A microbially produced AhR ligand promotes a Tph1-driven tolerogenic program in multiple sclerosis.

Multiple sclerosis is a debilitating autoimmune disease, characterized by chronic inflammation of the central nervous system. While the significance of the gut microbiome on multiple sclerosis pathogenesis is established, the underlining mechanisms are unknown. We found that serum levels of the microbial postbiotic tryptophan metabolite indole-3-carboxaldehyde (3-IAld) inversely correlated with disease duration in multiple sclerosis patients. Much like the host-derived tryptophan derivative L-Kynurenine, 3-IAld would bind and activate the Aryl hydrocarbon Receptor (AhR), which, in turn, controls endogenous tryptophan catabolic pathways. As a result, in peripheral lymph nodes, microbial 3-IAld, affected mast-cell tryptophan metabolism, forcing mast cells to produce serotonin via Tph1. We thus propose a protective role for AhR-mast-cell activation driven by the microbiome, whereby natural metabolites or postbiotics will have a physiological role in immune homeostasis and may act as therapeutic targets in autoimmune diseases.

Persisting IL-18 levels after COVID-19 correlate with markers of cardiovascular inflammation reflecting potential risk of CVDs development.

COVID-19 manifestation is associated with a strong immune system activation leading to inflammation and subsequently affecting the cardiovascular system. The objective of the study was to reveal possible interconnection between prolongated inflammation and the development or exacerbation of long-term cardiovascular complications after COVID-19. We investigated correlations between humoral and cellular immune system markers together with markers of cardiovascular inflammation/dysfunction during COVID-19 onset and subsequent recovery. We analyzed 22 hospitalized patients with severe COVID-19 within three timepoints (acute, 1 and 6 months after COVID-19) in order to track the impact of COVID-19 on the long-term decline of the cardiovascular system fitness and eventual development of CVDs. Among the cytokines dysregulated during COVID-19 changes, we showed significant correlations of IL-18 as a key driver of several pathophysiological changes with markers of cardiovascular inflammation/dysfunction. Our findings established novel immune-related markers, which can be used for the stratification of patients at high risk of CVDs for further therapy.

TGF-ß induces matrisome pathological alterations and EMT in patient-derived prostate cancer tumoroids.

Extracellular matrix (ECM) tumorigenic alterations resulting in high matrix deposition and stiffening are hallmarks of adenocarcinomas and are collectively defined as desmoplasia. Here, we thoroughly analysed primary prostate cancer tissues obtained from numerous patients undergoing radical prostatectomy to highlight reproducible structural changes in the ECM leading to the loss of the glandular architecture. Starting from patient cells, we established prostate cancer tumoroids (PCTs) and demonstrated they require TGF-ß signalling pathway activity to preserve phenotypical and structural similarities with the tissue of origin. By modulating TGF-ß signalling pathway in PCTs, we unveiled its role in ECM accumulation and remodelling in prostate cancer. We also found that TGF-ß-induced ECM remodelling is responsible for the initiation of prostate cell epithelial-to-mesenchymal transition (EMT) and the acquisition of a migratory, invasive phenotype. Our findings highlight the cooperative role of TGF-ß signalling and ECM desmoplasia in prompting prostate cell EMT and promoting tumour progression and dissemination.

Phenotype and oxidative burst of low-density neutrophil subpopulations are altered in common variable immunodeficiency patients.

Common variable immunodeficiency disorder (CVID) is the most common form of primary antibody immunodeficiency. Due to low antibody levels, CVID patients receive intravenous or subcutaneous immunoglobulin replacement therapy as treatment. CVID is associated with the chronic activation of granulocytes, including an increased percentage of low-density neutrophils (LDNs). In this study, we examined changes in the percentage of LDNs and the expression of their surface markers in 25 patients with CVID and 27 healthy donors (HD) after in vitro stimulation of whole blood using IVIg. An oxidative burst assay was used to assess the functionality of LDNs. CVID patients had increased both relative and absolute LDN counts with a higher proportion of mLDNs compared to iLDNs, distinguished based on the expression of CD10 and CD16. Immature LDNs in the CVID and HD groups had significantly reduced oxidative burst capacity compared to mature LDNs. Interestingly we observed reduced oxidative burst capacity, reduced expression of CD10 after stimulation of WB, and higher expression of PD-L1 in mature LDNs in CVID patients compared to HD cells. Our data indicate that that the functional characteristics of LDNs are closely linked to their developmental stage. The observed reduction in oxidative burst capacity in mLDNs in CVID patients could contribute to an increased susceptibility to recurrent bacterial infections among CVID patients.

