Tumor-neutrophil cross talk orchestrates the tumor microenvironment to determine the bladder cancer progression.

The immune landscape of bladder cancer progression is not fully understood, and effective therapies are lacking in advanced bladder cancer. Here, we visualized that bladder cancer cells recruited neutrophils by secreting interleukin-8 (IL-8); in turn, neutrophils played dual functions in bladder cancer, including hepatocyte growth factor (HGF) release and CCL3highPD-L1high super-immunosuppressive subset formation. Mechanistically, c-Fos was identified as the mediator of HGF up-regulating IL-8 transcription in bladder cancer cells, which was central to the positive feedback of neutrophil recruitment. Clinically, compared with serum IL-8, urine IL-8 was a better biomarker for bladder cancer prognosis and clinical benefit of immune checkpoint blockade (ICB). Additionally, targeting neutrophils or hepatocyte growth factor receptor (MET) signaling combined with ICB inhibited bladder cancer progression and boosted the antitumor effect of CD8+ T cells in mice. These findings reveal the mechanism by which tumor-neutrophil cross talk orchestrates the bladder cancer microenvironment and provide combination strategies, which may have broad impacts on patients suffering from malignancies enriched with neutrophils.

The splicing factor U2AF1 contributes to cancer progression through a noncanonical role in translation regulation.

Somatic mutations in the genes encoding components of the spliceosome occur frequently in human neoplasms, including myeloid dysplasias and leukemias, and less often in solid tumors. One of the affected factors, U2AF1, is involved in splice site selection, and the most common change, S34F, alters a conserved nucleic acid-binding domain, recognition of the 3' splice site, and alternative splicing of many mRNAs. However, the role that this mutation plays in oncogenesis is still unknown. Here, we uncovered a noncanonical function of U2AF1, showing that it directly binds mature mRNA in the cytoplasm and negatively regulates mRNA translation. This splicing-independent role of U2AF1 is altered by the S34F mutation, and polysome profiling indicates that the mutation affects translation of hundreds of mRNA. One functional consequence is increased synthesis of the secreted chemokine interleukin 8, which contributes to metastasis, inflammation, and cancer progression in mice and humans.

CD9 and Aryl Hydrocarbon Receptor Are Markers of Human CD19+CD14+ Atypical B Cells and Are Dysregulated in Systemic Lupus Erythematous Disease.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor whose expression regulates immune cell differentiation. Single-cell transcriptomic profiling was used to ascertain the heterogeneity of AHR expression in human B cell subpopulations. We identified a unique population of B cells marked by expression of AHR, CD9, and myeloid genes such as CD14 and CXCL8. Results were confirmed directly in human PBMCs and purified B cells at the protein level. TLR9 signaling induced CD14, CD9, and IL-8 protein expression in CD19+ B cells. CD14-expressing CD9+ B cells also highly expressed AHR and atypical B cell markers such as CD11c and TBET. In patients with active lupus disease, CD14+ and CD9+ B cells are dysregulated, with loss of CD9+ B cells strongly predicting disease severity and demonstrating the relevance of CD9+ B cells in systemic lupus erythematosus and autoimmune disease.

Structural basis of CXC chemokine receptor 2 activation and signalling.

Chemokines and their receptors mediate cell migration, which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer1. Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor-chemokine recognition2-4, less is known about endogenous chemokine-induced receptor activation and G-protein coupling. Here we present the cryo-electron microscopy structures of interleukin-8 (IL-8, also known as CXCL8)-activated human CXC chemokine receptor 2 (CXCR2) in complex with Gi protein, along with a crystal structure of CXCR2 bound to a designed allosteric antagonist. Our results reveal a unique shallow mode of binding between CXCL8 and CXCR2, and also show the interactions between CXCR2 and Gi protein. Further structural analysis of the inactive and active states of CXCR2 reveals a distinct activation process and the competitive small-molecule antagonism of chemokine receptors. In addition, our results provide insights into how a G-protein-coupled receptor is activated by an endogenous protein molecule, which will assist in the rational development of therapeutics that target the chemokine system for better pharmacological profiles.

Mechanistic computational modeling of monospecific and bispecific antibodies targeting interleukin-6/8 receptors.

