Search Results (44)

Polycystic kidney disease (PKD), the most common inherited kidney disorder, is characterized by progressive cyst growth and eventual organ failure. Although aberrant innate immune activation is a recognized contributor to PKD progression, the underlying molecular mechanisms remain incompletely defined. Here, we showed that Pkd1 deletion increased TLR2 and MyD88 mRNA expression in renal epithelial cells, indicating enhanced innate immune priming. In vivo, administration of Pam3CSK4 (PAM), a synthetic TLR2 agonist, preferentially amplified pro-inflammatory and pro-fibrotic responses in Pkd1^RC/RC mice compared with wild-type controls, despite inducing similar signaling responses in vitro. Acute PAM treatment for one week rapidly enhanced NF-κB activation in cyst-lining epithelial cells, increased renal inflammation and cell proliferation, and was associated with activation of mTOR signaling and upregulation of c-Myc and Wnt proteins. Sustained PAM treatment further accelerated cyst expansion and renal fibrosis in PKD mice. Importantly, the endogenous TLR2 ligands decorin and biglycan were markedly elevated in human PKD kidneys, supporting the translational relevance of enhanced TLR2 signaling in disease progression. Together, these findings suggest that TLR2 signaling is an important contributor to PKD progression and a potential therapeutic target. Full article

The oil extract derived from black soldier fly (Hermetia illucens) larvae (BSFL) is characterized by a distinctive fatty acid composition and bioactive compounds with demonstrated anti-inflammatory properties, as shown in our previous work. The present study aims to mechanistically explore the immunomodulatory effects of a saponified form of BSFL oil (MBSFL) and its potential interaction with metabolic signaling pathways. Using Pam3CSK4-polarized M1 primary human peripheral blood mononuclear cells (PBMCs), we demonstrate that MBSFL phenotypically suppressed the secretion of pro-inflammatory cytokines TNFα, IL-6, IL-17, and GM-CSF (p < 0.01) without altering anti-inflammatory cytokine levels (TGFβ1, IL-13, and IL-4). A phosphoproteomic analysis of Pam3CSK4-stimulated THP-1 macrophages revealed MBSFL-mediated downregulation of CK2 and ERK kinases (p < 0.05), key regulators of NF-κB signaling activation. We confirmed that MBSFL directly inhibits NF-κB p65 nuclear translocation (p < 0.05), using both immunofluorescence staining and a western blot analysis of nuclear and cytoplasmic fractions. In the context of metabolism, using a luciferase reporter assay, we demonstrate that MBSFL functions as a weak agonist of PPARγ and PPARδ (p < 0.05), which are nuclear receptors involved in lipid metabolism and immune regulation. However, subsequent immunoblotting revealed a macrophage polarization-dependent regulation: MBSFL upregulated PPARγ in M0 macrophages but did not prevent its suppression upon Pam3CSK4 stimulation, whereas it specifically enhanced PPARδ expression during M1 polarization (p < 0.05). This study provides novel experimental evidence supporting our hypothesis of MBSFL’s role in immunometabolism. We demonstrate for the first time that MBSFL acts as a dual regulator by suppressing NF-κB-mediated inflammation while promoting PPARδ activity—an inverse relationship with potential relevance to immunometabolic disorders. Full article

Background: Peptide amphiphile micelles (PAMs) are a promising lipid-based nanotechnology currently in development for a variety of applications ranging from atherosclerosis to cancer therapy. Especially relevant for immune applications, PAMs improve trafficking through lymphatic vessels, enhance uptake by antigen-presenting cells, and inhibit the protease-mediated degradation of cargo. However, the creation of the peptide amphiphiles (PAs) necessary to induce micellization often requires modifying an immunotarget peptide with non-native moieties, which can induce the production of off-target antibodies. Methods: PAs containing different linkers between the antigen and non-native flanking regions were synthesized and physically characterized. BALB/c mice were then subcutaneously immunized on days 0 and 14 with these formulations and ELISAs were conducted on the sera collected from vaccinated mice on day 35 to evaluate antibody responses. Results: We determined that Palm₂K-M2_2–16-(KE)₄ PAMs elicited off-target antibody responses and sought to avoid these unintended responses by adding linkers in between the M2_2–16 antigen and the non-native flanking regions (i.e., Palm₂K- and -(KE)₄) of the PA. Most significantly, the addition of diproline linkers on either side of the M2_2–16 antigen conferred a loss of β-sheet structure, whereas changing the method of lipid attachment from Palm₂K- to Pam₂CS-induced the formation of primarily spherical micelles compared to a mixture of spherical and short cylindrical micelles. Despite these morphological changes, all linker-containing PAMs still induced the production of off-target antibodies. Excitingly, however, the formulation containing a Pam₂CS moiety (intended to mimic the adjuvanticity of the TLR2 agonist adjuvant Pam₂CSK₄) elicited high on-target antibody titers similar to those induced by PAMs co-delivered with Pam₂CSK₄. Conclusions: While the linkers tested did not completely eliminate the production of off-target antibodies elicited by the PAMs, the inclusion of a Pam₂CS moiety both increased the amount of on-target antibodies and improved the ratio of on-target to off-target antibodies in response to the M2_2–16 vaccine. Full article

