine/threonine kinase 3 (AKT3), Toll-like receptor 7 (TKR7), and interferon gamma receptor 1 (IFNGR1), was changed by LPC. Altered influenza A pathways were linked with MAPK and PI3K/AKT signaling. Treatment with inhibitors of MAPK or PI3K attenuated viral gene expression changes induced by LPCs. The present study shows that LPCs stimulated virus reproduction by modifying the cellular environment to one in which viruses proliferated better. This was mediated by the MAPK, JNK, and PI3K/AKT pathways. Further animal studies are needed to confirm the link between LPCs from serum or the respiratory system and IAV proliferation. Full article

Leptin regulates lipid metabolism, maximizing insulin sensitivity; however, peripheral leptin resistance is not fully understood, and its contribution to metabolic dysfunction-associated steatotic liver disease (MASLD) is unclear. This study evaluated the contribution of the leptin axis to MASLD in humans. Forty-three participants, mostly female (86.04%), who underwent cholecystectomy were biopsied. Of the participants, 24 were healthy controls, 8 had MASLD, and 11 had metabolic dysfunction-associated steatohepatitis (MASH). Clinical and biochemical data and the gene expression of leptin, leptin receptor (LEPR), suppressor of cytokine signaling 3 (SOCS3), sterol regulatory element-binding transcription factor 1 (SREBF1), stearoyl-CoA desaturase-1 (SCD1), and patatin-like phospholipase domain-containing protein 2 (PNPLA2), were determined from liver and adipose tissue. Higher serum leptin and LEPR levels in the omental adipose tissue (OAT) and liver with MASH were found. In the liver, LEPR was positively correlated with leptin expression in adipose tissue, and SOCS3 was correlated with SREBF1-SCD1. In OAT, SOCS3 was correlated with insulin resistance and transaminase enzymes (p < 0.05 for all. In conclusion, we evidenced the correlation between the peripheral leptin resistance axis in OAT–liver crosstalk and the complications of MASLD in humans. Full article

Fluid overload in hemodialysis patients (HD) has been proven to be associated with inflammation. Elevated levels of the pro-inflammatory cytokine interleukin-6 (IL-6) appear to be inadequately counterbalanced by the anti-inflammatory cytokine interleukin-10 (IL-10). We initiated a cross-sectional study enrolling 40 HD patients who were categorized by a bioimpedance measurement in normovolemic (N; 23) and hypervolemic (H; 17) groups to test whether IL-10- and IL-6-related signal transduction pathways (signal transducer of transcript 3: STAT3) and/or a post-transcriptional regulating mechanism (miR-142) are impaired by hypervolemia. IL-10/IL-6 transcript and protein production by PBMCs (peripheral blood mononuclear cells) were determined. Phospho-flow cytometry was used to detect the phosphorylated forms of STAT3 (pY705 and pS727). miR-142-3p/5p levels were detected by qPCR. Hypervolemic patients were older, more frequently had diabetes, and showed higher CRP levels. IL-10 transcripts were elevated in H patients but not IL-10 protein levels. In spite of the elevated mRNA expression of the suppressor of cytokine expression 3 (SOCS3), IL-6 mRNA and protein expression were increased in immune cells of H patients. The percentage of cells staining positive for STAT3 (pY705) were comparable in both groups; in STAT3 (pS727), however, the signal needed for full transactivation was decreased in H patients. miR-142-3p, a proven target of IL-10 and IL-6, was significantly elevated in H patients. Insufficient phosphorylation of STAT3 may impair inflammatory and anti-inflammatory cytokine signaling. How far degradative mechanisms induced by elevated miR-142-3p levels contribute to an inefficient anti-inflammatory IL-10 signaling remains elusive. Full article

