Background and Objectives Trained immunity (TI) refers to a non-specific, long-lasting protective immune response that occurs following initial stimulation of the immune system and thought to be largely mediated by functional reprogramming of myeloid cells. TI has been demonstrated in BCG-vaccinated infants and can be induced in human cells and adult mice via agonists of pattern recognition receptors (PRR), such as β-glucan or MDP (a muropeptide that activates NOD2). However, its induction in neonates remains poorly understood. Previously, we demonstrated that the synthetic TLR2-NOD2 dual agonist CL429 enhances antimicrobial functions in adult mice and protects against subsequent infection with Leptospira interrogans , a zoonotic pathogen. We also demonstrated the immediate protective benefits of NOD2 stimulation in neonates against Cryptosporidium, a zoonotic pathogen that affects young animals in livestock herds. However, whether NOD2 agonists can induce TI in neonates and protect them at adulthood is unknown. Methods and Results Here, we investigated whether exposure of neonatal mice to PRR agonists (CL429, MDP or β-glucan), administered intraperitoneally at one week interval at 7/14 or 14/21 days of age, could enhance inflammatory cytokines production after ex vivo restimulation and confer long-term protection into adulthood. Surprisingly, none of the treatments enhanced ex vivo cytokine responses in adulthood after restimulation, nor did they confer protection against experimental leptospirosis. Instead, MDP-treated neonates exhibited 50% mortality following adult infection, revealing an unexpected detrimental effect. Conclusion These findings demonstrate that these PRR agonists fail to induce protective TI against Leptospira when administered to neonatal mice, challenging assumptions derived from adult models. Furthermore, our study reveals the risks of administering immunostimulants during the early stages of life, and highlights unanticipated and potentially harmful, PRR- and age-specific mechanisms of immune system modulation.
Dendritic cells (DCs) orchestrate antitumor immunity by integrating signals from the tumor microenvironment to prime effective T cell responses. Many tumors display altered glycosylation patterns, including tumor-associated carbohydrate antigens (TACAs) such as the Tn antigen (GalNAcα1- O -Ser/Thr); yet how these structures influence DC function is not well defined. Here, we investigated how Tn-bearing glycoconjugates modulate DC activation and shape adaptive immunity. Bone marrow-derived DCs (BMDCs) efficiently internalized fluorescently labeled Tn-glycoconjugates, whereas uptake of non-glycosylated counterparts was negligible. Although Tn-glycoconjugates alone did not induce DC maturation, co-stimulation with the Toll-like receptor 9 (TLR9) agonist CpG markedly increased CD86 expression and the secretion of IL-12/23p40 and IL-6, with the multivalent construct MAG:Tn3-PV eliciting the strongest response. These conditioned BMDCs promoted strong IFN-γ production by allogeneic splenocytes, consistent with a Th1-polarizing phenotype. Mechanistically, both uptake and CpG-enhanced activation required the C-type lectin receptor MGL2, as blockade of MGL or competition with GalNAc abrogated glycoconjugate uptake and CpG-enhanced cytokine induction. Pharmacological inhibition revealed that MGL2 signaling synergizes with TLR9 through the Syk–Raf-1–NF-κB axis. In vivo , mice immunized with DCs conditioned with Tn-glycoconjugate- plus CpG displayed enhanced splenocyte proliferation, increased IFN-γ secretion, and elevated cytotoxic activity without IL-10 induction, confirming a Th1-skewed response. Collectively, these findings identify MGL2 as a critical mediator of Tn-glycoconjugate sensing and unveil a synergistic C-type lectin receptor (CLR)–TLR9 cross-talk that amplifies DC maturation and cytotoxic immunity. This study provides mechanistic insight into how specific glycan–lectin interactions fine-tune innate receptor signaling, highlighting the potential of Tn-based glycoconjugates as immunomodulatory tools for vaccine design and cancer immunotherapy.
