Chemokine Levels Research Articles

Silibinin-loaded chitosan-capped silver nanoparticles exhibit potent antimicrobial, antibiofilm, and anti-inflammatory activity against drug-resistant nosocomial pathogens

Nanoparticles capped with natural products can be a cost-effective alternative to treat drug-resistant nosocomial infections. Therefore, silibinin-loaded chitosan-capped silver nanoparticles (S-C@AgNPs) were synthesized to evaluate their antimicrobial and anti-inflammatory potential. The S-C@AgNPs plasmon peak was found at 430 nm and had a particle size distribution of about 130 nm with an average hydrodynamic diameter of 101.37 nm. The Scanning Electron Microscopy images showed the presence of sphere-shaped homogeneous nanoparticles. The Fourier Transform Infrared Spectroscopy analysis confirmed the loading of silibinin and chitosan on the AgNPs surface. The minimum inhibitory concentration of the S-C@AgNPs was reported between 3.12 μg/ml to 12.5 μg/ml and a minimum bactericidal concentration between 6.25 μg/ml to 25 μg/ml against drug-resistant nosocomial pathogens. Moreover, concentration-dependent significant inhibition of the biofilm formation was reported against P. aeruginosa (70.21%) and K. pneumoniae (71.02%) at 30 μg/ml, and the highest destruction of preformed biofilm was observed at 100 μg/ml against P. aeruginosa (89.74%) and K. pneumoniae (77.65%) as compared to individual bacterial control. Additionally, the fluorescence live/dead assay for bacterial biofilm confirmed that 100 µg/ml effectively inhibits the biofilm formed by these pathogens. S-C@AgNPs also showed anti-inflammatory activity, which is evident by the significant decrease in the proinflammatory cytokines and chemokines level in THP1 cells treated with LPS. This study concluded that S-C@AgNPs have potent antimicrobial, antibiofilm, and anti-inflammatory properties and could be a potential option for treating drug resistant nosocomial infections.

Innate and Adaptive Immune Parameters following mRNA Vaccination in Mice.

The COVID-19 pandemic has raised the standard regarding the current vaccine development pace, as several messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines have proved their ability to induce strong immunogenicity and protective efficacy. We developed 1-methylpseudouridine-containing mRNA-LNP vaccines, expressing either the more conserved SARS-CoV-2 nucleoprotein (mRNA-N) or spike protein (mRNA-S), both based on the prototypic viral sequences. When combining both mRNA-S and mRNA-N together (mRNA-S+N), the vaccine showed high immunogenicity and broad protection against different SARS-CoV-2 variants, including wildtype, Delta, BA.1, BA.5, and BQ.1. To better understand the mechanisms behind this broad protection obtained by mRNA-S+N, we analyzed innate and adaptive immune parameters following vaccination in mice. Compared to either mRNA-S or mRNA-N alone, mice vaccinated with mRNA-S+N exhibited an increase in the innate immune response, as depicted by the higher cytokine (IL-6 and chemokine (MCP-1) levels. In addition, lymph node immunophenotyping showed the maturation and activation of dendritic cells and natural killer cells, respectively. To understand the adaptive immune response, RNA-Seq analyses of the lung and spleen samples of the vaccinated mice were performed in parallel and revealed a stronger immune gene-expression profile in the lung than that in the spleen. Compared to mRNA-S alone, mRNA-S+N vaccination elicited higher levels of expression for genes involved in multiple immune pathways, including T cells, cytokine signaling, antigen presentation, B cells, and innate immunity. Together, our studies provide immunological insights into the mechanisms of broad protection conferred by dual mRNA vaccination against SARS-CoV-2 variants.

Treponema pallidum recombinant protein Tp0768 enhances the ability of HUVECs to promote neutrophil chemotaxis through the TLR2/ER stress signaling pathway.

Neutrophils are essential cells involved in inflammation. However, the specific mechanism of neutrophil chemotaxis induced by Treponema pallidum remains unknown. In this study, human umbilical vein endothelial cells (HUVECs) were utilized as target cells to investigate the expression levels of chemokines when stimulated with different concentrations of Tp0768 (also known as TpN44.5 or TmpA, a T. pallidum infection dependent antigen). The results indicated that Tp0768 treatment enhanced neutrophil chemotaxis in HUVECs, which was closely associated with the expression levels of CXCL1, CXCL2, and CXCL8 (also known as interleukin-8). At the same time, the results show that the Toll-like receptor 2 (TLR2) signaling pathway is activated and that endoplasmic reticulum (ER) stress occurs. Furthermore, the findings revealed that the use of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and immunoglobulin-regulated enhancer 1 (IRE1) inhibitors reduced the expression levels of CXCL1, CXCL2, and CXCL8. Additionally, inhibiting TLR2 significantly decreased the expression levels of ER stress-related proteins (PERK and IRE1), CXCL1, CXCL2, and CXCL8. Consequently, neutrophil chemotaxis was significantly inhibited after treatment with TLR2, PERK, and IRE1 inhibitors. These findings shed light on the role of Tp0768 in enhancing neutrophil chemotaxis in endothelial cells, providing a foundation for further exploration of syphilis pathogenesis and offering a new direction for the diagnosis and treatment of T. pallidum infection.