Epigenetic modifications, including aberrant DNA methylation occurring at the promoters of oncogenes and oncosuppressor genes and histone modifications, can contribute to carcinogenesis. Aberrant methylation mediated by histone methylatransferases, alongside histones, can affect methylation of proteins involved in the regulation of pro-survival pathways such as JAK/STAT and contribute to their activation. In this study, we used DNA or histone demethylating agents, 5-Azacytidine (5-AZA) or DS-3201 (valemetostat), respectively, to treat primary effusion lymphoma (PEL) cells, alone or in combination with AG490, a Signal transducer and activator of transcription 3 (STAT3) inhibitor. Cell viability was investigated by trypan blue assay and FACS analysis. The molecular changes induced by 5-AZA and/or AG490 treatments were investigated by Western blot analysis, while cytokine release by PEL cells treated by these drugs was evaluated by Luminex. Statistical analyses were performed with Graphpad Prism^® software (version 9) and analyzed by Student’s t test or a nonparametric one-way ANOVA test. The results obtained in this study suggest that 5-AZA upregulated molecules that inhibit STAT3 tyrosine phosphorylation, namely Suppressor of Cytokine Signaling 3 (SOCS3) and tyrosine–protein phosphatase non-receptor type (PTPN) 6/Src homology region 2 domain-containing phosphatase-1 (SHP-1), reducing STAT3 activation and downregulating several STAT3 pro-survival targets in PEL cells. As this lymphoma is highly dependent on the constitutive activation of STAT3, 5-AZA impaired PEL cell survival, and when used in combination with AG490 JAK2/STAT3 inhibitor, it potentiated its cytotoxic effect. Differently from 5-AZA, the inhibition of the EZH1/2 histone methyltransferase by DS-3201, reported to contribute to STAT3 activation in other cancers, slightly affected STAT3 phosphorylation or survival in PEL cells, either alone or in combination with AG490. This study suggests that 5-AZA, by upregulating the expression level of SOCS3 and PTPN6/SHP1, reduced STAT3 activation and improved the outcome of treatment targeting this transcription factor in PEL cells. Full article

This study aimed to identify and evaluate drug candidates targeting the kinase inhibitory region of suppressor of cytokine signaling (SOCS) 3 for the treatment of allergic rhinitis (AR). Utilizing an artificial intelligence (AI)-based new drug development platform, virtual screening was conducted to identify compounds inhibiting the SH2 domain binding of SOCS3. Luminescence assays assessed the ability of these compounds to restore JAK-2 activity diminished by SOCS3. Jurkat T and BEAS-2B cells were utilized to investigate changes in SOCS3 and STAT3 expression, along with STAT3 phosphorylation in response to the identified compounds. In an OVA-induced allergic rhinitis mouse model, we measured serum levels of total IgE and OVA-specific IgE, performed real-time PCR on nasal mucosa samples to quantify Th2 cytokines and IFN-γ expression, and conducted immunohistochemistry to analyze eosinophil levels. Screening identified 20 hit compounds with robust binding affinities. As the concentration of SOCS3 increased, a corresponding decrease in JAK2 activity was observed. Compounds 5 and 8 exhibited significant efficacy in restoring JAK2 activity without toxicity. Treatment with these compounds resulted in reduced SOCS3 expression and the reinstatement of STAT3 phosphorylation in Jurkat T and BEAS-2B cells. In the OVA-induced allergic rhinitis mouse model, compounds 5 and 8 effectively alleviated nasal symptoms and demonstrated lower levels of immune markers compared to the allergy group. This study underscores the promising nonclinical efficacy of compounds identified through the AI-based drug development platform. These findings introduce innovative strategies for the treatment of AR and highlight the potential therapeutic value of targeting SOCS3 in managing AR. Full article

Depression is linked to changes in GABAergic inhibitory neurons, especially parvalbumin (PV) interneurons, which are susceptible to redox dysregulation. Olanzapine (Olz) is an atypical antipsychotic whose mode of action remains unclear. We determined the effect of Olz on PV-positive (+) and glutamate decarboxylase 67 (GAD67) + cell numbers in the layers of dorsal hippocampus (dHIPP) cornu ammonis (CA1–CA3) and dentate gyrus (DG) subregions in rats exposed to chronic social isolation (CSIS), which is an animal model of depression. Antioxidative enzymes and proinflammatory cytokine levels were also examined. CSIS decreased the PV+ cell numbers in the Stratum Oriens (SO) and Stratum Pyramidale (SP) of dCA1 and dDG. It increased interleukin-6 (IL-6), suppressor of cytokine signaling 3 (SOCS3), and copper–zinc superoxide dismutase (CuZnSOD) levels, and it decreased catalase (CAT) protein levels. Olz in CSIS increased the number of GAD67+ cells in the SO and SP layers of dCA1 with no effect on PV+ cells. It reduced the PV+ and GAD67+ cell numbers in the Stratum Radiatum of dCA3 in CSIS. Olz antagonizes the CSIS-induced increase in CuZnSOD, CAT and SOCS3 protein levels with no effect on IL-6. Data suggest that the protective Olz effects in CSIS may be mediated by altering the number of PV+ and GAD67+ cells in dHIPP subregional layers. Full article

