Spatiotemporal regulation of type I interferon expression determines the antiviral polarization of CD4+ T cells.

Differentiation of CD4+ T cells into either follicular helper T (TFH) or type 1 helper T (TH1) cells influences the balance between humoral and cellular adaptive immunity, but the mechanisms whereby pathogens elicit distinct effector cells are incompletely understood. Here we analyzed the spatiotemporal dynamics of CD4+ T cells during infection with recombinant vesicular stomatitis virus (VSV), which induces early, potent neutralizing antibodies, or recombinant lymphocytic choriomeningitis virus (LCMV), which induces a vigorous cellular response but inefficient neutralizing antibodies, expressing the same T cell epitope. Early exposure of dendritic cells to type I interferon (IFN), which occurred during infection with VSV, induced production of the cytokine IL-6 and drove TFH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into TH1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4+ T cell differentiation and might instruct vaccine design strategies.

Kynurenine importation by SLC7A11 propagates anti-ferroptotic signaling.

IDO1 oxidizes tryptophan (TRP) to generate kynurenine (KYN), the substrate for 1-carbon and NAD metabolism, and is implicated in pro-cancer pathophysiology and infection biology. However, the mechanistic relationships between IDO1 in amino acid depletion versus product generation have remained a longstanding mystery. We found an unrecognized link between IDO1 and cell survival mediated by KYN that serves as the source for molecules that inhibit ferroptotic cell death. We show that this effect requires KYN export from IDO1-expressing cells, which is then available for non-IDO1-expressing cells via SLC7A11, the central transporter involved in ferroptosis suppression. Whether inside the "producer" IDO1+ cell or the "receiver" cell, KYN is converted into downstream metabolites, suppressing ferroptosis by ROS scavenging and activating an NRF2-dependent, AHR-independent cell-protective pathway, including SLC7A11, propagating anti-ferroptotic signaling. IDO1, therefore, controls a multi-pronged protection pathway from ferroptotic cell death, underscoring the need to re-evaluate the use of IDO1 inhibitors in cancer treatment.

The obesity-autophagy-cancer axis: Mechanistic insights and therapeutic perspectives.

Autophagy, a self-degradative process vital for cellular homeostasis, plays a significant role in adipose tissue metabolism and tumorigenesis. This review aims to elucidate the complex interplay between autophagy, obesity, and cancer development, with a specific emphasis on how obesity-driven changes affect the regulation of autophagy and subsequent implications for cancer risk. The burgeoning epidemic of obesity underscores the relevance of this research, particularly given the established links between obesity, autophagy, and various cancers. Our exploration delves into hormonal influence, notably INS (insulin) and LEP (leptin), on obesity and autophagy interactions. Further, we draw attention to the latest findings on molecular factors linking obesity to cancer, including hormonal changes, altered metabolism, and secretory autophagy. We posit that targeting autophagy modulation may offer a potent therapeutic approach for obesity-associated cancer, pointing to promising advancements in nanocarrier-based targeted therapies for autophagy modulation. However, we also recognize the challenges inherent to these approaches, particularly concerning their precision, control, and the dual roles autophagy can play in cancer. Future research directions include identifying novel biomarkers, refining targeted therapies, and harmonizing these approaches with precision medicine principles, thereby contributing to a more personalized, effective treatment paradigm for obesity-mediated cancer.

A fluorescent reporter model for the visualization and characterization of TDC.

TDC are hematopoietic cells that combine dendritic cell (DC) and conventional T-cell markers and functional properties. They were identified in secondary lymphoid organs (SLOs) of naïve mice as cells expressing CD11c, major histocompatibility molecules (MHC)-II, and the T-cell receptor (TCR). Despite thorough characterization, a physiological role for TDC remains to be determined. Unfortunately, using CD11c as a marker for TDC has the caveat of its upregulation on different cells, including T cells, upon activation. Here, we took advantage of Zbtb46-GFP reporter mice to explore the frequency and localization of TDC in different tissues at steady state and upon viral infection. RNA sequencing analysis confirmed that TDC sorted from Zbtb46-GFP mice have a gene signature that is distinct from conventional T cells and DC. In addition, this reporter model allowed for identification of TDC in situ not only in SLOs but also in the liver and lung of naïve mice. Interestingly, we found that TDC numbers in the SLOs increased upon viral infection, suggesting that TDC might play a role during viral infections. In conclusion, we propose a visualization strategy that might shed light on the physiological role of TDC in several pathological contexts, including infection and cancer.