Bronchom assuages airway hyperresponsiveness in house dust mite-induced mouse model of allergic asthma and moderates goblet cell metaplasia, sub-epithelial fibrosis along with changes in Th2 cytokines and chemokines.

Asthma is a common obstructive airway disease with an inflammatory etiology. The main unmet need in the management of asthma is inadequate adherence to pharmacotherapy, leading to a poorly-controlled disease state, necessitating the development of novel therapies. Bronchom is a calcio-herbal formulation, which is purported to treat chronic asthma. The objective of the current study was to examine the in-vivo efficacy of Bronchom in mouse model of allergic asthma. Ultra high performance liquid chromatography was utilized to analyze the phytocompounds in Bronchom. Further, the in-vivo efficacy of Bronchom was evaluated in House dust mite (HDM)-induced allergic asthma in mice. Mice were challenged with aerosolized methacholine to assess airway hyperresponsiveness. Subsequently, inflammatory cell influx was evaluated in bronchoalveolar lavage fluid (BALF) followed by lung histology, wherein airway remodeling features were studied. Simultaneously, the levels of Th2 cytokines and chemokines in the BALF was also evaluated. Additionally, the mRNA expression of pro-inflammatory and Th2 cytokines was also assessed in the lung along with the oxidative stress markers. Phytocompounds present in Bronchom included, gallic acid, protocatechuic acid, methyl gallate, rosmarinic acid, glycyrrhizin, eugenol, 6-gingerol and piperine. Bronchom effectively suppressed HDM-induced airway hyperresponsiveness along with the influx of leukocytes in the BALF. Additionally, Bronchom reduced the infiltration of inflammatory cells in the lung and it also ameliorated goblet cell metaplasia, sub-epithelial fibrosis and increase in α-smooth muscle actin. Bronchom decreased Th2 cytokines (IL-4 and IL-5) and chemokines (Eotaxin and IP-10) in the BALF. Likewise, it could also suppress the mRNA expression of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6 and IL-33), and IL-13. Moreover, Bronchom restored the HDM-induced diminution of endogenous anti-oxidants (GSH and SOD) and the increase in pro-oxidants (GSSG and MDA). Furthermore, Bronchom could also decrease the nitrosative stress by lowering the observed increase in nitrite levels. Taken together, the results of the present study data convincingly demonstrate that Bronchom exhibits pharmacological effects in an animal model of allergic asthma. Bronchom mitigated airway hyperresponsiveness, inflammation and airway remodeling evoked by a clinically relevant allergen and accordingly it possesses therapeutic potential for the treatment of asthma.

The Effects of Convalescent Plasma Transfusion on Serum Levels of Macrophage-Associated Inflammatory Biomarkers in Patients with Severe COVID-19.

As an antibody-based therapy, plasma therapy has been used as an emergency therapeutic strategy against severe acute respiratory syndrome coronavirus type 2 infection. Due to the critical role of macrophages in coronavirus disease-19 (COVID-19)-associated hyperinflammation, the main objective of this study was to assess the effect of plasma transfusion on the expression levels of the inflammatory biomarkers involved in activation and pulmonary infiltration of macrophages. The target population included 50 severe hospitalized COVID-19 patients who were randomly assigned into 2 groups, including intervention and control. Serum levels of chemokine (C-C motif) ligand (CCL)-2, CCL-3, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were measured by enzyme-linked immunosorbent assay. Moreover, quantitative real-time polymerase chain reaction (PCR) was carried out to assess the relative expression of nuclear factor (NF)-κB1, NF-κB2, nuclear factor erythroid 2 p45-related factor 2 (NRF-2), and thioredoxin-interacting protein genes. Sampling was done at baseline and 72 h after receiving plasma. The intervention group demonstrated significantly lower serum levels of IL-6, TNF-α, and CCL-3. In addition, real-time PCR data analyses showed that the relative expression of NF-κB2 was significantly declined in the patients who received plasma. The use of convalescent plasma probably has a significant inhibitory effect on the cytokines, chemokines, and inflammatory genes related to macrophage activation, which are closely associated with the worsening of clinical outcomes in severe COVID-19.

The atypical chemokine receptor 2 reduces T cell expansion and tertiary lymphoid tissue but does not limit autoimmune organ injury in lupus-prone B6lpr mice.