Hydrogen sulfide (H₂S) emerged recently as an anti-oxidative signaling molecule that contributes to gastrointestinal (GI) mucosal defense and repair. Indomethacin belongs to the class of non-steroidal anti-inflammatory drugs (NSAIDs) and is used as an effective intervention in the treatment of gout- or osteoarthritis-related inflammation. However, its clinical use is strongly limited since indomethacin inhibits gastric mucosal prostaglandin (PG) biosynthesis, predisposing to or even inducing ulcerogenesis. The H₂S moiety was shown to decrease the GI toxicity of some NSAIDs. However, the GI safety and anti-oxidative effect of a novel H₂S-releasing indomethacin derivative (ATB-344) remain unexplored. Thus, we aimed here to compare the impact of ATB-344 and classic indomethacin on gastric mucosal integrity and their ability to counteract the development of oxidative gastric mucosal injuries. Wistar rats were pretreated intragastrically (i.g.) with vehicle, ATB-344 (7–28 mg/kg i.g.), or indomethacin (5–20 mg/kg i.g.). Next, animals were exposed to microsurgical gastric ischemia-reperfusion (I/R). Gastric damage was assessed micro- and macroscopically. The volatile H₂S level was assessed in the gastric mucosa using the modified methylene blue method. Serum and gastric mucosal PGE₂ and 8-hydroxyguanozine (8-OHG) concentrations were evaluated by ELISA. Molecular alterations for gastric mucosal barrier-specific targets such as cyclooxygenase-1 (COX)-1, COX-2, heme oxygenase-1 (HMOX)-1, HMOX-2, superoxide dismutase-1 (SOD)-1, SOD-2, hypoxia inducible factor (HIF)-1α, xanthine oxidase (XDH), suppressor of cytokine signaling 3 (SOCS3), CCAAT enhancer binding protein (C/EBP), annexin A1 (ANXA1), interleukin 1 beta (IL-1β), interleukin 1 receptor type I (IL-1R1), interleukin 1 receptor type II (IL-1R2), inducible nitric oxide synthase (iNOS), tumor necrosis factor receptor 2 (TNFR2), or H₂S-producing enzymes, cystathionine γ-lyase (CTH), cystathionine β-synthase (CBS), or 3-mercaptopyruvate sulfur transferase (MPST), were assessed at the mRNA level by real-time PCR. ATB-344 (7 mg/kg i.g.) reduced the area of gastric I/R injuries in contrast to an equimolar dose of indomethacin. ATB-344 increased gastric H₂S production, did not affect gastric mucosal PGE₂ content, prevented RNA oxidation, and maintained or enhanced the expression of oxidation-sensitive HMOX-1 and SOD-2 in line with decreased IL-1β and XDH. We conclude that due to the H₂S-releasing ability, i.g., treatment with ATB-344 not only exerts dose-dependent GI safety but even enhances gastric mucosal barrier capacity to counteract acute oxidative injury development when applied at a low dose of 7 mg/kg, in contrast to classic indomethacin. ATB-344 (7 mg/kg) inhibited COX activity on a systemic level but did not affect cytoprotective PGE₂ content in the gastric mucosa and, as a result, evoked gastroprotection against oxidative damage. Full article