The spread of cancer from organ to organ (metastasis) is responsible for the vast majority of cancer deaths; however, most current anti-cancer drugs are designed to arrest or reverse tumor growth without directly addressing disease spread. It was recently discovered that tumor cell-secreted interleukin-6 (IL-6) and interleukin-8 (IL-8) synergize to enhance cancer metastasis in a cell-density dependent manner, and blockade of the IL-6 and IL-8 receptors (IL-6R and IL-8R) with a novel bispecific antibody, BS1, significantly reduced metastatic burden in multiple preclinical mouse models of cancer. Bispecific antibodies (BsAbs), which combine two different antigen-binding sites into one molecule, are a promising modality for drug development due to their enhanced avidity and dual targeting effects. However, while BsAbs have tremendous therapeutic potential, elucidating the mechanisms underlying their binding and inhibition will be critical for maximizing the efficacy of new BsAb treatments. Here, we describe a quantitative, computational model of the BS1 BsAb, exhibiting how modeling multivalent binding provides key insights into antibody affinity and avidity effects and can guide therapeutic design. We present detailed simulations of the monovalent and bivalent binding interactions between different antibody constructs and the IL-6 and IL-8 receptors to establish how antibody properties and system conditions impact the formation of binary (antibody-receptor) and ternary (receptor-antibody-receptor) complexes. Model results demonstrate how the balance of these complex types drives receptor inhibition, providing important and generalizable predictions for effective therapeutic design.

A Microphysiological Device to Model the Choriodecidual Interface Immune Status during Pregnancy.

During human pregnancy the chorion (fetal) lines decidua (maternal) creating the feto-maternal interface. Despite their proximity, resident decidual immune cells remain quiescent during gestation and do not invade the chorion. Infection and infiltration of activated immune cells toward the chorion are often associated with preterm birth. However, the mechanisms that maintain choriodecidual immune homeostasis or compromise immune barrier functions remain unclear. To understand these processes, a two-chamber microphysiological system (MPS) was created to model the human choriodecidual immune interface under normal and infectious conditions in vitro. This MPS has outer (fetal chorion trophoblast cells) and inner chambers (maternal decidual + CD45+ cells [70:30 ratio]) connected by microchannels. Decidual cells were treated with LPS to mimic maternal infection, followed by immunostaining for HLA-DR and HLA-G, immune panel screening by imaging cytometry by time of flight, and immune regulatory factors IL-8 and IL-10, soluble HLA-G, and progesterone (ELISA). LPS induced a proinflammatory phenotype in the decidua characterized by a decrease in HLA-DR and an increase in IL-8 compared with controls. LPS treatment increased the influx of immune cells into the chorion, indicative of chorionitis. Cytometry by time of flight characterized immune cells in both chambers as active NK cells and neutrophils, with a decrease in the abundance of nonproinflammatory cytokine-producing NK cells and T cells. Conversely, chorion cells increased progesterone and soluble HLA-G production while maintaining HLA-G expression. These results highlight the utility of MPS to model choriodecidual immune cell infiltration and determine the complex maternal-fetal crosstalk to regulate immune balance during infection.

Fever-like temperature impacts on Staphylococcus aureus and Pseudomonas aeruginosa interaction, physiology, and virulence both in vitro and in vivo.

BACKGROUND: Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39 °C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37 °C, by using RNAseq and physiological assays including in vivo experiments. RESULTS: In general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes in both species. Altered metabolic features at 39 °C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39 °C, they upregulated ethanol oxidation-related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pmsα, and pmsß genes at 39 °C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphyloxantin production, and a decrease in biofilm formation at 39 °C. On the side of PA monocultures, we observed an increase in extracellular lipase and protease and biofilm formation at 39 °C along with a decrease in the motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both in vitro and in vivo. S. aureus monocultured at 39οC showed a decrease in cellular invasion and an increase in IL-8-but not in IL-6-production by A549 cell line. PA also decreased its cellular invasion when monocultured at 39 °C and did not induce any change in IL-8 or IL-6 production. PA strongly increased cellular invasion when co-cultured at 37 and 39 °C. Finally, we observed increased lethality in mice intranasally inoculated with S. aureus monocultures pre-incubated at 39 °C and even higher levels when inoculated with co-cultures. The bacterial burden for P. aeruginosa was higher in liver when the mice were infected with co-cultures previously incubated at 39 °C comparing with 37 °C. CONCLUSIONS: Our results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.

Chlamydia trachomatis T3SS Effector CT622 Induces Proinflammatory Cytokines Through TLR2/TLR4-Mediated MAPK/NF-κB Pathways in THP-1 Cells.