The atypical chemokine receptor 2 (ACKR2) is a chemokine scavenger receptor, which limits inflammation and organ damage in several experimental disease models including kidney diseases. However, potential roles of ACKR2 in reducing inflammation and tissue injury in autoimmune disorders like systemic lupus erythematosus (SLE) and lupus nephritis are unknown, as well as its effects on systemic autoimmunity. To characterize functional roles of ACKR2 in SLE, genetic Ackr2 deficiency was introduced into lupus-prone C57BL/6lpr (Ackr2-/- B6lpr) mice. Upon inflammatory stimulation in vitro, secreted chemokine levels increased in Ackr2 deficient tubulointerstitial tissue but not glomeruli. Moreover, Ackr2 expression was induced in kidneys and lungs of female C57BL/6lpr mice developing SLE. However, female Ackr2-/- B6lpr mice at 28 weeks of age showed similar renal functional parameters as wildtype (WT)-B6lpr mice. Consistently, assessment of activity and chronicity indices for lupus nephritis revealed comparable renal injury. Interestingly, Ackr2-/- B6lpr mice showed significantly increased renal infiltrates of CD3+ T and B cells, but not neutrophils, macrophages or dendritic cells, with T cells predominantly accumulating in the tubulointerstitial compartment of Ackr2-/- B6lpr mice. In addition, histology demonstrated significantly increased peribronchial lung infiltrates of CD3+ T cells in Ackr2-/- B6lpr mice. Despite this, protein levels of pro-inflammatory chemokines and mRNA expression of inflammatory mediators were not different in kidneys and lungs of WT- and Ackr2-/- B6lpr mice. This data suggests compensatory mechanisms for sufficient chemokine clearance in Ackr2-deficient B6lpr mice in vivo. Analysis of systemic autoimmune responses revealed comparable levels of circulating lupus-associated autoantibodies and glomerular immunoglobulin deposition in the two genotypes. Interestingly, similar to kidney and lung CD4+ T cell numbers and activation were significantly increased in spleens of Ackr2-deficient B6lpr mice. In lymph nodes of Ackr2-/- B6lpr mice abundance of activated dendritic cells decreased, but CD4+ T cell numbers were comparable to WT. Moreover, increased plasma levels of CCL2 were present in Ackr2-/- B6lpr mice, which may facilitate T cell mobilization into spleens and peripheral organs. In summary, we show that ACKR2 prevents expansion of T cells and formation of tertiary lymphoid tissue, but is not essential to limit autoimmune tissue injury in lupus-prone B6lpr mice.

Discovery of a brain penetrant small molecule antagonist targeting LPA1 receptors to reduce neuroinflammation and promote remyelination in multiple sclerosis

Multiple sclerosis (MS) is a chronic neurological disease characterized by inflammatory demyelination that disrupts neuronal transmission resulting in neurodegeneration progressive disability. While current treatments focus on immunosuppression to limit inflammation and further myelin loss, no approved therapies effectively promote remyelination to mitigate the progressive disability associated with chronic demyelination. Lysophosphatidic acid (LPA) is a pro-inflammatory lipid that is upregulated in MS patient plasma and cerebrospinal fluid (CSF). LPA activates the LPA1 receptor, resulting in elevated CNS cytokine and chemokine levels, infiltration of immune cells, and microglial/astrocyte activation. This results in a neuroinflammatory response leading to demyelination and suppressed remyelination. A medicinal chemistry effort identified PIPE-791, an oral, brain-penetrant, LPA1 antagonist. PIPE-791 was characterized in vitro and in vivo and was found to be a potent, selective LPA1 antagonist with slow receptor off-rate kinetics. In vitro, PIPE-791 induced OPC differentiation and promoted remyelination following a demyelinating insult. PIPE-791 further mitigated the macrophage-mediated inhibition of OPC differentiation and inhibited microglial and fibroblast activation. In vivo, the compound readily crossed the blood–brain barrier and blocked LPA1 in the CNS after oral dosing. Direct dosing of PIPE-791 in vivo increased oligodendrocyte number, and in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS, we observed that PIPE-791 promoted myelination, reduced neuroinflammation, and restored visual evoked potential latencies (VEP). These findings support targeting LPA1 for remyelination and encourage development of PIPE-791 for treating MS patients with advantages not seen with current immunosuppressive disease modifying therapies.