Scar formation resulting from overly active wound healing is a critical factor in the success rate of glaucoma filtration surgery (GFS). IL-6 and TGF-β have been implicated in the pathogenesis of fibrogenesis. In addition, the signal transducer and activator of transcription 3 (STAT3) can be activated by numerous cytokines and growth factors, including IL-6 and TGF-β1. Thus, STAT3 activation may integrate common profibrotic pathways to promote fibrosis. In this study, an increase in p-STAT3 was observed in activated HTFs. Inhibiting STAT3 in cultured HTFs by pharmacological inactivation reversed the fibrotic responses, such as fibroblast migration, the differentiation of resting fibroblasts into myofibroblasts and the deposition of ECM, mediated by IL-6 and TGF-β1. Moreover, the expression of suppressor of cytokine signaling 3 (SOCS3) was decreased in HTFs cultured with IL-6 and TGF-β1, and SOCS3 overexpression rescued ECM deposition, α-SMA expression and migration in IL-6- and TGF-β1-stimulated HTFs by inactivating STAT3. Finally, S3I-201 treatment inhibited profibrotic gene expression and subconjunctival fibrosis in a rat model of GFS. In conclusion, our data suggests that STAT3 plays a central role in fibrosis induced by different profibrotic pathways and that STAT3 is a potential target for antifibrotic therapies following GFS. Full article

Recent studies by us and others have shown that enhancer of zeste homolog-2 (EZH2), a histone methyltransferase, in glial cells regulates the genesis of neuropathic pain by modulating the production of proinflammatory cytokines and chemokines. In this review, we summarize recent advances in this research area. EZH2 is a subunit of polycomb repressive complex 2 (PRC2), which primarily serves as a histone methyltransferase to catalyze methylation of histone 3 on lysine 27 (H3K27), ultimately resulting in transcriptional repression. Animals with neuropathic pain exhibit increased EZH2 activity and neuroinflammation of the injured nerve, spinal cord, and anterior cingulate cortex. Inhibition of EZH2 with DZNep or GSK-126 ameliorates neuroinflammation and neuropathic pain. EZH2 protein expression increases upon activation of Toll-like receptor 4 and calcitonin gene-related peptide receptors, downregulation of miR-124-3p and miR-378 microRNAs, or upregulation of Lncenc1 and MALAT1 long noncoding RNAs. Genes suppressed by EZH2 include suppressor of cytokine signaling 3 (SOCS3), nuclear factor (erythroid-derived 2)-like-2 factor (NrF2), miR-29b-3p, miR-146a-5p, and brain-specific angiogenesis inhibitor 1 (BAI1). Pro-inflammatory mediators facilitate neuronal activation along pain-signaling pathways by sensitizing nociceptors in the periphery, as well as enhancing excitatory synaptic activities and suppressing inhibitory synaptic activities in the CNS. These studies collectively reveal that EZH2 is implicated in signaling pathways known to be key players in the process of neuroinflammation and genesis of neuropathic pain. Therefore, targeting the EZH2 signaling pathway may open a new avenue to mitigate neuroinflammation and neuropathic pain. Full article

The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. Transcriptomic analysis on LPS-activated wild-type macrophages demonstrated an alteration of several epigenetic enzymes. Although the Ezh2-silencing macrophages (RAW264.7), using small interfering RNA (siRNA), indicated a non-different response to the control cells after a single LPS stimulation, the Ezh2-reducing cells demonstrated a less severe LPS tolerance, after two LPS stimulations, as determined by the higher supernatant TNF-α. With a single LPS stimulation, Ezh2 null (Ezh2^flox/flox; LysM-Cre^cre/−) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2^fl/fl; LysM-Cre^−/−), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. On the other hand, there were similar serum cytokines after LPS tolerance and the non-reduction of serum cytokines after the second dose of LPS, indicating less severe LPS tolerance in Ezh2 null mice compared with control mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3. Full article