BACKGROUND: The pathogenesis of Chlamydia trachomatis is associated with the induction of the host inflammatory response; however, the precise underlying molecular mechanisms remain poorly understood. METHODS: CT622, a T3SS effector protein, has an important role in the pathogenesis of C trachomatis; however, whether CT622 can induce a host inflammatory response is not understood. Our findings demonstrate that CT622 induces the expression of interleukins 6 and 8 (IL-6 and IL-8). Mechanistically, these effects involve the activation of the MAPK/NF-κB signaling pathways (mitogen-activated protein kinase/nuclear factor κB). RESULTS: Interestingly, we demonstrated that the suppression of toll-like receptor 4 using small interfering RNA markedly reduced the phosphorylation of ERK, p38, JNK, and IκBα, concomitant with a significant decrease in IL-6 and IL-8 secretion. Conversely, disruption of toll-like receptor 2 abrogated the CT622-induced upregulation of IL-8 and activation of ERK, whereas IL-6 expression and p38, JNK, and IκBα phosphorylation were unaffected. CONCLUSIONS: Taken together, these results indicate that CT622 contributes to the inflammatory response through the toll-like receptor 2/4-mediated MAPK/NF-κB pathways, which provides insight into the molecular pathology of C trachomatis infection.

Olink Proteomics-Based Exploration of Immuno-Oncology-Related Biomarkers Leading to Lung Adenocarcinoma Progression.

INTRODUCTION: It is crucial to investigate the distinct proteins that contribute to the advancement of lung cancer. MATERIAL AND METHODS: We analyzed the expression levels of 92 immuno-oncology-related proteins in 96 pairs of lung adenocarcinoma tissue samples using Olink proteomics. The differentially expressed proteins (DEPs) were successively screened in tumor and paraneoplastic groups, early and intermediate-late groups by a nonparametric rank sum test, and the distribution and expression levels of DEPs were determined by volcano and heat maps, etc., and the area under the curve was calculated. RESULTS: A total of 24 DEPs were identified in comparisons between tumor and paracancerous tissues. Among them, interleukin-8 (IL8) and chemokine (C-C motif) ligand 20 (CCL20) as potential markers for distinguishing tumor tissues. Through further screening, it was found that interleukin-6 (IL6) and vascular endothelial growth factor A (VEGFA) may be able to lead to tumor progression through the JaK-STAT signaling pathway, Toll-like receptor signaling pathway and PI3K/AKT signaling pathway. Interestingly, our study revealed a down-regulation of IL6 and VEGFA in tumor tissues compared to paracancerous tissues. CONCLUSIONS: IL8 + CCL20 (AUC: 0.7056) have the potential to differentiate tumor tissue from paracancerous tissue; IL6 + VEGFA (AUC: 0.7531) are important protein markers potentially responsible for tumor progression.

TLR8-activating miR-146a-3p is an intermediate signal contributing to fetal membrane inflammation in response to bacterial LPS.

Preterm birth is the largest contributor to neonatal morbidity and is often associated with chorioamnionitis, defined as inflammation/infection of the fetal membranes (FMs). Chorioamnionitis is characterised by neutrophil infiltration of the FMs and is associated with elevated levels of the neutrophil chemoattractant, interleukin (IL)-8 and the proinflammatory cytokine, IL-1ß. While FMs can respond to infections through innate immune sensors, such as toll-like receptors (TLRs), the downstream mechanisms by which chorioamnionitis arises are not fully understood. A novel group of non-classical microRNAs (miR-21a, miR-29a, miR-146a-3p, Let-7b) function as endogenous danger signals by activating the ssRNA viral sensors TLR7 and TLR8. In this study, the pro-inflammatory roles of TLR7/TLR8-activating miRs were examined as mediators of FM inflammation in response to bacterial lipopolysaccharide (LPS) using an in vitro human FM explant system, an in vivo mouse model of pregnancy, and human clinical samples. Following LPS exposure, miR-146a-3p was significantly increased in both human FM explants and wild-type mouse FMs. Expression of miR-146a-3p was also significantly elevated in FMs from women with preterm birth and chorioamnionitis. FM IL-8 and inflammasome-mediated IL-1ß production in response to LPS was dependent on miR-146a-3p and TLR8 downstream of TLR4 activation. In wild-type mice, LPS exposure increased FM IL-8 and IL-1ß production and induced preterm birth. In TLR7-/-/TLR8-/- mice, LPS exposure was able to initiate but not sustain preterm birth, and FM inflammation was reduced. Together, we demonstrate a novel signalling mechanism at the maternal-fetal interface in which TLR8-activating miR-146a-3p acts as an intermediate danger signal to drive FM inflammasome-dependent and -independent mechanisms of inflammation and, thus, may play a role in chorioamnionitis and subsequent preterm birth.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

Enhanced lipid biosynthesis in oral squamous cell carcinoma cancer-associated fibroblasts contributes to tumor progression: Role of IL8/AKT/p-ACLY axis.