Activation of TLRs by Opportunistic Fungi in Lung Organoids.

Organoid cultures may express several types of pattern-recognition receptors and in particular toll-like receptors, representing an extremely efficient and innovative system to understand how pathogen-associated molecular patterns exposure may affect the immunity, the growth, or differentiation of complex tissues. Here, we describe how to generate lung organoids from human-induced pluripotent stem cells. Three-dimensional (3D) cultures are then stimulated with different toll-like receptor ligands derived from fungi or with Aspergillus fumigatus. RNA sequencing may be performed upon organoid cultures to understand host-pathogen innate immune interactions.

Expression of circular RNAs in myelodysplastic neoplasms and their association with mutations in the splicing factor gene SF3B1.

Mutations in the splicing factor 3b subunit 1 (SF3B1) gene are frequent in myelodysplastic neoplasms (MDS). Because the splicing process is involved in the production of circular RNAs (circRNAs), we investigated the impact of SF3B1 mutations on circRNA processing. Using RNA sequencing, we measured circRNA expression in CD34+ bone marrow MDS cells. We defined circRNAs deregulated in a heterogeneous group of MDS patients and described increased circRNA formation in higher-risk MDS. We showed that the presence of SF3B1 mutations did not affect the global production of circRNAs; however, deregulation of specific circRNAs was observed. Particularly, we demonstrated that strong upregulation of circRNAs processed from the zinc finger E-box binding homeobox 1 (ZEB1) transcription factor; this upregulation was exclusive to SF3B1-mutated patients and was not observed in those with mutations in other splicing factors or other recurrently mutated genes, or with other clinical variables. Furthermore, we focused on the most upregulated ZEB1-circRNA, hsa_circ_0000228, and, by its knockdown, we demonstrated that its expression is related to mitochondrial activity. Using microRNA analyses, we proposed miR-1248 as a direct target of hsa_circ_0000228. To conclude, we demonstrated that mutated SF3B1 leads to deregulation of ZEB1-circRNAs, potentially contributing to the defects in mitochondrial metabolism observed in SF3B1-mutated MDS.

Pathogenesis of Alzheimer's disease: Involvement of the choroid plexus.

The choroid plexus (ChP) produces and is bathed in the cerebrospinal fluid (CSF), which in aging and Alzheimer's disease (AD) shows extensive proteomic alterations including evidence of inflammation. Considering inflammation hampers functions of the involved tissues, the CSF abnormalities reported in these conditions are suggestive of ChP injury. Indeed, several studies document ChP damage in aging and AD, which nevertheless remains to be systematically characterized. We here report that the changes elicited in the CSF by AD are consistent with a perturbed aging process and accompanied by aberrant accumulation of inflammatory signals and metabolically active proteins in the ChP. Magnetic resonance imaging (MRI) imaging shows that these molecular aberrancies correspond to significant remodeling of ChP in AD, which correlates with aging and cognitive decline. Collectively, our preliminary post-mortem and in vivo findings reveal a repertoire of ChP pathologies indicative of its dysfunction and involvement in the pathogenesis of AD. HIGHLIGHTS: Cerebrospinal fluid changes associated with aging are perturbed in Alzheimer's disease Paradoxically, in Alzheimer's disease, the choroid plexus exhibits increased cytokine levels without evidence of inflammatory activation or infiltrates In Alzheimer's disease, increased choroid plexus volumes correlate with age and cognitive performance.