A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling.

mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.

Prolonged exposure to simulated microgravity promotes stemness impairing morphological, metabolic and migratory profile of pancreatic cancer cells: a comprehensive proteomic, lipidomic and transcriptomic analysis.

BACKGROUND: The impact of the absence of gravity on cancer cells is of great interest, especially today that space is more accessible than ever. Despite advances, few and contradictory data are available mainly due to different setup, experimental design and time point analyzed. METHODS: Exploiting a Random Positioning Machine, we dissected the effects of long-term exposure to simulated microgravity (SMG) on pancreatic cancer cells performing proteomic, lipidomic and transcriptomic analysis at 1, 7 and 9 days. RESULTS: Our results indicated that SMG affects cellular morphology through a time-dependent activation of Actin-based motility via Rho and Cdc42 pathways leading to actin rearrangement, formation of 3D spheroids and enhancement of epithelial-to-mesenchymal transition. Bioinformatic analysis reveals that SMG may activates ERK5/NF-κB/IL-8 axis that triggers the expansion of cancer stem cells with an increased migratory capability. These cells, to remediate energy stress and apoptosis activation, undergo a metabolic reprogramming orchestrated by HIF-1α and PI3K/Akt pathways that upregulate glycolysis and impair ß-oxidation, suggesting a de novo synthesis of triglycerides for the membrane lipid bilayer formation. CONCLUSIONS: SMG revolutionizes tumor cell behavior and metabolism leading to the acquisition of an aggressive and metastatic stem cell-like phenotype. These results dissect the time-dependent cellular alterations induced by SMG and pave the base for altered gravity conditions as new anti-cancer technology.

TOWARD A CONTINGENCY MODEL FOR HOSPITAL-BASED HEALTH TECHNOLOGY ASSESSMENT: EVIDENCE FROM ADHOPHTA PROJECT.

OBJECTIVES: Hospital-based health technology assessment (HB-HTA) is becoming increasingly relevant because of its role in managing the introduction and withdrawal of health technologies. The organizational arrangement in which HB-HTA activities are conducted depends on several contextual factors, although the dominant models have several similarities. The aims of this study were to explore, describe, interpret, and explain seven cases of the application of HB-HTA logic and to propose a classification for HB-HTA organizational models which may be beneficial for policy makers and HTA professionals. METHODS: The study was part of the AdHopHTA Project, granted under the European 7th Framework Research Programme. A case study methodology was applied to analyze seven HB-HTA initiatives in seven countries, with collection of qualitative and quantitative data. Cross-case analysis was performed within the framework of contingent organizational theory. RESULTS: Evidence showed that some organizational or "structural" variables, namely the level of procedure formalization/structuration and the level of integration with other HTA bodies at the national, regional, and provincial levels, predominantly shape the HB-HTA approach, determining a contingency model of HB-HTA. Crossing the two variables, four options have emerged: integrated specialized HTA unit, stand-alone HTA unit, integrated-essential HTA, independent group unit. CONCLUSIONS: No one-best-way approach can be used for HTA at the hospital level. Rather, the characteristics of HTA models depend on many contextual factors. Such conceptualization may aid the diffusion of HB-HTA to inform managerial decision making and clinical practice.

Methylsulfonylmethane and boswellic acids versus glucosamine sulfate in the treatment of knee arthritis: Randomized trial.