Lipid metabolic reprogramming of tumor cells has been proven to play a critical role in tumor initiation and development. However, lipid metabolism in cancer-associated fibroblasts (CAFs) has rarely been studied, particularly in CAFs of oral squamous cell carcinoma (OSCC). Additionally, the molecular mechanism by which tumor cells regulate lipid metabolism in fibroblasts is unclear. In this study, we found that phosphorylated ATP citrate lyase (p-ACLY), a key lipid metabolic enzyme, was upregulated in OSCC CAFs. Compared to paracancerous normal fibroblasts, CAFs showed enhanced lipid synthesis, such as elevated cytosolic acetyl-CoA level and accumulation of lipid droplets. Conversely, reduction of p-ACLY level blocked this biological process. In addition, blocking lipid synthesis in CAFs or inhibiting fatty acid uptake by OSCC cells reduced the promotive effects of CAFs on OSCC cell proliferation, invasion, and migration. These findings suggested that CAFs are one of lipid sources required for OSCC progression. Mechanistically, AKT signaling activation was involved in the upregulation of p-ACLY level and lipid synthesis in CAFs. Interleukin-8 (IL8), an exocrine cytokine of OSCC cells, could activate AKT and then phosphorylate ACLY in fibroblasts. This study suggested that the IL8/AKT/p-ACLY axis could be considered as a potential target for OSCC treatment.

Tumour cell-derived serglycin promotes IL-8 secretion of CAFs in gastric cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs)-derived IL-8 plays important roles in chemoresistance, immunosuppression, and lymph node metastasis of gastric cancer. However, the mechanisms underlying IL-8 production in CAFs remains unclear. METHODS: DNA pulldown assay was performed to identify the transcription factors responsible for IL-8 expression in CAFs, which was further verified using CHIP-qPCR and DNA agarose gel electrophoresis assays. The cellular localisation of IL-8 was analysed using multiplex immunofluorescence (MxIF). RESULTS: MxIF demonstrated that IL-8 was mainly produced by CAFs in gastric cancer. Lysine[K]-specific demethylase 5B (KDM5B) was identified as an IL-8 transcription factor in CAFs, and the binding of KDM5B to phosphorylated RB1 limited the transcriptional regulation of IL-8 in gastric cancer cells. Serglycin (SRGN) secreted by tumour cells activated the CD44/c-Myc pathway to upregulate KDM5B expression, thereby promoting IL-8 production in CAFs. Furthermore, tumour-associated neutrophils (TANs)-derived regenerating family member 4 (REG4) upregulates SRGN expression by activating cAMP-responsive element binding protein 1 (CREB1) in gastric cancer cells. Thus, the SRGN-IL-8-TANs-SRGN loop, which facilitates tumour progression, has been explored in gastric cancer. CONCLUSIONS: This study revealed the mechanisms of the preferential production of IL-8 by CAFs in gastric cancer, and paves the way for potential new therapeutic strategies for gastric cancer.

Tumor cell-derived LC3B+extracellular vesicles mediate the crosstalk between tumor microenvironment and immunotherapy efficacy in hepatocellular carcinoma via the HSP90α-IL-6/IL-8 signaling axis.