Combination therapy with oncolytic virus and T cells or mRNA vaccine amplifies antitumor effects

Antitumor therapies based on adoptively transferred T cells or oncolytic viruses have made significant progress in recent years, but the limited efficiency of their infiltration into solid tumors makes it difficult to achieve desired antitumor effects when used alone. In this study, an oncolytic virus (rVSV-LCMVG) that is not prone to induce virus-neutralizing antibodies was designed and combined with adoptively transferred T cells. By transforming the immunosuppressive tumor microenvironment into an immunosensitive one, in B16 tumor-bearing mice, combination therapy showed superior antitumor effects than monotherapy. This occurred whether the OV was administered intratumorally or intravenously. Combination therapy significantly increased cytokine and chemokine levels within tumors and recruited CD8+ T cells to the TME to trigger antitumor immune responses. Pretreatment with adoptively transferred T cells and subsequent oncolytic virotherapy sensitizes refractory tumors by boosting T-cell recruitment, down-regulating the expression of PD-1, and restoring effector T-cell function. To offer a combination therapy with greater translational value, mRNA vaccines were introduced to induce tumor-specific T cells instead of adoptively transferred T cells. The combination of OVs and mRNA vaccine also displays a significant reduction in tumor burden and prolonged survival. This study proposed a rational combination therapy of OVs with adoptive T-cell transfer or mRNA vaccines encoding tumor-associated antigens, in terms of synergistic efficacy and mechanism.

Gut Microbiome and Cytokine Profiles in Post-COVID Syndrome.

Recent studies highlight the crucial role of the gut microbiome in post-infectious complications, especially in patients recovering from severe COVID-19. Our research aimed to explore the connection between gut microbiome changes and the cytokine profile of patients with post-COVID syndrome. Using 16S rRNA amplicon sequencing, we analyzed the composition of the gut microbiome in 60 COVID-19 patients over the course of one year. We also measured the levels of serum cytokines and chemokines using the Milliplex system. Our results showed that severe SARS-CoV-2 infection cases, especially those complicated by pneumonia, induce a pro-inflammatory microbial milieu with heightened presence of Bacteroides, Faecalibacterium, and Prevotella_9. Furthermore, we found that post-COVID syndrome is characterized by a cross-correlation of various cytokines and chemokines MDC, IL-1b, Fractalkine, TNFa, FGF-2, EGF, IL-1RA, IFN-a2, IL-10, sCD40L, IL-8, Eotaxin, IL-12p40, and MIP-1b as well as a shift in the gut microbiome towards a pro-inflammatory profile. At the functional level, our analysis revealed associations with post-COVID-19 in homolactic fermentation, pentose phosphate, NAD salvage, and flavin biosynthesis. These findings highlight the intricate interplay between the gut microbiota, their metabolites, and systemic cytokines in shaping post-COVID symptoms. Unraveling the gut microbiome's role in post-infectious complications opens avenues for new treatments for those patients with prolonged symptoms.

Abstract PS18-09: The DARC side of Breast Cancer - DARC, Duffy-null and African ancestry influence in the Triple Negative Breast Cancer tumor microenvironment

Abstract Background. Despite a recent convergence of breast cancer (BC) incidence rates between African American (AA) and European American (EA) women, AA women continue to have ~40% higher mortality rates. BC is a heterogeneous collection of diseases, where African ancestry women are disproportionately burdened with the most aggressive subtype of BC, triple negative BC (TNBC). We have previously reported that the African ancestry-specific Duffy-null allele (FY-) is a risk factor for TNBC diagnoses. FY- is a promoter variant that removes expression of the Duffy Antigen Receptor for Chemokines (DARC/ACKR1), from red blood cells (RBCs). DARC serves as a portal of entry for Plasmodium vivax malaria parasite, and FY- confers immunity, leading to fixation of FY- among Sub-Saharan African populations. DARC is an atypical chemokine receptor that functions to modulate chemokine levels in circulation and tissues to aid immune cell recruitment. DARC is also expressed on breast tumor epithelial cells, however the significance of DARC in the TNBC tumor microenvironment (TME) has not fully been explored. Leveraging our African ancestry enriched International Study for the Center of Breast Cancer Subtypes cohort and in vivo murine models, we employed multi-omics approaches to explore the function of FY- and DARC expression across cell types in the TNBC TME. Genomics. Using transcriptomic data from the TCGA BC cohort, we have previously reported that DARC expression is highly correlated with deconvoluted immune cell abundance using CIBERSORT. To build upon this finding among TNBC patients, we have analyzed RNAseq data from two cohorts of African ancestry women to explore correlation of DARC expression status and immune cell abundance. Among TNBC cases, we report that higher DARC expression was significantly associated with increased immune cell abundance, with increases in B cell, T cell and monocyte populations. Using whole genome sequencing data across 86 African ancestry cases, we are currently investigating germline FY- status with mutational signatures. Circulating biomarkers. Patient plasma was collected prior to surgery, where Luminex multiplex assays were performed to quantify levels of >40 circulating chemokines and cytokines. FY- status has been determined for ~550 patients using single-plex or multiplex PCR genotyping methods from germline saliva DNA. Our univariate analyses show several circulating chemokines, including CCL2, CXCL2, CXCL6 and CXCL11, have significant differences between FY- and non-FY- BC patients. Curation of clinicopathological variables for multivariate analysis is ongoing. Murine models. We crossed C3(1)Tag BC transgenic mice with a DARC knock-out (KO) line to generate mice lacking DARC expression that spontaneously develop tumors in the mammary fat pads. The C3(1)Tag model is reported to have similar gene expression profiles to basal-like TNBC tumors. Tumors were collected at pre-determined time points of 25 and 30 weeks, where DARC KO mice had increased tumor size and tumor burden at 30 weeks. Initial fluorescent staining revealed an increase in immune cell infiltration in the TME of DARC expressing mice compared to DARC KO mice. To further explore this, we employed multiplex staining across a representative cohort of DARC expressing and KO mice at 25 and 30 weeks to capture the specific immune cell populations infiltrating the TME. Conclusions. TNBC tumors have worse prognosis due to lack of targeted therapeutic options. Increased immunogenicity of TNBC tumors, especially among African ancestry women may be driven in part by DARC expression. Characterization of FY- status, in coordination with DARC among other cell types in the TNBC TME could present new opportunities for biomarker and/or therapeutic development to improve prognosis for these underserved populations. Citation Format: Rachel Martini, Stevens Patino, Emma Guyonnet, Brian Stonaker, Isra Elhussein, Julie Sahler, Avery August, Nancy Manley, Rick Kittles, Clayton Yates, Lisa Newman, Melissa Davis. The DARC side of Breast Cancer - DARC, Duffy-null and African ancestry influence in the Triple Negative Breast Cancer tumor microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS18-09.