IgE may lead to life-threatening anaphylaxis. Currently, no satisfactory treatment to inhibit IgE production exists. This study aims to explore the anti-IgE effect of berberine (BBR) and possible mechanisms using human tonsil cells. Tonsil cells were treated with BBR at different doses following stimulation with anti-CD40/IL4 alone or in combination with poly I:C and Pam3CSK4 for 10 or 4 days. IgE and IgG levels were determined by ELISA and cell viability by trypan blue exclusion. Gene expression was analyzed by qRT-PCR and affinity binding assay was performed by chromatin immunoprecipitation assay (ChIP). BBR showed dose-dependent inhibition of IgE production following anti-CD40/IL4 stimulation without affecting cell viability and IgG levels. BBR (10 µg/mL) completely inhibited IgE production by B cells stimulated with anti-CD40/IL4 in combination with vaccine adjuvants—poly I:C and Pam3CSK4 without affecting IgG levels and cell viability. BBR inhibited IgE heavy chain (IgEh), epsilon germline-transcript (εGLT), STAT6, and NFκB1 and enhanced IFN-γ, NFκB1A, and BCL6 gene expression. ChIP assay showed that BBR inhibited STAT6 binding in the IgEh promoter region by enhancing BCL6 binding. This study shows BBR regulates IgE in human tonsil cells by inhibiting STAT6 binding through BCL6 at the IgEh promoter showing its potential for treating IgE-mediated allergies. Full article

Oligosaccharides and glycoconjugates play essential roles in various biological processes such as cellular recognition and signaling, and thus have attracted tremendous attention in the synthetic and biological communities over the past few decades. Contributing to this field, we have achieved the synthesis of the aminoxyglycoside pentasaccharide subunit of Pseudomonas aeruginosa polysaccharide synthesis locus (Psl) exopolysaccharide through an efficient 23 step process. This pentasaccharide was designed with an aminooxy derivative at the reducing end, which was used in a 2-step oxime-based bioconjugation to the protein carrier CRM197, with an epitope ratio of 1:4. The conjugate vaccine could generate anti-Psl antibodies that could recognize P. aeruginosa PAO1 bacteria and initiate opsonophagocytic killing of the bacteria. In addition, the aminoxyglycoside could be conveniently conjugated to a bifunctional aldehyde-biotin reagent, which can be used for quantifying antibody titers in vaccination studies. Full article

(1) Background: Toll-like receptor 2 (TLR2) on platelets is increasingly recognized as a pivotal mediator in infection-induced platelet activation and aggregation, contributing to both inflammatory and thrombotic diseases. Targeting TLR2 on platelets offers a promising therapeutic strategy for inflammatory and thrombotic-related disorders. However, inhibitors targeting platelet TLR2 have not yet been reported. (2) Methods: Platelet aggregation was assessed using a light transmission aggregometer. Platelet activation was evaluated by measuring the release of P-selectin and von Willebrand factor (vWF) via ELISA. Intracellular Ca²⁺ mobilization was quantified using Fluo 3-AM fluorescence, recorded by flow cytometry. Static platelet adhesion was visualized under a microscope, and the formation of platelet–granulocyte aggregates in human whole blood was analyzed by flow cytometry. (3) Results: Fucosylated glycosaminoglycan (FG) tetradecasaccharide HS14 inhibited the activation and aggregation of human platelets induced by the synthetic bacterial lipopeptide Pam3CSK4 in a concentration-dependent manner. This inhibitory effect gives rise to significant anti-inflammatory and anti-thrombotic activities, as evidenced by reduced platelet adhesion and decreased platelet–granulocyte aggregates formation in human whole blood. (4) Conclusions: This study is the first to identify FG oligosaccharide HS14 as a promising inhibitor of platelet TLR2/TLR1, demonstrating significant therapeutic potential for inflammatory and thrombotic-related diseases. Full article