Introduction Lupus nephritis (LN) is characterized by significant heterogeneity and a complex pathophysiology, which traditional methods struggle to fully resolve. Advanced multi-omics approaches are essential to disentangle its cellular and molecular drivers. Methods We employed an integrative strategy combining single-cell RNA sequencing (scRNA-seq) profiling of LN biopsies with large-scale bulk RNA-seq cohorts. We applied non-negative matrix factorization (NMF) to scRNA-seq data to define robust immune meta-programs and utilized CellChat to decode cell-cell communication networks. Leveraging these insights to overcome sample size limitations, we prioritized key pathways and developed 399 machine learning predictive models using bulk transcriptomics, validated on independent cohorts. Results ScRNA-seq analysis revealed a distinct cellular landscape, including a rare population of plasmacytoid dendritic cells (pDCs) and an expanded population of CD56dimCD16 + natural killer (NK) cells expressing high levels of IFN-γ and perforin, suggesting a role in inflammatory pathology. Macrophage subpopulation CM2 emerged as a central pro-inflammatory hub, potentially driving fibrosis via autocrine signaling and epithelial activation. We observed reduced Treg-B cell interactions, suggesting a regulatory collapse. Our machine learning models, based on innate immunity, circadian rhythms, apoptosis, and NF-κB signaling, achieved high diagnostic accuracy (AUC = 0.929 for innate immunity). Hub genes, including CYBB , CSF2RB , and IRF8 , were confirmed to be upregulated in LN and correlated with clinical severity in external validation datasets. Molecular docking simulations suggested a potential structural basis for CYBB-dexamethasone interaction, providing a hypothesis for future verification. Discussion This study identifies CM2 macrophages and dysregulated pDC-NK axes as key drivers of LN. By bridging cellular interactomes with clinical predictive modeling, we provide a robust roadmap for precision detection and identifying potential therapeutic targets in LN.
Objective Endothelial progenitor cells (EPCs) originate from hematopoietic stem cells and can be quantified in peripheral blood using flow cytometry. The anti-GM-CSFα antibody (CD116) may serve as a specific marker for EPC enumeration. This study aimed to quantify peripheral EPCs expressing CD116 and compare the results with other specific antibodies in newborns and adults. Materials and Methods EPC enumeration was performed by flow cytometric analysis of peripheral blood leukocytes (PBLs) obtained from 50 individuals, including 25 newborns and 25 adults. A CD34-specific antibody was used to identify hematopoietic stem/progenitor cells, while an antibody panel consisting of CD116, CD146, CD31, and CD45 was employed for EPC identification. Results Enumeration of CD34 + hematopoietic progenitor cells (HPCs) demonstrated that the mean CD34 + HPC count per 10 6 PBLs was 1643 (935–1458) in newborns and 242.7 (163–190) in adults, with a statistically significant difference between the groups ( p < 0.001). Using CD146 staining, the mean number of circulating EPCs per 10 6 PBLs was 94.2 (90.5–129.0) in newborns and 9.2 (7.4–12.4) in adults ( p < 0.001). Similarly, enumeration based on CD31 staining revealed mean EPC counts of 19.0 (12.5–28.0) in newborns and 6.0 (5.0–7.0) in adults ( p < 0.001). Enumeration using CD116 staining showed mean EPC numbers of 29.0 (23.0–34.0) in newborns and 3.0 (2.0–4.0) in adults, also indicating a significant difference between the two groups ( p < 0.001). Conclusion EPC numbers are significantly higher in newborns than in adults, suggesting an important developmental role for these cells. The GM-CSFα-specific antibody (CD116) may serve as a novel auxiliary marker for the identification and quantification of circulating EPCsubpopulations. Furthermore, EPC numbers appear to vary across different life stages, with higher numbers in newborns potentially reflecting the presence of a highly regenerative microenvironment.