BACKGROUND: Inflammatory factors are being recognized as critical modulators of host antitumor immunity in liver cancer. We have previously shown that tumor cell-released LC3B positive extracellular vesicles (LC3B+ EVs) are responsible for malignant progression by dampening antitumor immunity. However, the relationship between LC3B+ EVs and inflammatory factors in the regulation of the liver cancer microenvironment remains unclear. METHODS: Flow cytometry analyses were performed to examine the panel of 12 cytokines, the main source of positive cytokines, and plasma LC3B+ EVs carrying HSP90α in peripheral blood of liver cancer patients. We correlated the levels of plasma IL-6, IL-8 with LC3B+ EVs carrying HSP90α and with prognosis. In vitro culture of healthy donor leukocytes with liver cancer-derived LC3B+ EVs was performed to evaluate the potential effect of blocking HSP90α, IL-6 or IL-8 alone or in combination with PD-1 inhibitor on CD8+ T cell function. We also investigated the potential associations of MAP1LC3B, HSP90AA1, IL6 or IL8 with immunotherapy efficacy using the TCGA databases. RESULTS: In liver cancer patients, plasma IL-6 and IL-8 levels were significantly higher than in healthy controls and associated with poor clinical outcome. In peripheral blood, levels of plasma LC3B+ EVs carrying HSP90α were significantly elevated in HCC patients and positively associated with IL-6 and IL-8 levels, which are predominantly secreted by monocytes and neutrophils. Moreover, LC3B+ EVs from human liver cancer cells promoted the secretion of IL-6 and IL-8 by leukocytes through HSP90α. Besides, we show that the cytokines IL-6 and IL-8 secreted by LC3B+ EVs-induced leukocytes were involved in the inhibition of CD8+ T-cell function, while blockade of the HSP90α on the LC3B+ EVs, IL-6, or IL-8 could enhance anti-PD-1-induced T cell reinvigoration. Finally, patients who received anti-PD-1/PD-L1 immunotherapy with high MAP1LC3B, HSP90AA1, IL6, or IL8 expression had a lower immunotherapy efficacy. CONCLUSIONS: Our data suggest that liver cancer-derived LC3B+ EVs promote a pro-oncogenic inflammatory microenvironment by carrying membrane-bound HSP90α. Targeting HSP90α on the LC3B+ EVs, IL-6, or IL-8 may synergize with anti-PD-1 treatment to enhance the CD8+ T-cell functions, which may provide novel combination strategies in the clinic for the treatment of liver cancer.

Evaluating nephrotoxicity reduction in a novel polymyxin B formulation: insights from a 3D kidney-on-a-chip model.

This study aimed to assess the nephrotoxicity associated with VRP-034 (novel formulation of polymyxin B [PMB]) compared to marketed PMB in a three-dimensional (3D) kidney-on-a-chip model. To model the human kidney proximal tubule for analysis, tubular structures were established using 23 triple-channel chips seeded with RPTEC/hTERT1 cells. These cells were exposed to VRP-034 or PMB at seven concentrations (1-200 µM) over 12, 24, and 48 h. A suite of novel kidney injury biomarkers, cell health, and inflammatory markers were quantitatively assessed in the effluent. Additionally, caspase and cytochrome C levels were measured, and cell viability was evaluated using calcein AM and ethidium homodimer-1 (EthD-1). Exposure to marketed PMB resulted in significantly elevated levels (P < 0.05) of four key biomarkers (KIM-1, cystatin C, clusterin, and OPN) compared to VRP-034, particularly at clinically relevant concentrations of ≥10 µM. At 25 µM, all biomarkers demonstrated a significant increase (P < 0.05) with marketed PMB exposure compared to VRP-034. Inflammatory markers (interleukin-6 and interleukin-8) increased significantly (P < 0.05) with marketed PMB at concentrations of ≥5 µM, relative to VRP-034. VRP-034 displayed superior cell health outcomes, exhibiting lower lactate dehydrogenase release, while ATP levels remained comparable. Morphological analysis revealed that marketed PMB induced more severe damage, disrupting tubular integrity. Both treatments activated cytochrome C, caspase-3, caspase-8, caspase-9, and caspase-12 in a concentration-dependent manner; however, caspase activation was significantly reduced (P < 0.05) with VRP-034. This study demonstrates that VRP-034 significantly reduces nephrotoxicity compared to marketed PMB within a 3D microphysiological system, suggesting its potential to enable the use of full therapeutic doses of PMB with an improved safety profile, addressing the need for less nephrotoxic polymyxin antibiotics.

Structure-based design of small molecule inhibitors of the cagT4SS ATPase Cagα of Helicobacter pylori.

We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori. The secretion system is encoded by the cag pathogenicity island, and we chose Cagα, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cagα in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cagα subunits and mutagenesis of the binding site identified Cagα residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889, and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cagα, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect.

Ischemic cardiac stromal fibroblast-derived protein mediators in the infarcted myocardium and transcriptomic profiling at single cell resolution.