A comprehensive analysis of the immune system in healthy Vietnamese people

Lifestyle, diet, socioeconomic status and genetics all contribute to heterogeneity in immune responses. Vietnam is plagued with a variety of health problems, but there are no available data on immune system values in the Vietnamese population. This study aimed to establish reference intervals for immune cell parameters specific to the healthy Vietnamese population by utilizing multi-color flow cytometry (MCFC). We provide a comprehensive analysis of total leukocyte count, quantitative and qualitative shifts within lymphocyte subsets, serum and cytokine and chemokine levels and functional attributes of key immune cells including B cells, T cells, natural killer (NK) cells and their respective subpopulations. By establishing these reference values for the Vietnamese population, these data contribute significantly to our understanding of the human immune system variations across diverse populations. These data will be of substantial comparative value and be instrumental in developing personalized medical approaches and optimizing diagnostic strategies for individuals based on their unique immune profiles.

Metabolic modulation unleashes immune vigor in murine pancreas tumors

Abstract Our study investigates the transformation of the pancreatic ductal adenocarcinoma tumor microenvironment from an immune-excluded to an immune-inflamed state using metabolic inhibitors metformin and mitochondria-selective conjugates, MMe and pCF3-MMe. In murine models of pancreas cancer, MMe and pCF3-MMe exhibited potent inhibition of tumor growth. Flow cytometry indicated increased levels of total T cells, activated CD8+, and NK T cells in MMe and pCF3-MMe treated mice. The anti-tumor effects of these metabolic inhibitors were reversed upon CD8+ T cell immunodepletion. In vitro experiments demonstrated elevated levels of pro-inflammatory chemokines in MMe-treated tumor cells, aligned with increased CD8+ T cell infiltration. Furthermore, metabolic inhibitor treatment activated both helper CD4+ and cytolytic CD8+ T cells, with the latter demonstrating increased glycolysis and acidification rates indicative of enhanced activation. Flow cytometry corroborated these findings, revealing heightened proliferation and activation markers on treated T cells. In addition to the direct effects on T cells, immunofluorescence microscopy suggested the activation of the mitochondrial antiviral-signaling protein (MAVS) pathway in tumor cells and an increase in apoptosis as indicated by the significant increase in caspase 3/7. Our findings highlight the potential of metabolic inhibitors to activate anti-tumor immune responses directly and indirectly in murine pancreas tumors.

Pre-Weaning Overfeeding Promotes Prepubertal Obesity and Evokes Sexual Dysfunction in Young Adult Rats