IntroductionThe gut microbiota modulates dementia pathogenesis through immune interactions. Using Mendelian randomization, we investigate immune mediated mechanisms linking microbial dysbiosis to four dementia subtypes (Alzheimer's disease, Frontotemporal dementia, Vascular dementia, Parkinson's disease dementia . Our study tests whether gut microbiome effects on dementia are transmitted via immunoregulatory pathways.MethodsGenome wide association studies data included gut microbiota, 731 immune traits, and dementia cohorts (Alzheimer's disease, Frontotemporal dementia, Vascular dementia, Parkinson's disease dementia). Two step Mendelian randomization with Inverse Variance Weighted analyses assessed mediation effects, controlled by F-statistics >10 and Steiger filtering. Sensitivity analyses addressed pleiotropy.ResultsA total of 37 gut microbiome species demonstrated potential causal effects relationships with four types of dementia, and 137 immune cell subsets exhibited potential causal effects associations with these four dementia subtypes. In the Two step Mendelian randomization analysis, CD45RA + CD28- CD8+ T cells, CD19 on IgD- CD38dim B cells, and BAFF-R on CD20- B cells were shown to exert mediating effects between class/order/family.Deltaproteobacteria and Alzheimer's disease. CD4+ CD8+ T cells were found to exert a mediating effect between genus.Roseburia and Parkinson's disease dementia . CD20- CD38- B cells, CD19 on CD20- B cells, and IgD on unswitched memory B cells were found to exert a mediating effect between class/order/family.Coriobacteriales,genus.Lactococcus and Vascular dementia.ConclusionThis Mendelian randomization study revealed that certain immune cells serve as mediators in the pathway by which the gut microbiome contributes to the onset of dementia.
Background Periodontitis is one of the most common inflammatory diseases in humans, mostly caused by bacterial infection and with diverse populations of immune cells involved. Myeloid-derived suppressor cells (MDSCs), a heterogeneous group of immature myeloid cells derived from hematopoietic precursor cells, have exhibited immunomodulatory functions by production of different molecules such as inducible NO synthase (iNOS) and it is thought to be involved in periodontitis. However, reports of characterization of cells with MDSC phenotypes in gingival tissues are very scarce. This study aimed to characterize gingival cells with MDSC phenotypes in healthy gingiva and periodontitis tissues.Methods and Results Human healthy gingival tissues and those with periodontitis were included to analyze cells with MDSC phenotypes by flow cytometry. Additionally, a mouse model of experimental periodontitis was used to identify cells with MDSC phenotypes and production of iNOS. Results showed an increased accumulation of CD45+HLA-DRneg/lowCD11b+CD33+ cells in human gingival tissues with periodontitis. Experimental periodontitis promotes accumulation of CD45+CD11b+Gr-1+ and CD45+CD11b+Ly6G+ cells in gingival tissues. Experimental periodontitis did not promote accumulation of these subpopulations in other tissues as spleen. Additionally, gingival CD45+Gr-1+ iNOS+ cells were identified.Conclusions cells with MDSC phenotypes are resident in healthy gingival tissues and their accumulation is locally triggered by periodontitis. Cells with capacity of iNOS production could be implicated in generation of reactive nitrogen species, suggesting immunomodulatory properties.
High-molecular-weight kininogen (HK) is known to bind lipopolysaccharides (LPS) with high affinity and serves as a crucial LPS carrier in circulation, supporting endotoxemia. However, its role in host defense against Gram-negative bacterial infection remains unclear. Here we demonstrate that HK directly binds to Escherichia coli ( E. coli ) via LPS and rapidly localizes to sites of infection. HK-deficient mice ( Kng 1 – /– ) showed increased susceptibility to infection, with increased bacterial dissemination, lung injury, and proinflammatory cytokine production. In contrast, endogenous expression of human HK in Kng 1 – /– mice restored survival, limited bacterial spread, and reduced tissue damage. Mechanistically, HK promoted neutrophil antimicrobial responses by enhancing reactive oxygen species production and microbicidal activity. Consistently, liver-specific HK deficiency recapitulated the impaired bacterial clearance and reduced survival upon E. coli challenge, highlighting the importance of plasma HK. Together, these findings identify HK as a new soluble pattern recognition molecule that senses E. coli invasion and initiates neutrophil-mediated antimicrobial responses, revealing a previously unrecognized protective function of the contact system in innate immunity.