Hepcidin and ferritin levels as markers of immune cell activation during septic shock, severe COVID-19 and sterile inflammation.

Introduction: Major clinically relevant inflammatory events such as septic shock and severe COVID-19 trigger dynamic changes in the host immune system, presenting promising candidates for new biomarkers to improve precision diagnostics and patient stratification. Hepcidin, a master regulator of iron metabolism, has been intensively studied in many pathologies associated with immune system activation, however these data have never been compared to other clinical settings. Thus, we aimed to reveal the dynamics of iron regulation in various clinical settings and to determine the suitability of hepcidin and/or ferritin levels as biomarkers of inflammatory disease severity. Cohorts: To investigate the overall predictive ability of hepcidin and ferritin, we enrolled the patients suffering with three different diagnoses - in detail 40 patients with COVID-19, 29 patients in septic shock and eight orthopedic patients who were compared to nine healthy donors and all cohorts to each other. Results: We showed that increased hepcidin levels reflect overall immune cell activation driven by intrinsic stimuli, without requiring direct involvement of infection vectors. Contrary to hepcidin, ferritin levels were more strongly boosted by pathogen-induced inflammation - in septic shock more than four-fold and in COVID-19 six-fold in comparison to sterile inflammation. We also defined the predictive capacity of hepcidin-to-ferritin ratio with AUC=0.79 and P = 0.03. Discussion: Our findings confirm that hepcidin is a potent marker of septic shock and other acute inflammation-associated pathologies and demonstrate the utility of the hepcidin-to-ferritin ratio as a predictor of mortality in septic shock, but not in COVID-19.

Lactate from the tumor microenvironment - A key obstacle in NK cell-based immunotherapies.

Recent findings about the new roles of lactate have changed our understanding of this end product of glycolysis or fermentation that was once considered only a waste product. It is now well accepted that lactate acts as a signaling molecule and fuel source for cancer cells in a glucose-restricted environment. Moreover, lactate and lactate dehydrogenase are markers of poor prognosis of many cancers and regulate many functions of immune cells. The presence of lactate in the tumor microenvironment (TME) leads to polarization of the immunosuppressive phenotypes of dendritic cells and impairs the cytotoxic abilities of T cells and NK cells, and as such lactate is a major obstacle to immune-cell effector functions and the efficacy of cell-based immunotherapies. Emerging evidence suggests that lactate in the TME might be a novel therapeutic target to enhance the immunotherapeutic potential of cell-based therapies. This review describes our current understanding of the role of lactate in tumor biology, including its detrimental effects on cell-based immunotherapy in cancer. We also highlight how the role of lactate in the TME must be considered when producing cell therapies designed for adoptive transfer and describe how targeted modulation of lactate in the TME might boost immune-cell functions and positively impact cellular immunotherapy, with a focus on NK cell.

Prognostic value of soluble endoglin in patients with septic shock and severe COVID-19.

Sepsis is a clinical syndrome characterized by a dysregulated response to infection. It represents a leading cause of mortality in ICU patients worldwide. Although sepsis is in the point of interest of research for several decades, its clinical management and patient survival are improving slowly. Monitoring of the biomarkers and their combinations could help in early diagnosis, estimation of prognosis and patient's stratification and response to the treatment. Circulating soluble endoglin (sEng) is the cleaved extracellular part of transmembrane glycoprotein endoglin. As a biomarker, sEng has been tested in several pathologic conditions where its elevation was associated with endothelial dysfunction. In this study we have tested the ability of sEng to predict mortality and its correlation with other clinical characteristics in the cohort of septic shock patients (n = 37) and patients with severe COVID-19 (n = 40). In patients with COVID-19 sEng did not predict mortality or correlate with markers of organ dysfunction. In contrast, in septic shock the level of sEng was significantly higher in patients with early mortality (p = 0.019; AUC = 0.801). Moreover, sEng levels correlated with signs of circulatory failure (required dose of noradrenalin and lactate levels; p = 0.002 and 0.016, respectively). The predominant clinical problem in patients with COVID-19 was ARDS, and although they often showed signs of other organ dysfunction, circulatory failure was exceptional. This potentially explains the difference between sEng levels in COVID-19 and septic shock. In conclusion, we have confirmed that sEng may reflect the extent of the circulatory failure in septic shock patients and thus could be potentially used for the early identification of patients with the highest degree of endothelial dysfunction who would benefit from endothelium-targeted individualized therapy.