Heterodimeric β2 integrin surface receptors (CD11a-d/CD18) are specifically expressed by leukocytes that contribute to pathogen uptake, cell migration, immunological synapse formation and cell signaling. In humans, the loss of CD18 expression results in leukocyte adhesion deficiency syndrome (LAD-)1, largely characterized by recurrent severe infections. All available mouse models display the constitutive and ubiquitous knockout of either α or the common β2 (CD18) subunit, which hampers the analysis of the cell type-specific role of β2 integrins in vivo. To overcome this limitation, we generated a CD18 gene floxed mouse strain. Offspring generated from crossing with CD11c-Cre mice displayed the efficient knockdown of β2 integrins, specifically in dendritic cells (DCs). Stimulated β2-integrin-deficient splenic DCs showed enhanced cytokine production and the concomitantly elevated activity of signal transducers and activators of transcription (STAT) 1, 3 and 5, as well as the impaired expression of suppressor of cytokine signaling (SOCS) 2–6 as assessed in bone marrow-derived (BM) DCs. Paradoxically, these BMDCs also showed the attenuated expression of genes involved in inflammatory signaling. In line, in experimental autoimmune encephalomyelitis mice with a conditional DC-specific β2 integrin knockdown presented with a delayed onset and milder course of disease, associated with lower frequencies of T helper cell populations (Th)1/Th17 in the inflamed spinal cord. Altogether, our mouse model may prove to be a valuable tool to study the leukocyte-specific functions of β2 integrins in vivo. Full article

Leptin resistance is a hallmark of obesity. Treatments aiming to improve leptin sensitivity are considered a promising therapeutical approach against obesity. However, leptin receptor (LepR) signaling also modulates several neurovegetative aspects, such as the cardiovascular system and hepatic gluconeogenesis. Thus, we investigated the long-term consequences of increased leptin sensitivity, considering the potential beneficial and deleterious effects. To generate a mouse model with increased leptin sensitivity, the suppressor of cytokine signaling 3 (SOCS3) was ablated in LepR-expressing cells (LepR^∆SOCS3 mice). LepR^∆SOCS3 mice displayed reduced food intake, body adiposity and weight gain, as well as improved glucose tolerance and insulin sensitivity, and were protected against aging-induced leptin resistance. Surprisingly, a very high mortality rate was observed in aging LepR^∆SOCS3 mice. LepR^∆SOCS3 mice showed cardiomyocyte hypertrophy, increased myocardial fibrosis and reduced cardiovascular capacity. LepR^∆SOCS3 mice exhibited impaired post-ischemic cardiac functional recovery and middle-aged LepR^∆SOCS3 mice showed substantial arhythmic events during the post-ischemic reperfusion period. Finally, LepR^∆SOCS3 mice exhibited fasting-induced hypoglycemia and impaired counterregulatory response to glucopenia associated with reduced gluconeogenesis. In conclusion, although increased sensitivity to leptin improved the energy and glucose homeostasis of aging LepR^∆SOCS3 mice, major autonomic/neurovegetative dysfunctions compromised the health and longevity of these animals. Consequently, these potentially negative aspects need to be considered in the therapies that increase leptin sensitivity chronically. Full article

Maternal obesity is associated with inflammation and oxidative stress, strongly impacting the intrauterine environment with detrimental consequences for both mother and offspring. The saliva is a non-invasive biofluid reflecting both local and systemic health status. This observational study aimed to profile the epigenetic signature in the saliva of Obese (OB) and Normal-Weight (NW) pregnant women. Sixteen NW and sixteen OB Caucasian women with singleton spontaneous pregnancies were enrolled. microRNAs were quantified by the OpenArray Platform. The promoter region methylation of Suppressor of Cytokine Signaling 3 (SOCS3) and Transforming Growth Factor Beta 1 (TGF-Beta1) was assessed by pyrosequencing. There were 754 microRNAs evaluated: 20 microRNAs resulted in being differentially expressed between OB and NW. microRNA pathway enrichment analysis showed a significant association with the TGF-Beta signaling pathway (miTALOS) and with fatty acids biosynthesis/metabolism, lysine degradation, and ECM–receptor interaction pathways (DIANA–miRPath). Both SOCS3 and TGF-Beta1 were significantly down-methylated in OB vs. NW. These results help to clarify impaired mechanisms involved in obesity and pave the way for the understanding of specific damaged pathways. The characterization of the epigenetic profile in saliva of pregnant women can represent a promising tool for the identification of obesity-related altered mechanisms and of possible biomarkers for early diagnosis and treatment of pregnancy-adverse conditions. Full article