Malaria, a globally prevalent disease caused by Plasmodium species, significantly impacts the immune system, particularly affecting splenic function. This study investigates the therapeutic potential of Indigofera oblongifolia leaf extracts (IOLE) with silver nanoparticles (AgNPs) against Plasmodium chabaudi-induced splenic damage in a female C57BL/6 mice model. Fifty female mice were infected with P. chabaudi and subsequently treated with IOLE AgNPs or chloroquine phosphate. Histopathological and immunohistochemical analyses revealed that IOLE AgNPs effectively restored splenic architecture, reduced inflammatory markers, and improved immune responses compared to the control and chloroquine-treated groups. These findings suggest that IOLE AgNPs may offer a novel therapeutic approach to mitigate splenic dysfunction associated with malaria and provide comparative analysis between established therapies like chloroquine and innovative combination of traditional medical plant and modern nano technology.
Background: To determine whether (i) altered levels of acute-phase response (APR)-, inflammation-, and extracellular matrix (ECM)-related proteins in the amniotic fluid (AF) were associated with spontaneous preterm delivery (SPTD) in asymptomatic women with midtrimester short cervix (SCX) and (ii) if SPTD risk severity was related to the expression levels of inflammation-related proteins in the AF. Methods: This retrospective cohort study included 70 singleton pregnant women diagnosed with a SCX (<25 mm) at 17–25 weeks, who were subjected to amniocentesis to exclude intraamniotic inflammation (IAI; defined as AF interleukin [IL]-6 ≥ 2.6 ng/mL). APR ( i.e., h epcidin, kallistatin, MBL, pentraxin-2, RBP4 , and serpin A1) , inflammatory ( i.e., IL-6, IL-8, and resistin) , and ECM-related ( i.e., lumican, MMP-8, TGFBI, and uPA) molecules were assayed in the AF by ELISA. The primary outcome measure was SPTD at <34 weeks. The levels of each identified dysregulated inflammatory mediator were divided into quartiles to assess the correlation between their AF expression profiles and SPTD risk severity. Results: Multivariable Firth logistic regression analyses revealed that elevated AF levels of IL-6, IL-8, kallistatin, pentraxin-2, resistin, and serpin A1, and IAI presence were independently associated with SPTD at <34 weeks after adjusting for baseline covariates. The areas under the curves of the aforementioned mediators ranged from 0.67 to 0.79 for outcome prediction. The odds of SPTD at <34 weeks, even after adjusting for confounders, significantly increased with each increasing quartile of baseline AF levels of IL-6/8, pentraxin-2, and resistin. Conclusions: APR (kallistatin, pentraxin-2, and serpin A1)- and inflammation (IL-6/8 and resistin)-, but not ECM-related mediators in the AF are involved in SPTD development in asymptomatic women with a midtrimester SCX. In particular, SPTD risk (especially risk severity) is associated with the degree of the inflammatory response in the AF, as categorized by inflammatory protein expression profiles, as well as IAI presence.
Bronchial cell pyroptosis and IL-17 respectively contribute- to the pathogenesis of steroid-insensitive asthma. In this study, we aim to explore the relationship between bronchial cell pyroptosis and Th17 in airway inflammation of steroid-insensitive asthma. The steroid-insensitive asthma model of mice was induced by toluene diisocyanate (TDI), which was also intraperitoneally injected with NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inhibitor MCC950. The bronchial epithelial cell pyroptosis was identified in morphology by transmission electron microscope. Protein expressions of pyroptosis cytokines (pro-Caspase-1, Caspase-1 p20, pro-GSDMD, cleaved-GSDMD and HMGB1), IL-17A, IL-17F and phosphorylated STAT3 (p-STAT3) in lung tissues were assessed by western blotting. Th17 in lung tissues was measured by flow cytometry. IL-17A + and p-STAT3 + cells in airway were identified by immunohistochemistry. In steroid-insensitive asthma mice, bronchial epithelial cell pyroptosis was confirmed in morphology using transmission electron microscope. Compared with controls, the protein expressions of Caspase-1 p20, cleaved-GSDMD and HMGB1 in lung tissues were increased in mice with steroid-insensitive asthma, which could be attenuated by MCC950. Th17 cells precentage and proteins expressions of p-STAT3, IL-17A and IL-17F were also increased in lung of steroid-insensitive asthmatic mice, which were also attenuated by MCC950. Similarly, the counts of IL-17A + cell and p-STAT3 + cell were more in airway of steroid-insensitive asthmatic mice than controls, and was attenuated by MCC950. In conclusion, bronchial epithelial cell pyroptosis could promote Th17 inflammation in airway of steroid-insensitive asthma mouse, which will provide further understanding on the interaction between innate immunity and acquired immunity in the pathogenesis of steroid-insensitive asthma.