Lung Organoids-The Ultimate Tool to Dissect Pulmonary Diseases?

Organoids are complex multicellular three-dimensional (3D) in vitro models that are designed to allow accurate studies of the molecular processes and pathologies of human organs. Organoids can be derived from a variety of cell types, such as human primary progenitor cells, pluripotent stem cells, or tumor-derived cells and can be co-cultured with immune or microbial cells to further mimic the tissue niche. Here, we focus on the development of 3D lung organoids and their use as disease models and drug screening tools. We introduce the various experimental approaches used to model complex human diseases and analyze their advantages and disadvantages. We also discuss validation of the organoids and their physiological relevance to the study of lung diseases. Furthermore, we summarize the current use of lung organoids as models of host-pathogen interactions and human lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, or SARS-CoV-2 infection. Moreover, we discuss the use of lung organoids derived from tumor cells as lung cancer models and their application in personalized cancer medicine research. Finally, we outline the future of research in the field of human induced pluripotent stem cell-derived organoids.

Influence of protein corona on the interaction of glycogen-siRNA constructs with ex vivo human blood immune cells.

Glycogen-nucleic acid constructs i.e., glycoplexes are emerging promising platforms for the alteration of gene expression and transcription. Understanding the interaction of glycoplexes with human blood components, such as serum proteins and peripheral blood mononuclear cells (PBMCs), is important to overcome immune cell activation and control biodistribution upon administration of the glycoplexes in vivo. Herein, we investigated the interactions of polyethylene glycol (PEG)ylated and non-PEGylated glycoplexes carrying siRNA molecules with PBMCs isolated from the blood of healthy donors. We found that both types of glycoplexes were non-toxic and were primarily phagocytosed by monocytes without triggering a pro-inflammatory interleukin 6 cytokine production. Furthermore, we investigated the role of the protein corona on controlling the internalization efficiency in immune cells - we found that the adsorption of serum proteins, in particular haptoglobin, alpha-1-antitrypsin and apolipoprotein A-II, onto the non-PEGylated glycoplexes, significantly reduced the uptake of the glycoplexes by PBMCs. Moreover, the non-PEGylated glycoplexes were efficient in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) knockdown in monocytic THP-1 cell line. This study provides an insight into the rational design of glycogen-based nanocarriers for the safe delivery of siRNA without eliciting unwanted immune cell activation and efficient siRNA activity upon its delivery.

RUNX1 mutations contribute to the progression of MDS due to disruption of antitumor cellular defense: a study on patients with lower-risk MDS.

Patients with lower-risk myelodysplastic syndromes (LR-MDS) have a generally favorable prognosis; however, a small proportion of cases progress rapidly. This study aimed to define molecular biomarkers predictive of LR-MDS progression and to uncover cellular pathways contributing to malignant transformation. The mutational landscape was analyzed in 214 LR-MDS patients, and at least one mutation was detected in 137 patients (64%). Mutated RUNX1 was identified as the main molecular predictor of rapid progression by statistics and machine learning. To study the effect of mutated RUNX1 on pathway regulation, the expression profiles of CD34 + cells from LR-MDS patients with RUNX1 mutations were compared to those from patients without RUNX1 mutations. The data suggest that RUNX1-unmutated LR-MDS cells are protected by DNA damage response (DDR) mechanisms and cellular senescence as an antitumor cellular barrier, while RUNX1 mutations may be one of the triggers of malignant transformation. Dysregulated DDR and cellular senescence were also observed at the functional level by detecting γH2AX expression and ß-galactosidase activity. Notably, the expression profiles of RUNX1-mutated LR-MDS resembled those of higher-risk MDS at diagnosis. This study demonstrates that incorporating molecular data improves LR-MDS risk stratification and that mutated RUNX1 is associated with a suppressed defense against LR-MDS progression.