Obesity in early life can contribute to several health issues in adulthood, including diabetes, high blood pressure, and high cholesterol, and these conditions are associated with sexual dysfunction. Here, we hypothesized that pre-weaning overfeeding can have long lasting effects on body fat distribution and erectile function that will be observed in young adult males. On postnatal day (PND 1), Wistar rats were separated into two groups: normal litter (NL) with 10 pups per dam, or small litter (SL) with 3 pups per dam. Male rats (n = 8-10 per group) were then assessed at two different time points: during the prepubertal stage (PND 40) and in adulthood (PND 120). Erectile function was assessed only after sexual maturation (i.e., PND 120). Body weight, retroperitoneal and perigonadal adipose tissue (fat pad weight/100g of the body) were evaluated. Following euthanasia at PND 120, the corpus cavernosum (CC) was removed to perform functional studies using a strip myograph. Contractile and relaxation responses were evaluated by electrical field stimulation (EFS) and concentration-response curves (CRC) to phenylephrine [Phe, 1nM-30μM, millinewton (mN)] or acetylcholine (Ach 1nM-30μM, % relaxation) were performed. At the prepubertal stage, SL rats had higher body weight (NL 149±3 vs. SL 186±5, g, p<0.05), and increased retroperitoneal (NL 0.17±0.01 vs. SL 0.32±0.03, g/100g, p<0.05) and perigonadal (NL 0.38± 0.02 vs. SL 0.45±0.03, g/100g, p<0.05) adipose tissue. At PND 120, SL group presented increased body weight (NL 534.2.7±13 vs. SL 631.6±10, g, p<0.05), retroperitoneal (NL 1.31±0.11 vs. SL 1.82±0.14, g/100g, p<0.05) and perigonadal (NL 1.74±0.10 vs. SL 2.18±0.15, g/100g, p<0.05) adipose tissue when compared with NL group. The CC of SL rats at PND 120 had increased contraction induced by EFS for all frequencies (1 Hz: NL 0.5 ± 0.2 mN vs. SL 0.9 ± 0.2 mN; 4 Hz: NL 1.2 ± 0.3 mN vs. SL 1.8 ± 0.2 mN; 16 Hz: NL 1.9 ± 0.4 mN vs. SL 3 ± 0.4 mN), whereas the relaxation was decreased in SL (1 Hz: NL 0.77 ± 0.18% vs. SL 0.27 ± 0.09%; 4 Hz: NL 2.48 ± 0.26% vs. SL 1.36 ± 0.41%; 16 Hz: NL 3.06 ± 0.28% vs. SL 2.39 ± 0.53%). CRC showed increased contractile response to Phe (Emax NL 2.3±0.3 vs. SL 3.8± 0.6, mN, p=0.05) and reduced potency of ACh (pEC50 NL 6.4±0.6 vs. SL 4.9±0.7, mN, p=0.1) in the CC of SL. Incubation with indomethacin (10μM) abolished the differences in the contractile response induced by EFS or Phe between SL and NL, suggesting a heightened inflammatory response. Moreover, the CC of SL rats presented increased expression of Phospho-p44/42 MAPK (NL 0.9±0.25 vs. SL 1.3±0.11, p=0.2). Finally, proteome profiler cytokine array of serum samples from SL animals showed increased circulating levels of cytokines and chemokines associated with obesity, aging, and vascular dysfunction. Together, these data suggest that early prepubertal obesity has long-lasting negative implications on sexual function in young adulthood. National Institutes of Health (NIH); American Heart Association (AHA); Fulbright Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MnO2 nanoparticle improves the immunosuppressive characteristics of tumor-associated mesenchymal stromal cells

Tumor-associated marrow mesenchymal stem/stromal cells have immunosuppressive effects on immune cells within the tumor microenvironment in various cancers. However, effective treatments targeting these cells are currently limited. Here, we developed a nanoparticle of MnO2 aiming to inhibit the immunosuppressive properties of Tumor-Associated Marrow Mesenchymal stem/stromal cells. We investigated the physicochemical characteristics of MnO2 and its potential for alleviating hypoxia in IL17 and IFNγ transformed Tumor-Associated Marrow Mesenchymal stem/stromal cells. The Tumor-Associated Marrow Mesenchymal stem/stromal cells had higher expression level of chemokines and immunosuppressors, which promotes the progression of tumors and inhibits the immune cells, leading to tumor metastasis and angiogenesis. After treatment of the nanoparticle of MnO2, the expression level of chemokines had significantly decreased due to the unique properties of MnO2. Furthermore, the nanoparticle decreased the expression level of immunosuppressors of Tumor-Associated Marrow Mesenchymal stem/stromal cells and rescued the proliferation of T lymphocytes cocultured with the cells. In conclusion, our findings demonstrate that MnO2 nanoparticles effectively improve the hypoxic microenvironment and alleviate immunosuppression caused by Tumor-Associated Marrow Mesenchymal stem/stromal cells. The utilization of this novel approach exhibits promising potential as a therapeutic strategy for addressing diverse types of cancers.

Methamphetamine and the Synthetic Cathinone 3,4-Methylenedioxypyrovalerone (MDPV) Produce Persistent Effects on Prefrontal and Striatal Microglial Morphology and Neuroimmune Signaling Following Repeated Binge-like Intake in Male and Female Rats.