Mastitis in cattle poses a significant health challenge and results in substantial economic losses for the dairy industry. This study aimed to extend the existing precision-cut bovine udder slices (PCBUS) model as an in vitro model to explore the potential of inducing trained immunity in the udder with the goal to use the resulting knowledge for potential new treatment strategies. Interestingly, incubation of PCBUS with 10% fetal calf serum (FCS), but no 2% or FCS-free, negatively affected the production of some of the chemokines/cytokines analysed. When trained immunity was induced by zymosan, followed by stimulation with E. coli -derived lipopolysaccharide (LPS), production of interleukin (IL)-1β, IL-6, tumor necrosis factor α and interferon (IFNγ) was downregulated while production of IL-17A and pro-resolving lipid mediators (leukotrienes and prostaglandins) was upregulated. While the current experimental setup did not definitively confirm the induction of trained immunity for all parameters analysed in PCBUS, it validated the utility of PCBUS as a robust in vitro model for studying bovine udder inflammation. This model offers a promising platform for developing innovative mastitis treatments, particularly given the growing concern over antimicrobial resistance, as well as offering alternatives to the use of live animals in experimental studies in line with the 3Rs principles. It also provides a valuable tool for advancing our understanding of immune responses in the bovine udder. By adapting the precision-cut tissue slice technique to bovine udders, this model enables extensive research into new therapeutic approaches and supports basic research efforts to characterise complex pathophysiological processes associated with mastitis. Furthermore, our data highlight the potential limitations of FCS in in vitro studies. Our data should not only stimulate the discussion about FCS in homologues or heterologues species, but should also be kept in mind regarding the need for foetal calves to generate FCS in line with the 3Rs guideline.
ObjectiveTo research whether radiation-induced liver damage and fibrosis could be mitigated by resveratrol (RSV) and to elucidate its underlying mechanism.MethodsA radiation-induced liver damage (RILD) model of murine was constructed. RSV was used as an intervention agent. The effects of RSV on inflammatory reaction, apoptosis, senescence, fibrosis, survival, and liver functions were detected by β-Gal, Sirius red, Masson's trichrome, and Tunnel staining using an automated biochemistry analyzer. The protein expression levels of P16 and P21 were detected by Western blot.ResultsRSV alleviated inflammatory injury of RILD mice. RSV decreased the serum pro-inflammatory cytokines of RILD mice. RSV alleviated radiation-induced hepatocellular senescence. The protein expression levels of P16 and P21 in RILD mice were decreased with RSV administration. RSV decreased the number of apoptotic cells in the early stage of RILD. RSV alleviated liver fibrosis and liver function in RILD mice.ConclusionsRSV reduces RILD and fibrosis, and may be related to inhibiting cellular aging and reducing inflammation.
Objective To investigate the important active sites within the NTFs to affect the in vitro interaction of oxidized low-density lipoprotein (ox-LDL) with its receptor, OLR1. Methods Simulation analysis online was performed to generate various OLR1 chimeras, truncation mutants, and site-specific mutations. They were transfected in COS-7 cells and subjected to ox-LDL stimulations to observe the different reactions. Immunoprecipitation-mass spectrometry (IP-MS) was performed to show what proteins combined with OLR1 mutants in reaction to ox-LDL. Lipid uptake in human monocytes (THP-1) originated foam cells overexpressing somatic mutant of OLR1 were also determined. Further studies focusing on these regions were conducted using truncation mutants and site-specific mutants such as G43A, V44A, L45A, C46A, and L47A. Results Amino acids within the TM were highly conserved, spanning amino acids 35 to 57. The induction of intracellular p-ERK1/2 in response to ox-LDL stimulation was highly promoted in Chimera 3 possessing the TM from OLR1 like OLR1/WT ( p < 0.05). Sequence alignment revealed two conserved regions within the TM of OLR1, Leu45-Cys46-Leu47 and Val55-Leu56-Gly57. Western blot showed that most of the TM changes ablated ERK1/2 activation in response to ox-LDL stimulation ( p < 0.05). One human somatic mutation at L45F revealed significantly lower p-ERK1/2 levels with enhanced intake of ox-LDL in THP-1-derived foam cells than the control cells ( p < 0.05). L45A and C46A molecular complexes were identified. After ox-LDL stimulation, these underlined interactions with keratins, namely KRT2 and KRT6A. Conclusion These findings emphasize the vital role of the TM in the interactions between OLR1 and ox-LDL and point to an exciting possibility that signal transduction induced by ox-LDL through its receptor OLR1 may involve complex interactions with cytoskeletal proteins.