This article focuses on screening the major secreted proteins by the ischemia-challenged cardiac stromal fibroblasts (CF), the assessment of their expression status and functional role in the post-ischemic left ventricle (LV) and in the ischemia-challenged CF culture and to phenotype CF at single cell resolution based on the positivity of the identified mediators. The expression level of CRSP2, HSP27, IL-8, Cofilin-1, and HSP90 in the LV tissues following coronary artery bypass graft (CABG) and myocardial infarction (MI) and CF cells followed the screening profile derived from the MS/MS findings. The histology data unveiled ECM disorganization, inflammation and fibrosis reflecting the ischemic pathology. CRSP2, HSP27, and HSP90 were significantly upregulated in the LV-CABG tissues with a concomitant reduction ion LV-MI whereas Cofilin-1, IL8, Nrf2, and Troponin I were downregulated in LV-CABG and increased in LV-MI. Similar trends were exhibited by ischemic CF. Single cell transcriptomics revealed multiple sub-phenotypes of CF based on their respective upregulation of CRSP2, HSP27, IL-8, Cofilin-1, HSP90, Troponin I and Nrf2 unveiling pathological and pro-healing phenotypes. Further investigations regarding the underlying signaling mechanisms and validation of sub-populations would offer novel translational avenues for the management of cardiac diseases.

Interleukin-8 in HepG2 cells: Enhancing antiviral proteins in uninfected cells but promoting HBV replication in infected cells.

In vitro studies have revealed that hepatitis B virus (HBV) infection upregulates interleukin-8 (IL-8), which enhances HBV replication. Clinically, elevated IL-8 levels in chronic HBV patients are associated with diminished therapeutic efficacy of interferon-α (IFN-α). Our study advances these findings by demonstrating that IL-8 promotes the expression of myxovirus resistance A (MxA) and protein kinase R (PKR) in HepG2 cells via the PI3K-AKT pathway. However, HBV-infected cells fail to exhibit IL-8-induced upregulation of MxA and PKR, likely due to HBV's upregulation of PP2A that inhibits the PI3K-AKT pathway. Notably, IL-8 targets the C/EBPα transcription factor, increasing HBV promoter activity and viral replication, which in turn partially suppresses the expression of MxA and PKR induced by IFN-α. Our findings uncover a mechanism by which HBV may evade immune responses, suggesting potential new strategies for immunotherapy against chronic HBV infection.

Microbiome-derived metabolite effects on intestinal barrier integrity and immune cell response to infection.

The gut microbiota exerts a significant influence on human health and disease. While compositional changes in the gut microbiota in specific diseases can easily be determined, we lack a detailed mechanistic understanding of how these changes exert effects at the cellular level. However, the putative local and systemic effects on human physiology that are attributed to the gut microbiota are clearly being mediated through molecular communication. Here, we determined the effects of gut microbiome-derived metabolites l-tryptophan, butyrate, trimethylamine (TMA), 3-methyl-4-(trimethylammonio)butanoate (3,4-TMAB), 4-(trimethylammonio)pentanoate (4-TMAP), ursodeoxycholic acid (UDCA), glycocholic acid (GCA) and benzoate on the first line of defence in the gut. Using in vitro models of intestinal barrier integrity and studying the interaction of macrophages with pathogenic and non-pathogenic bacteria, we could ascertain the influence of these metabolites at the cellular level at physiologically relevant concentrations. Nearly all metabolites exerted positive effects on barrier function, but butyrate prevented a reduction in transepithelial resistance in the presence of the pathogen Escherichia coli, despite inducing increased apoptosis and exerting increased cytotoxicity. Induction of IL-8 was unaffected by all metabolites, but GCA stimulated increased intra-macrophage growth of E. coli and tumour necrosis-alpha (TNF-α) release. Butyrate, 3,4-TMAB and benzoate all increased TNF-α release independent of bacterial replication. These findings reiterate the complexity of understanding microbiome effects on host physiology and underline that microbiome metabolites are crucial mediators of barrier function and the innate response to infection. Understanding these metabolites at the cellular level will allow us to move towards a better mechanistic understanding of microbiome influence over host physiology, a crucial step in advancing microbiome research.

iPSC-derived PSEN2 (N141I) astrocytes and microglia exhibit a primed inflammatory phenotype.

BACKGROUND: Widescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimer's disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells. METHODS: iPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and Aß42 and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently-labelled fibrillar Aß42. RESULTS: AD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100ß and increased secretion and phagocytosis of Aß42 while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells showed exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia. CONCLUSION: Our study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a 'primed' phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aß42 production and phagocytosis.