Until now glucosamine sulfate (GS) has been the most widely used supplement and has been shown to be efficacious in the treatment of osteoarthritis (OA). Methylsulfonylmethane (MSM) and boswellic acids (BA) are new effective supplements for the management of inflammation and joint degeneration, according to previous experimental studies. The aim of our study is to test the effectiveness of association of MSM and BA in comparison with GS in knee arthritis.In this prospective randomized clinical trial, MEBAGA (Methylsulfonylmethane and Boswellic Acids versus Glucosamine sulfate in the treatment of knee Arthritis), 120 participants affected by arthritis of the knee were randomly assigned to an experimental group (MB group) or a control group (GS group) treated for 60 days with 5 g of MSM and 7.2 mg of BA or with 1500 mg of GS daily, respectively. At the 2-month (T1) and 6-months (T2) follow-up , the efficacy of these two nutraceuticals was assessed using the visual analog pain scale (VAS) and the Lequesne Index (LI) for joint function, along with the use of anti-inflammatory drugs (non-steroidal anti-inflammatory drugs and anti-cyclooxygenase-2).The repeated measures ANOVA analysis shows that for VAS, LI, and the use of anti-inflammatory drugs scores there are improvements due to the time in the two groups (respectively, F=26.0; P<0.0001; F=4.15; P=0.02; F=3.38; P=0.04), with a tendency to better values for the MB group at T2.On the basis of these preliminary data, we could support the efficacy of the MSM in association with BA in the treatment of OA. These results are consistent with the anti-inflammatory and chondroprotective effects previously occurred in experimental studies. This new combination of integration (MSM and BS) has presented good results and satisfactory in comparison with GS, until now the cornerstone of the treatment of arthritis in according to guidelines.

Comparison of shock wave therapy and nutraceutical composed of Echinacea angustifolia, alpha lipoic acid, conjugated linoleic acid and quercetin (perinerv) in patients with carpal tunnel syndrome.

Even though the initial treatment of carpal tunnel syndrome (CTS) is conservative, knowledge of the clinical effects of supplements and of some methods of physiotherapy is still preliminary. Many biological mechanisms can support the administration of shock wave therapy (ESWT) or of alpha lipoic acid (ALA) based nutraceutical, conjugated linoleic acid (GLA), anti-oxidants and Echinacea angustifolia for CTS. The shock waves reduce the nerve compression, produce an anti-inflammatory action, and accelerate the regeneration of neuropathy. ALA and GLA induce antioxidant protective actions, reduce inflammation, promote neuroregeneration, and decrease pain. The Echinacea modulates the endogenous cannabinoid system.The aim of study is to verify the efficiency of shock wave therapy versus nutraceutical composed of ALA, GLA, and Echinacea in CTS. Sixty patients were enrolled in this study and they were randomly assigned to one of two treatments. Both groups showed significant improvements in pain, symptoms' severity and functional scores, and electrodiagnostic results until the sixth month. We verified a trend to a better pain regression in the nutraceutical group. The presence of the medicinal Echinacea represents an added value to the antioxidant effect in ALA and GLA, which can justify this result. ESWT or the association of ALA, GLA, and Echinacea proved to be two effective treatments for controlling symptoms and improving the evolution of CTS.

Characterization of a genetic mouse model of lung cancer: a promise to identify Non-Small Cell Lung Cancer therapeutic targets and biomarkers.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 81% of all cases of lung cancer and they are often fatal because 60% of the patients are diagnosed at an advanced stage. Besides the need for earlier diagnosis, there is a high need for additional effective therapies. In this work, we investigated the feasibility of a lung cancer progression mouse model, mimicking features of human aggressive NSCLC, as biological reservoir for potential therapeutic targets and biomarkers. RESULTS: We performed RNA-seq profiling on total RNA extracted from lungs of a 30 week-old K-ras(LA1)/p53(R172HΔg) and wild type (WT) mice to detect fusion genes and gene/exon-level differential expression associated to the increase of tumor mass. Fusion events were not detected in K-ras(LA1)/p53(R172HΔg) tumors. Differential expression at exon-level detected 33 genes with differential exon usage. Among them nine, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of more than 500 NSCLC RNA-seq transcriptomes. None of the genes showed a significant correlation between exon-level expression and disease prognosis. Differential expression at gene-level allowed the identification of 1513 genes with a significant increase in expression associated to tumor mass increase. 74 genes, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of two transcriptomics datasets of human NSCLC samples, encompassing more than 900 samples. SPP1 was the only molecule whose over-expression resulted statistically related to poor outcome regarding both survival and metastasis formation. Two other molecules showed over-expression associated to poor outcome due to metastasis formation: GM-CSF and ADORA3. GM-CSF is a secreted protein, and we confirmed its expression in the supernatant of a cell line derived by a K-ras(LA1)/p53(R172HΔg) mouse tumor. ADORA3 is instead involved in the induction of p53-mediated apoptosis in lung cancer cell lines. Since in our model p53 is inactivated, ADORA3 does not negatively affect tumor growth but remains expressed on tumor cells. Thus, it could represent an interesting target for the development of antibody-targeted therapy on a subset of NSCLC, which are p53 null and ADORA3 positive. CONCLUSIONS: Our study provided a complete transcription overview of the K-ras(LA1)/p53(R172HΔg) mouse NSCLC model. This approach allowed the detection of ADORA3 as a potential target for antibody-based therapy in p53 mutated tumors.