A previous study found that repeated intrauterine infusions of lipopolysaccharide (LPS) followed by an LPS infusion into the mammary glands attenuated the mammary inflammatory response. This suggests that repeated LPS infusion into the uterus promotes endotoxin tolerance (ET) in the mammary gland. However, the specific changes in mammary glands under ET conditions remain unclear. We hypothesized that ET affects leukocyte function in milk. This study aimed to investigate leukocyte function in milk under ET conditions induced through repeated LPS infusions into the uterus for three days followed by LPS infusion into the mammary glands of goats. Goats in the IU group (n = 17) received an infusion of 100 μg LPS in 5 ml saline into the uterus for three consecutive days (d −3, −2, and −1), whereas the goats in the control group did not receive this infusion (n = 19). On d 0, 1 μg LPS in 5 ml saline was infused into the mammary glands of both groups. Milk was collected 0, 4, 8, 12, 24, 48, 72, and 120 h after LPS intramammary infusion. The IU group decreased cytokine production (interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-1Ra) in milk following intramammary LPS infusion. Moreover, leukocyte activation, measured by phagocytic activity and CD11b expression, was higher in the IU group than in the control group. These findings suggest that goats exhibit enhanced leukocyte function in mammary glands under ET conditions, induced by repeated intrauterine infusion of LPS.
BackgroundPrevious studies suggest that transient receptor potential melastatin 2 (TRPM2) plays a protective role in sepsis by enhancing bacterial clearance. This effect is mediated through the modulation of macrophage phenotypic changes, which strengthen the immune response against infection. However, the specific role and underlying mechanism of TRPM2 in macrophage polarization during sepsis remain unclear.MethodCecal ligation and puncture (CLP) was used to establish a mouse sepsis model, and bone marrow-derived macrophages (BMDMs) and peritoneal macrophages were prepared from C57BL/6 wild-type and TRPM2 knockout (trpm2-/-) mice. IPI549 was utilized as a specific inhibitor of PI3K. Macrophage polarization, bactericidal ability, and the PI3K/protein kinase B (AKT)/cyclic adenosine monophosphate response element-binding protein signaling pathway were assessed. In addition, survival rate, bacterial burden, lung wet/dry weight ratio, lung and liver injury scores, and cytokine levels were measured in CLP-induced septic mice.ResultsIn lipopolysaccharide (LPS)-stimulated BMDMs, trpm2 deficiency increased the expression of characteristic markers associated with the M2b phenotype, reduced the bactericidal ability, and activated the PI3K/AKT/CREB signaling pathway. Consequently, both trpm2-/- BMDMs and trpm2-/- mice exhibited impaired bactericidal clearance during CLP-induced sepsis. Furthermore, IPI549 attenuated TRPM2 deletion-induced M2b polarization and restored the bactericidal function of BMDMs. Notably, IPI549 preconditioning reversed the increased susceptibility of the trpm2-/- mice to sepsis. The 7-day mortality rate was 92% in trpm2-/- mice, compared to 42% in IPI549-pretreated trpm2-/- mice. Moreover, IPI549-treated mice exhibited improved lung wet/dry ratios, reduced lung and liver injury scores, reversed M2b polarization and decreased bacterial load.ConclusionThe PI3K/AKT/CREB pathway mediates the effect of TRPM2 by inhibiting M2b macrophage polarization and promoting bacterial clearance during sepsis.