Psychostimulants alter cellular morphology and activate neuroimmune signaling in a number of brain regions, yet few prior studies have investigated their persistence beyond acute abstinence or following high levels of voluntary drug intake. In this study, we examined the effects of the repeated binge-like self-administration (96 h/week for 3 weeks) of methamphetamine (METH) and 21 days of abstinence in female and male rats on changes in cell density, morphology, and cytokine levels in two addiction-related brain regions-the prefrontal cortex (PFC) and dorsal striatum (DStr). We also examined the effects of similar patterns of intake of the cocaine-like synthetic cathinone derivative 3,4-methylenedioxypyrovalerone (MDPV) or saline as a control. Robust levels of METH and MDPV intake (~500-1000 infusions per 96 h period) were observed in both sexes. We observed no changes in astrocyte or neuron density in either region, but decreases in dendritic spine densities were observed in PFC pyramidal and DStr medium spiny neurons. The microglial cell density was decreased in the PFC of METH self-administering animals, accompanied by evidence of microglial apoptosis. Changes in microglial morphology (e.g., decreased territorial volume and ramification and increased cell soma volume) were also observed, indicative of an inflammatory-like state. Multiplex analyses of PFC and DStr cytokine content revealed elevated levels of various interleukins and chemokines only in METH self-administering animals, with region- and sex-dependent effects. Our findings suggest that voluntary binge-like METH or MDPV intake induces similar cellular perturbations in the brain, but they are divergent neuroimmune responses that persist beyond the initial abstinence phase.

Inflammatory Protein Signatures as Predictive Disease-Specific Markers for Non-Alcoholic Steatohepatitis (NASH).

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic disease worldwide, consisting of a broad spectrum of diseases such as simple steatosis (NAFL), non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatic inflammation plays a key role in the pathophysiology of NAFLD. Inflammatory mediators such as cytokines and chemokines are considered as contributing factors to NAFLD development and progression. In the present study, we aimed to investigate the inflammatory protein signatures as predictive disease-specific markers for non-alcoholic fatty liver disease (NAFLD). This cross-sectional study included healthy control (n = 64), NAFL (n = 109), and NASH (n = 60) human subjects. Serum concentrations of various cytokines and chemokines were evaluated using sensitive multiplex assays. We used principal component analysis (PCoA) to reveal distinct differences in the levels of cytokines and chemokines between each of the study groups. Further, a random forest classification model was developed to identify the panel of markers that could predict diseases. The protein-protein network analysis was performed to determine the various signaling pathways associated with the disease-specific panel of markers. Serum concentrations of TNF-α, IL-1β, IL-1ra, G-CSF, PDGF-BB, MCP-1, MIP-1a, MIP-1b, RANTES, eotaxin, IL-8 and IP-10 were significantly increased in NASH group as compared to control group. Furthermore, serum concentrations of IL-9 and IL-13 were significantly lower in the NASH group, whereas IL-2 levels were significantly decreased in the NAFL group when compared to the control group. PCoA results demonstrated statistically significant differences in cytokines and chemokines between each of the study groups (PERMANOVA p = 0.001; R2 = 0.102). RANTES, IL-1ra, MIP-1b, IL-2, and G-CSF could differentiate the NAFL group from the controls; G-CSF, IL-1ra, TNF-α, RANTES, and IL-9 could differentiate the NASH group from the controls; and G-CSF, IL-9, IL-13, eotaxin, and TNF- α could differentiate the NASH group from the NAFL group. Our protein-protein network revealed that these markers are involved in cytokine-cytokine receptor interaction, Th1 and Th2 cell differentiation, TNF, chemokine, JAK/STAT, P13K/Akt, TLR, NOD-like receptor, NF-kB, and adipocytokine signaling pathways which might be responsible for disease pathogenesis.Our study findings revealed a set of distinct cytokine and chemokine markers and they might be considered as biomarkers in distinguishing NASH from NAFL. Future multicentre studies with larger sample size are recommended to determine the potential utility of these panels of markers.

Interferon-γ+ Th1 activates intrahepatic resident memory T cells to promote HBsAg loss by inducing M1 macrophage polarization.

The immune mechanism underlying hepatitis B surface antigen (HBsAg) loss, particularly type I inflammatory response, during pegylated interferon-α (PEG-IFN) therapy remains unclear. In this study, we aimed to elucidate such immune mechanisms. Overall, 82 patients with chronic hepatitis B (CHB), including 41 with HBsAg loss (cured group) and 41 uncured patients, received nucleos(t)ide analogue and PEG-IFN treatments. Blood samples from all patients, liver tissues from 14 patients with CHB, and hepatic perfusate from 8 liver donors were collected for immune analysis. Jurkat, THP-1 and HepG2.2.15 cell lines were used in cell experiments. The proportion of IFN-γ+ Th1 cells was higher in the cured group than in the uncured group, which was linearly correlated with HBsAg decline and alanine aminotransferase (ALT) levels during treatment. However, CD8+ T cells were weakly associated with HBsAg loss. Serum and intrahepatic levels of Th1 cell-associated chemokines (C-X-C motif chemokine ligand [CXCL] 9, CXCL10, CXCL11, IFN-γ) were significantly lower in the cured patients than in patients with a higher HBsAg quantification during therapy. Serum from cured patients induced more M1 (CD68+CD86+ macrophage) cells than that from uncured patients. Patients with chronic HBV infection had significantly lower proportions of CD86+ M1 and CD206+ M2 macrophages in their livers than healthy controls. M1 polarization of intrahepatic Kupffer cells promoted HBsAg loss by upregulating the effector function of tissue-resident memory T cells with increased ALT levels. IFN-γ+ Th1 activates intrahepatic resident memory T cells to promote HBsAg loss by inducing M1 macrophage polarization.