Several natural products are being studied to identify new bioactive molecules with therapeutic potential for infections, immune modulation, and other pathologies. TLRs are a family of receptors that play a crucial role in the immune system, constituting the first line of immune defense. They recognize specific products derived from microorganisms that activate multiple pathways and transcription factors in target cells, which are vital for producing immune mediators. Mygalin is a synthetic acylpolyamine derived from hemocytes of the spider Acanthoscurria gomesiana. This molecule negatively regulates macrophage response to LPS stimulation by interacting with MD2 in the TLR4/MD2 complex. Here, we investigated the activity of Mygalin mediated by TLR2 agonists in cells treated with Pam3CSK4 (TLR2/1), Pam2CSK4, Zymosan (TLR2/6), and IFN-γ. Our data showed that Mygalin significantly inhibited stimulation with agonists and IFN-γ, reducing NO and IL-6 synthesis, regardless of the stimulation. There was also a significant reduction in the phosphorylation of proteins NF-κB p65 and STAT-1 in cells treated with Pam3CSK4. Molecular docking assays determined the molecular structure of Mygalin and agonists Pam3CSK4, Pam2CSK4, and Zymosan, as well as their interaction and free energy with the heterodimeric complexes TLR2/1 and TLR2/6. Mygalin interacted with the TLR1 and TLR2 dimer pathway through direct interaction with the agonists, and the ligand-binding domain was similar in both complexes. However, the binding of Mygalin was different from that of the agonists, since the interaction energy with the receptors was lower than with the agonists for their receptors. In conclusion, this study showed the great potential of Mygalin as a potent natural inhibitor of TLR2/1 and TLR2/6 and a suppressor of the inflammatory response induced by TLR2 agonists, in part due to its ability to interact with the heterodimeric complexes. Full article

Inflammation is a protective mechanism against harmful stimuli. There are two types of inflammation, acute and chronic, and severe diseases such as cardiovascular disease and cancer can be caused by chronic inflammation. Therefore, this research was conducted to discover new anti-inflammatory drugs. Meriania hexamera Sprague is a common herb in the Amazon region in South America. It is used as a traditional medical herb by natives, but no studies to date have investigated its anti-inflammatory activity. Using lipopolysaccharide (LPS), pam3CSK4 (Pam3), and poly(I:C), we studied the M. hexamera Sprague–Methanol Extract’s (Mh-ME) in vitro anti-inflammatory functions. Using RAW264.7 cells, we detected the released nitric oxide (NO) and mRNA expression extent of inducible nitric oxide synthase (iNOS) with pro-inflammatory proteins like tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and iterleukin-1 beta (IL-1β). It was found that Mh-ME suppressed the inflammatory activities in a dose-dependent manner. In the luciferase assay, the nuclear factor kappa light chain enhancer of the activated B cells (NF-κB) pathway was inhibited by Mh-ME. Mh-ME especially acted as an inhibitor of Syk kinase according to the results from CETSA. We also confirmed that Mh-ME mitigates acute gastritis derived from HCl/EtOH in ICR mice, ameliorating the expression of IL-1β and tumor necrosis factor-alpha (TNF-α). In conclusion, Mh-ME is an herb with anti-inflammatory effects that targets Syk in the NF-κB pathway, suggesting that Mh-ME could be used as an anti-inflammatory herbal medicine. Full article

Neutrophils are an essential cellular component of innate immunity and control bacterial infections through a combination of intracellular and extracellular killing methods. Although the importance of neutrophils has been established, the exact methods used to handle particular bacterial challenges and the efficiency of bacterial killing remain not well understood. In this study, we addressed how neutrophils eliminate Streptococcus pneumoniae (Spn), a leading cause of community acquired and post-influenza bacterial pneumonia. We analyzed killing methods with variable bacterial:neutrophil concentrations and following priming with PAM3CSK4 (P3CSK), an agonist for Toll-like-receptor 2 (TLR2). Our results show that murine neutrophils display surprisingly weak bactericidal activity against Spn, employing a predominantly extracellular mode of killing at lower concentrations of bacteria, whereas challenges with higher bacterial numbers induce both extracellular and intracellular elimination modes but require TLR2 activation. TLR2 activation increased reactive oxygen species (ROS) and neutrophil extracellular trap (NET) formation in response to Spn. Despite this, supernatants from P3CSK-stimulated neutrophils failed to independently alter bacterial replication. Our study reveals that unstimulated neutrophils are capable of eliminating bacteria only at lower concentrations via extracellular killing methods, whereas TLR2 activation primes neutrophil-mediated killing using both intracellular and extracellular methods under higher bacterial burdens. Full article