Introduction Repeated injections of low-dose lipopolysaccharide (LPS preconditioning) augment the antibacterial activity of liver macrophages. In this study, a mouse model of acute kidney injury (AKI) induced by Staphylococcus aureus (S. aureus) bacteremia was used to investigate the effects of LPS preconditioning on renal macrophages. Methods Eight-week-old C57BL/6J mice were preconditioned with either low-dose LPS (5 μg/kg) or the vehicle for three consecutive days. Kidney immune cells were isolated, and the antibacterial activity of renal macrophages was assessed by pHrodo TM -labeled S. aureus in vitro . Twenty-four hours after the last LPS injection, the mice were intravenously challenged with S. aureus (2 × 10 7 CFU) and their renal function was evaluated to identify the changes. Results Mouse renal macrophages exhibited a weak antibacterial activity against S. aureus compared with the liver and spleen macrophages. LPS preconditioning elevated the count of F4/80 low CD11b high bone marrow-derived macrophages (BMDM) and augmented their antibacterial activities in the mouse kidney. It also enhanced the antibacterial activity of F4/80 high CD11b low tissue-resident macrophages (TRM) without altering their abundance. LPS preconditioning lowered the bacterial propagation in the kidney in the challenged mice and ameliorated sepsis-associated AKI compared with the control. LPS preconditioning upregulated the CD80/CD206 expression (M1/M2) ratio in BMDMs in the kidney before bacterial challenge and reduced their M1/M2 ratio following S. aureus challenge compared with the control. Conclusion LPS preconditioning enhanced the antibacterial activity of the renal macrophages against S. aureus and suppressed the excessive activation of M1 macrophages following S. aureus challenge, resulting in the amelioration of AKI caused by S. aureus bacteremia.
Background Four influenza pandemics have occurred during the past 100 years, and new variants of influenza viruses will continue to emerge. The nasal mucosa acts as the primary site of exposure to influenza A virus (IAV) infection, but viral recognition and host immune responses in the nasal mucosa are still poorly understood. Objectives This study aimed to evaluate the utility of non-invasive nasopharyngeal swabs for longitudinal monitoring of mucosal immune responses in pigs experimentally challenged with two swine-adapted and one human-adapted IAV. By tracking antiviral immune responses from disease onset to recovery, we sought to assess the feasibility of this method for capturing dynamic changes in viral load and host responses across different IAV strains. Methods Forty-two IAV-negative pigs were divided into four groups and housed separately for infection studies. Viral and host RNA from nasopharyngeal swabs was analyzed using microfluidic qPCR, while statistical analysis was performed with a Bayesian approach in R. Additionally, immunohistochemical staining was used to assess MUC5AC expression in the nasal mucosa of infected pigs. Results RNA was successfully isolated from nasopharyngeal swabs, enabling gene expression analysis to monitor innate immune responses to IAV infection. A classical innate antiviral immune response was demonstrated after the three virus infections including expression of pattern recognition receptors (PRRs), transcription factors, interferons (IFNs), interferon-stimulated genes (ISGs), cytokines, and chemokines. The kinetics and magnitude of immune responses varied between infections, with notable downregulation of mucins following infection with the Danish swine-adapted isolate. Further, the Danish isolate induced a fast but transient IFN-mediated response concurrent with high expression of cytokines and chemokines, while the other swine-adapted Mexican isolate induced a prolonged immune response of ISGs, cytokines, and chemokines. Conclusion This study highlights the significance of highly translational nasopharyngeal swabs as a non-invasive method for assessing mucosal antiviral immune responses. Utilizing microfluidic mRNA analysis, we gained valuable insights into antiviral mucosal responses across 216 swab samples collected from viral inoculation through recovery in three distinct influenza virus infections.