Dysregulation of interleukin-8 is involved in the onset and relapse of schizophrenia: An independent validation and meta-analysis

As a major mental health disorder, symptoms of schizophrenia (SCZ) include delusions, reduced motivation, hallucinations, reduced motivation and a variety of cognitive disabilities. Many of these symptoms are now known to be associated with abnormal regulation of the immune system. Low blood levels of cytokines and chemokines have been suggested to be one of the underlying causes of SCZ. However, their biological roles at different stages of SCZ remain unclear. Our objective was to investigate expression patterns of cytokines and chemokines at different stages of onset and relapse in SCZ patients and to conduct an analysis of their relationship to disease progression. We also aimed to identify immune features associated with different disease trajectories in patients with SCZ.Gene set enrichment analysis (GSEA) was used to interrogate the GSE27383 dataset and identify key genes associated with inflammation. These results led us to recruit 36 healthy controls, 40 patients with first-episode psychosis (FEP), and 39 patients with SCZ relapse. Meso Scale Discovery technology was used to independently validate serum levels of 35 cytokines and chemokines. This was followed by a meta-analysis to gain a more comprehensive understanding of the role of interleukin-8 (IL-8/CXCL8) in SCZ.Analysis of the GSE27383 database revealed 3596 genes with distinct expression patterns. A significant portion of these genes were identified as inflammation-related and showed remarkable enrichment in three key pathways: IL-17, cytokine-cytokine receptor, and AGE-RAGE signaling in diabetic complications. We observed co-expression of CXCL8 and IL-16 within these three pathways. In a subsequent analysis of independently validated samples, a notable discrepancy was detected in the inflammatory status between individuals experiencing FEP and those in relapse. In particular, expression of CXCL8 demonstrated superior predictive capability in FEP and relapsed patients. Notably, results of the meta-analysis confirmed that Chinese and European populations were consistent with the overall results (Z = 4.60, P < 0.001; Z = 3.70, P < 0.001). However, in the American subgroup, there was no significant difference in CXCL8 levels between patients with SCZ compared to healthy controls (Z = 1.09, P = 0.277). Our findings suggest that the inflammatory response in patients with SCZ differs across the different stages, with CXCL8 emerging as a potential predictive factor. Collectively, our data suggest that CXCL8 has the potential to serve as a significant immunological signature of SCZ subtypes.Trial registration: The clinical registration number for this trial is ChiCTR2100045240 (Registration Date: 2021/04/09).

Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells.

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa. Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

Crtc1 deficiency protects against sepsis-associated acute lung injury through activating akt signaling pathway

BackgroundInterplay between systemic inflammation and programmed cell death contributes to the pathogenesis of acute lung injury (ALI). cAMP-regulated transcriptional coactivator 1 (CRTC1) has been involved in the normal function of the pulmonary system, but its role in ALI remains unclear.Methods and resultsWe generated a Crtc1 knockout (KO; Crtc1−/−) mouse line. Sepsis-induced ALI was established by cecal ligation and puncture (CLP) for 24 h. The data showed that Ctrc1 KO substantially ameliorated CLP-induced ALI phenotypes, including improved lung structure destruction, reduced pulmonary vascular permeability, diminished levels of proinflammatory cytokines and chemokines, compared with the wildtype mice. Consistently, in lipopolysaccharide (LPS)-treated RAW264.7 cells, Crtc1 knockdown significantly inhibited the expression of inflammatory effectors, including TNF-α, IL-1β, IL-6 and CXCL1, whereas their expressions were significantly enhanced by Crtc1 overexpression. Moreover, both Crtc1 KO in mice and its knockdown in RAW264.7 cells dramatically reduced TUNEL-positive cells and the expression of pro-apoptotic proteins. In contrast, Crtc1 overexpression led to an increase in the pro-apoptotic proteins and LPS-induced TUNEL-positive cells. Mechanically, we found that the phosphorylation of Akt was significantly enhanced by Crtc1 knockout or knockdown, but suppressed by Crtc1 overexpression. Administration of Triciribine, an Akt inhibitor, substantially blocked the protection of Crtc1 knockdown on LPS-induced inflammation and cell death in RAW264.7 cells.ConclusionsOur study demonstrates that CRTC1 contribute to the pathological processes of inflammation and apoptosis in sepsis-induced ALI, and provides mechanistic insights into the molecular function of CRTC1 in the lung. Targeting CRTC1 would be a promising strategy to treat sepsis-induced ALI in clinic.