Hyperforin Enhances Heme Oxygenase-1 Expression Triggering Lipid Peroxidation in BRAF-Mutated Melanoma Cells and Hampers the Expression of Pro-Metastatic Markers.

Hyperforin (HPF) is an acylphloroglucinol compound found abundantly in Hypericum perforatum extract which exhibits antidepressant, anti-inflammatory, antimicrobial, and antitumor activities. Our recent study revealed a potent antimelanoma effect of HPF, which hinders melanoma cell proliferation, motility, colony formation, and induces apoptosis. Furthermore, we have identified glutathione peroxidase-4 (GPX-4), a key enzyme involved in cellular protection against iron-induced lipid peroxidation, as one of the molecular targets of HPF. Thus, in three BRAF-mutated melanoma cell lines, we investigated whether iron unbalance and lipid peroxidation may be a part of the molecular mechanisms underlying the antimelanoma activity of HPF. Initially, we focused on heme oxygenase-1 (HO-1), which catalyzes the heme group into CO, biliverdin, and free iron, and observed that HPF treatment triggered the expression of this inducible enzyme. In order to investigate the mechanism involved in HO-1 induction, we verified that HPF downregulates the BTB and CNC homology 1 (BACH-1) transcription factor, an inhibitor of the heme oxygenase 1 (HMOX-1) gene transcription. Remarkably, we observed a partial recovery of cell viability and an increase in the expression of the phosphorylated and active form of retinoblastoma protein when we suppressed the HMOX-1 gene using HMOX-1 siRNA while HPF was present. This suggests that the HO-1 pathway is involved in the cytostatic effect of HPF in melanoma cells. To explore whether lipid peroxidation is induced, we conducted cytofluorimetric analysis and observed a significant increase in the fluorescence of the BODIPY C-11 probe 48 h after HPF administration in all tested melanoma cell lines. To discover the mechanism by which HPF triggers lipid peroxidation, along with the induction of HO-1, we examined the expression of additional proteins associated with iron homeostasis and lipid peroxidation. After HPF administration, we confirmed the downregulation of GPX-4 and observed low expression levels of SLC7A11, a cystine transporter crucial for the glutathione production, and ferritin, able to sequester free iron. A decreased expression level of these proteins can sensitize cells to lipid peroxidation. On the other hand, HPF treatment resulted in increased expression levels of transferrin, which facilitates iron uptake, and LC3B proteins, a molecular marker of autophagy induction. Indeed, ferritin and GPX-4 have been reported to be digested during autophagy. Altogether, these findings suggest that HPF induced lipid peroxidation likely through iron overloading and decreasing the expression of proteins that protect cells from lipid peroxidation. Finally, we examined the expression levels of proteins associated with melanoma cell invasion and metastatic potential. We observed the decreased expression of CD133, octamer-4, tyrosine-kinase receptor AXL, urokinase plasminogen activator receptor, and metalloproteinase-2 following HPF treatment. These findings provide further support for our previous observations, demonstrating the inhibitory effects of HPF on cell motility and colony formation in soft agar, which are both metastasis-related processes in tumor cells.