Triggering the nanophase separation of albumin through multivalent binding to glycogen for drug delivery in 2D and 3D multicellular constructs.

Engineered nanoparticles for the encapsulation of bioactive agents hold promise to improve disease diagnosis, prevention and therapy. To advance this field and enable clinical translation, the rational design of nanoparticles with controlled functionalities and a robust understanding of nanoparticle-cell interactions in the complex biological milieu are of paramount importance. Herein, a simple platform obtained through the nanocomplexation of glycogen nanoparticles and albumin is introduced for the delivery of chemotherapeutics in complex multicellular 2D and 3D systems. We found that the dendrimer-like structure of aminated glycogen nanoparticles is key to controlling the multivalent coordination and phase separation of albumin molecules to form stable glycogen-albumin nanocomplexes. The pH-responsive glycogen scaffold conferred the nanocomplexes the ability to undergo partial endosomal escape in tumour, stromal and immune cells while albumin enabled nanocomplexes to cross endothelial cells and carry therapeutic agents. Limited interactions of nanocomplexes with T cells, B cells and natural killer cells derived from human blood were observed. The nanocomplexes can accommodate chemotherapeutic drugs and release them in multicellular 2D and 3D constructs. The drugs loaded on the nanocomplexes retained their cytotoxic activity, which is comparable with the activity of the free drugs. Cancer cells were found to be more sensitive to the drugs in the presence of stromal and immune cells. Penetration and cytotoxicity of the drug-loaded nanocomplexes in tumour mimicking tissues were validated using a 3D multicellular-collagen construct in a perfusion bioreactor. The results highlight a simple and potentially scalable strategy for engineering nanocomplexes made entirely of biological macromolecules with potential use for drug delivery.

Train the Trainer: Hematopoietic Stem Cell Control of Trained Immunity.

Recent evidence shows that innate immune cells, in addition to B and T cells, can retain immunological memory of their encounters and afford long-term resistance against infections in a process known as 'trained immunity'. However, the duration of the unspecific protection observed in vivo is poorly compatible with the average lifespan of innate immune cells, suggesting the involvement of long-lived cells. Accordingly, recent studies demonstrate that hematopoietic stem and progenitor cells (HSPCs) lay at the foundation of trained immunity, retaining immunological memory of infections and giving rise to a "trained" myeloid progeny for a long time. In this review, we discuss the research demonstrating the involvement of HSPCs in the onset of long-lasting trained immunity. We highlight the roles of specific cytokines and Toll-like receptor ligands in influencing HSPC memory phenotypes and the molecular mechanisms underlying trained immunity HSPCs. Finally, we discuss the potential benefits and drawbacks of the long-lasting trained immune responses, and describe the challenges that the field is facing.

High CD4-to-CD8 ratio identifies an at-risk population susceptible to lethal COVID-19.

Around half of people with severe COVID-19 requiring intensive care unit (ICU) treatment will survive, but it is unclear how the immune response to SARS-CoV-2 differs between ICU patients that recover and those that do not. We conducted whole-blood immunophenotyping of COVID-19 patients upon admission to ICU and during their treatment and uncovered marked differences in their circulating immune cell subsets. At admission, patients who later succumbed to COVID-19 had significantly lower frequencies of all memory CD8+ T cell subsets, resulting in increased CD4-to-CD8 T cell and neutrophil-to-CD8 T cell ratios. ROC and Kaplan-Meier analyses demonstrated that both CD4-to-CD8 and neutrophil-to-CD8 ratios at admission were strong predictors of in-ICU mortality. Therefore, we propose the use of the CD4-to-CD8 T cell ratio as a marker for the early identification of those individuals likely to require enhanced monitoring and/or pro-active intervention in ICU.