Background Acute Lung Injuries (ALI) are a severe consequence of influenza-induced cytokine storm that can cause respiratory failure and death. It has been demonstrated that Toll-like Receptor 4 (TLR4) is involved in cytokine storm and that TLR4 −/− mice are protected against ALI. Therefore, TLR4 is a prime target for protection against ALI. FP12 is a known TLR4 antagonist that reduces TLR4-dependent immune activation and it is a promising lead compound for the treatment of innate immunity related pathologies. Objectives We present here the preclinical development of FP12 as an anti-inflammatory lead compound acting on influenza-induced ALI. Methods In vitro: We pre-treated THP-1 cells with FP12 (10 μM) for 0.5 h, then exposed to LPS (100 ng/ml) for 0 to 16 h. In some experiments, cells were simultaneously incubated with FP12 and LPS, or FP12 was added 30 min after LPS. Cytokine levels were measured by Western blot and ELISA assays. In vivo: WT C57BL/6J mice were infected with mouse-adapted influenza virus (PR8). Two days after infection, mice received either vehicle, FP7 (200 µg/mouse), or FP12 (200 µg/mouse) once daily (Day 2 to Day 6). Mice were monitored daily for survival for 14 days. Data were collected through histological staining, qRT-PCR, and ELISA assay. Results FP12 treatment inhibited both LPS- and HMGB1-induced TLR4 intracellular pathways (MyD88 and TRIF) leading to significantly reduced levels of a variety of proinflammatory cytokines including Type I interferon (IFN-β), highlighting its effectiveness in controlling proinflammatory protein production and reducing inflammation. FP12 protected mice therapeutically from influenza virus-induced lethality and reduced both cytokine gene expression and High Mobility Group Box 1 (HMGB1) levels in the lungs as well as ALI. Conclusion FP12 can antagonize TLR4 activation in vitro and protects mice from severe influenza infection, most likely by reducing the TLR4-dependent cytokine storm mediated by danger-associated molecular patterns (DAMPs).
The formation of neutrophil extracellular traps (NETs) is known as an important part of the innate immune response. Still, some mechanisms regarding their formation and role during a disease are not completely understood yet. To visualize NETs by immunofluorescence microscopy, a chemical fixation is required. Therefore, this study focused on the effect of chemical fixatives on immunofluorescence staining of selected neutrophil and NET-markers, including myeloperoxidase (MPO), DNA/histone-1-complexes and citrullinated histone H3 (H3cit). Neutrophils isolated from fresh human blood were stimulated with phorbol-12-myristate 13-acetate (PMA) to induce NETs and fixed with paraformaldehyde (PFA, 4%), glutardialdehyde (GA, 5%) or methanol (MeOH, 100%) using different incubation times depending on the used fixative. We found that different fixation times with PFA had no effect on the staining intensity of MPO or DNA/histone-1-complex antibodies. For the staining of H3cit, fixation with PFA for 24 h decreased the signal intensity whereas 30 min fixation time had no effect. In contrast, glutardialdehyde induced a high amount of autofluorescence, and the fixation with 100% MeOH resulted in visible cellular damage. Therefore, we recommend 15-30 min PFA fixation for the respective stainings. Our results provide a solid basis for future experiments to study neutrophil activation and NET-formation.
This study aimed to investigate the impact of Cardamine violifolia on muscle protein degradation, the inflammatory response and antioxidant function in weaned piglets following LPS challenge. Twenty-four weaned piglets were used in a 2 × 2 factorial experiment with dietary treatment (sodium selenite or Cardamine violifolia ) and LPS challenge. After 28 days of feeding, pigs were injected intraperitoneally with 100 μg/kg LPS or saline. Dietary supplementation with Cardamine violifolia mitigated the reduction in insulin and growth hormone levels induced by LPS. It also curbed the LPS-induced elevation of plasma glucagon, urea nitrogen, and creatinine concentrations. Cardamine violifolia reduced muscle damage caused by LPS, as evidenced by increased protein content and protein/DNA ratio and decreased TNF-α and IL-1β mRNA expression. Furthermore, Cardamine violifolia modulated the expression of FOXO1, FOXO4, and MuRF1 in muscle, indicative of the protective effect against muscle protein degradation. Enhanced muscle antioxidant function was observed in the form of increased T-AOC, reduced MDA concentration, and decreased mRNA expression of GPX3, DIO3, TXNRD1, SELENOS, SELENOI, SELENOO, and SEPHS2 in LPS-treated piglets. The findings suggest that Cardamine violifolia supplementation can effectively alleviate muscle protein degradation induced by LPS and enhance the antioxidant capacity in piglets.