Dietary intervention in the treatment of ulcerative colitis involves, among other things, modifications in fatty acid content and/or profile. For example, replacing saturated long chain fatty acids with medium chain fatty acids (MCFAs) has been reported to ameliorate inflammation. The Black Soldier Fly Larvae’s (BSFL) oil is considered a sustainable dietary ingredient rich in the MCFA C12:0; however, its effect on inflammatory-related conditions has not been studied until now. Thus, the present study aimed to investigate the anti-inflammatory activity of BSFL oil in comparison to C12:0 using TLR4- or TLR2-activated THP-1 and J774A.1 cell lines and to assess its putative protective effect against dextran sulfate sodium (DSS)-induced acute colitis in mice. BSFL oil and C12:0 suppressed proinflammatory cytokines release in LPS-stimulated macrophages; however, only BSFL oil exerted anti-inflammatory activity in Pam3CSK4-stimulated macrophages. Transcriptome analysis provided insight into the possible role of BSFL oil in immunometabolism switch, involving mTOR signaling and an increase in PPAR target genes promoting fatty acid oxidation, exhibiting a discrepant mode of action compared to C12:0 treatment, which mainly affected cholesterol biosynthesis pathways. Additionally, we identified anti-inflammatory eicosanoids, oxylipins, and isoprenoids in the BSFL oil that may contribute to an orchestrated anti-inflammatory response. In vivo, a BSFL oil-enriched diet (20%) ameliorated the clinical signs of colitis, as indicated by improved body weight recovery, reduced colon shortening, reduced splenomegaly, and an earlier phase of secretory IgA response. These results indicate the novel beneficial use of BSFL oil as a modulator of inflammation. Full article

Turmeric (Curcuma longa) contains various compounds that potentially improve health. Bisacurone is a turmeric-derived compound but has been less studied compared to other compounds, such as curcumin. In this study, we aimed to evaluate the anti-inflammatory and lipid-lowering effects of bisacurone in high-fat diet (HFD)-fed mice. Mice were fed HFD to induce lipidemia and orally administered bisacurone daily for two weeks. Bisacurone reduced liver weight, serum cholesterol and triglyceride levels, and blood viscosity in mice. Splenocytes from bisacurone-treated mice produced lower levels of the pro-inflammatory cytokines IL-6 and TNF-α upon stimulation with a toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS), and TLR1/2 ligand, Pam3CSK4, than those from untreated mice. Bisacurone also inhibited LPS-induced IL-6 and TNF-α production in the murine macrophage cell line, RAW264.7. Western blot analysis revealed that bisacurone inhibited the phosphorylation of IKKα/β and NF-κB p65 subunit, but not of the mitogen-activated protein kinases, p38 kinase and p42/44 kinases, and c-Jun N-terminal kinase in the cells. Collectively, these results suggest that bisacurone has the potential to reduce serum lipid levels and blood viscosity in mice with high-fat diet-induced lipidemia and modulate inflammation via inhibition of NF-κB-mediated pathways. Full article

Developmentally regulated features of innate immunity are thought to place preterm and term infants at risk of infection and inflammation-related morbidity. Underlying mechanisms are incompletely understood. Differences in monocyte function including toll-like receptor (TLR) expression and signaling have been discussed. Some studies point to generally impaired TLR signaling, others to differences in individual pathways. In the present study, we assessed mRNA and protein expression of pro- and anti-inflammatory cytokines in preterm and term cord blood (CB) monocytes compared with adult controls stimulated ex vivo with Pam3CSK4, zymosan, polyinosinic:polycytidylic acid, lipopolysaccharide, flagellin, and CpG oligonucleotide, which activate the TLR1/2, TLR2/6, TLR3, TLR4, TLR5, and TLR9 pathways, respectively. In parallel, frequencies of monocyte subsets, stimulus-driven TLR expression, and phosphorylation of TLR-associated signaling molecules were analyzed. Independent of stimulus, pro-inflammatory responses of term CB monocytes equaled adult controls. The same held true for preterm CB monocytes—except for lower IL-1β levels. In contrast, CB monocytes released lower amounts of anti-inflammatory IL-10 and IL-1ra, resulting in higher ratios of pro-inflammatory to anti-inflammatory cytokines. Phosphorylation of p65, p38, and ERK1/2 correlated with adult controls. However, stimulated CB samples stood out with higher frequencies of intermediate monocytes (CD14⁺CD16⁺). Both pro-inflammatory net effect and expansion of the intermediate subset were most pronounced upon stimulation with Pam3CSK4 (TLR1/2), zymosan (TR2/6), and lipopolysaccharide (TLR4). Our data demonstrate robust pro-inflammatory and yet attenuated anti-inflammatory responses in preterm and term CB monocytes, along with imbalanced cytokine ratios. Intermediate monocytes, a subset ascribed pro-inflammatory features, might participate in this inflammatory state. Full article