Polymorphonuclear Cells Show Features of Dysfunctional Activation During Fatal Sepsis.

Sepsis and septic shock remain leading causes of morbidity and mortality for patients in the intensive care unit. During the early phase, immune cells produce various cytokines leading to prompt activation of the immune system. Polymorphonuclear leukocytes (PMNs) respond to different signals producing inflammatory factors and executing their antimicrobial mechanisms, resulting in the engulfment and elimination of invading pathogens. However, excessive activation caused by various inflammatory signals produced during sepsis progression can lead to the alteration of PMN signaling and subsequent defects in their functionality. Here, we analyzed samples from 34 patients in septic shock, focusing on PMNs gene expression and proteome changes associated with septic shock. We revealed that, compared to those patients who survived longer than five days, PMNs from patients who had fulminant sepsis were characterized by a dysfunctional hyper-activation, show altered metabolism, and recent exit from the cell cycle and signs of cellular lifespan. We believe that this multi-omics approach, although limited, pinpoints the alterations in PMNs' functionality, which may be rescued by targeted treatments.

Immunosuppression Affects Neutrophil Functions: Does Calcineurin-NFAT Signaling Matter?

Neutrophils are innate immune cells with important roles in antimicrobial defense. However, impaired or dysregulated neutrophil function can result in host tissue damage, loss of homeostasis, hyperinflammation or pathological immunosuppression. A central link between neutrophil activation and immune outcomes is emerging to be the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, which is activated by neutrophil detection of a microbial threat via pattern recognition receptors and results in inflammatory cytokine production. This potent pro-inflammatory pathway is also the target of several immunosuppressive drugs used for the treatment of autoimmune disorders, during solid organ and hematopoietic cell transplantations, and as a part of anti-cancer therapy: but what effects these drugs have on neutrophil function, and their broader consequences for immune homeostasis and microbial defense are not yet known. Here, we bring together the emerging literature describing pathology- and drug- induced neutrophil impairment, with particular focus on their effects on calcineurin-NFAT signaling in the innate immune compartment.

Targeted volatolomics of human monocytes: Comparison of 2D-GC/TOF-MS and 1D-GC/Orbitrap-MS methods.

Blood is a complex biological matrix providing valuable information on nutritional, metabolic, and immune status. The detection of blood biomarkers requires sensitive analytical methods because analytes are at very low concentrations. Peripheral blood monocytes play a crucial role in inflammatory processes, and the metabolites released by monocytes during these processes might serve as important signalling molecules and biomarkers of particular physiological states. Headspace solid-phase microextraction (HS-SPME) combined with two different mass spectrometric platforms, two-dimensional (2D) gas chromatography coupled to time-of-flight mass spectrometry (2D-GC/TOF-MS) and one-dimensional gas chromatography coupled to Orbitrap mass spectrometry (GC/Orbitrap-MS), were applied for the investigation of volatile organic compounds (VOCs) produced by human peripheral blood monocytes. An optimized method was subsequently applied for the characterization of changes in VOCs induced by lipopolysaccharides (LPS) and zymosan (ZYM) stimulation. Overall, the 2D-GC/TOF-MS and the 1D-GC/Orbitrap-MS analyses each yielded about 4000 and 400 peaks per sample, respectively. In total, 91 VOCs belonging to eight different chemical classes were identified. The samples were collected in two fractions, conditioned media for monitoring extracellularly secreted molecules and cell pellet samples to determine the intracellular composition of VOCs. Alcohols, ketones, and hydrocarbons were the main chemical classes of the metabolic profile identified in cell fractions. Aldehydes, acids and cyclic compounds were characteristic of the conditioned media fraction. Here we demonstrate that HS-SPME-2D-GC/TOF-MS is more suitable for the identification of specific VOC profiles produced by human monocytes than 1D-GC/Orbitrap-MS. We define the signature of VOCs occurring early after monocyte activation and characterise the signalling compounds released by immune cells into media.