Platelet-rich fibrin (PRF) has a potent anti-inflammatory activity but the components mediating this effect remain unknown. Blood lipids have anti-inflammatory properties. The question arises whether this is also true for the lipid fraction of PRF. To answer this question, lipid fractions of solid and liquid PRF were tested for their potential to lower the inflammatory response of ST2 bone marrow stromal cells and primary bone marrow macrophages exposed to IL1β and TNFα, and LPS, respectively. Cytokine production and the underlying signalling pathway were analysed by RT-PCR, immunoassays, and Western blotting. We report here that lipids from solid and liquid PRF substantially lowered cytokine-induced expression of IL6, CCL2 and CCL5 in ST2 cells. Moreover, the inflammatory response induced by Pam3CSK4, the agonist of Toll-like receptor (TLR) TLR2, was partially reduced by the lipid extracts in ST2 cells. The PRF lipids further reduced the LPS-induced expression of IL1β, IL6 and CCL5 in macrophages at the transcriptional level. This was confirmed by showing the ability of PRF lipids to diminish IL6 at the protein level in ST2 cells and macrophages. Likewise, PRF lipid extracts reduced the phosphorylation of p38 and JNK and moderately decreased the phosphorylation of NFκB-p65 in ST2 cells. These findings suggest that the lipid fraction is at least partially responsible for the anti-inflammatory activity of PRF in vitro. Full article

Innate recognition of pathogens depends on the interaction between microbial structures known as pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) in host cells. Toll-like receptors (TLR) are among the most important PRRs being expressed on and in a wide range of immune cell types. Studies on the interaction mechanisms between different pathogen species and the immune system of the dromedary camel are still scarce. The present study aimed to investigate the immunomodulatory effect of synthetic bacterial and viral TLR ligands on some phenotypic properties and selected functions of neutrophils purified from dromedary camel blood. Neutrophils were separated from camel blood (n = five animals) and were stimulated in vitro with the TLR ligands LPS, Pam3CSK4, R848 (Resiquimod), and Poly IC or were left without stimulation. Stimulation with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was used as a positive control stimulation. Shape change, phagocytosis activity, ROS production, the expression of cell surface markers, and cell vitality were compared between stimulated and non-stimulated cells. With exception of the TLR3 agonist Poly IC, all TLR ligands used showed the potential to stimulate camel neutrophils resulting in increased cell size and the upregulation of CD18 and CD14 on their surface. Similarly, the phagocytosis activity of camel neutrophils was significantly improved after priming with all TLR ligands, except Poly IC, which, in contrast, resulted in a reduced percentage of phagocytosis-positive cells. In contrast to stimulation with PMA, which induced a significant ROS production in camel neutrophils, none of the TLR ligands used stimulated ROS generation in neutrophils. Only stimulation with Pam3CSK4 increased the expression of MHCII molecules on camel neutrophils, resulting in an expanded MHCII^high fraction within camel neutrophils. Our study indicates selective immunomodulating effects of TLR agonists on purified camel neutrophils without affecting their vitality. Full article

(1) Toll-like receptors (TLR) are a family of pattern recognition receptors that sense distinct molecular patterns of microbial origin. Although the immune cell composition of camel milk has been recently described, host–pathogen interaction studies in the camel mammary gland are still scarce. The present study aimed to use a whole milk stimulation assay for investigating the modulatory effect of selected Toll-like receptor (TLR) ligands on the phenotype and function of milk immune cells. (2) Methods—camel milk samples (n = 7) were stimulated in vitro with the TLR4 ligand LPS or the TLR2/1 ligand Pam3CSK4, and separated milk cells were evaluated for stimulation-induced shape change, the expression of cell surface markers, phagocytosis, apoptosis, ROS production, and NETosis. Stimulation with PMA was used as a control stimulation. (3) Results—all stimulants induced shape change in milk cells, change in the expression of several cell markers, and increased cell apoptosis and NETosis. In addition, stimulation with Pam3CSK4 and PMA was associated with enhanced ROS production, while only PMA stimulation resulted in enhanced bacterial phagocytosis by milk immune cells. (4) Conclusions—our data indicates selective modulating effects of the TLR ligands LPS and Pam3CSK4 on camel milk phagocytes. These results may have implications for the use of synthetic TLR agonists as immunomodulatory adjuvants of the immune response to intra-mammary vaccines against mastitis pathogens. Full article