To metabolomics and beyond: a technological portfolio to investigate cancer metabolism.

Tumour cells have exquisite flexibility in reprogramming their metabolism in order to support tumour initiation, progression, metastasis and resistance to therapies. These reprogrammed activities include a complete rewiring of the bioenergetic, biosynthetic and redox status to sustain the increased energetic demand of the cells. Over the last decades, the cancer metabolism field has seen an explosion of new biochemical technologies giving more tools than ever before to navigate this complexity. Within a cell or a tissue, the metabolites constitute the direct signature of the molecular phenotype and thus their profiling has concrete clinical applications in oncology. Metabolomics and fluxomics, are key technological approaches that mainly revolutionized the field enabling researchers to have both a qualitative and mechanistic model of the biochemical activities in cancer. Furthermore, the upgrade from bulk to single-cell analysis technologies provided unprecedented opportunity to investigate cancer biology at cellular resolution allowing an in depth quantitative analysis of complex and heterogenous diseases. More recently, the advent of functional genomic screening allowed the identification of molecular pathways, cellular processes, biomarkers and novel therapeutic targets that in concert with other technologies allow patient stratification and identification of new treatment regimens. This review is intended to be a guide for researchers to cancer metabolism, highlighting current and emerging technologies, emphasizing advantages, disadvantages and applications with the potential of leading the development of innovative anti-cancer therapies.

People with intellectual and multiple disabilities access leisure, communication, and daily activities via a new technology-aided program.

People with mild to moderate intellectual or multiple disabilities may have serious difficulties in accessing leisure events, managing communication exchanges with distant partners, and performing functional daily activities. Recently, efforts were made to develop and assess technology-aided programs aimed at supporting people in all three areas (i.e., leisure, communication, and daily activities). This study assessed a new technology-aided program aimed at helping four participants with intellectual and multiple disabilities in the aforementioned areas. The program, which was implemented following a non-concurrent multiple baseline across participants design, relied on the use of a smartphone or tablet connected via Bluetooth to a two-switch device. This device served to select leisure and communication events and to control the smartphone or tablet's delivery of step instructions for the activities scheduled. Data showed that during the baseline phase (with only the smartphone or tablet available), three participants failed in each of the areas (i.e., leisure, communication and functional activities) while one participant managed to access a few leisure events. During the intervention phase (with the support of the technology-aided program), all participants managed to independently access leisure events, make telephone calls, and carry out activities. These results suggest that the program might be a useful tool for helping people with intellectual and multiple disabilities improve their condition in basic areas of daily life.

Agreement between In-Clinics and Virus Neutralization Tests in Detecting Antibodies against Canine Distemper Virus (CDV).

Core vaccinations and specific antibody titer evaluations are strongly recommended worldwide by all the vaccination guidelines. Virus neutralization (VN) is considered the gold standard for measuring antibody titer against canine distemper virus, but it is complex and time consuming, and the use of in-clinics tests would allow to obtain quicker results. The aim of this study was to evaluate the agreement of the commercial in-clinics VacciCheck test compared to VN. A total of 106 canine sera were analyzed using both methods. The best agreement was obtained using a protective threshold of ≥1:32. VacciCheck showed 95.5% sensitivity, 87.2% specificity, and 92.5% accuracy. The Cohen's kappa coefficient between methods was 0.84 (CI 95% 0.73 to 0.95), revealing an optimal agreement between the two methods (p = 0.0073). The evaluation of discordant results reveal that most samples had less than 1.5 dilution difference, and that usually did not affect the classification as protected or non-protected. Results also suggest that, in dubious cases, especially when a protective result is expected, retesting is advisable. In conclusion, VacciCheck may be considered as a reliable instrument that may help the clinician in identifying the best vaccine protocol, avoiding unnecessary vaccination, and thus reducing the incidence of adverse effects.

Tumor Suppressor Role of Wild-Type P53-Dependent Secretome and Its Proteomic Identification in PDAC.

The study of the cancer secretome is gaining even more importance in cancers such as pancreatic ductal adenocarcinoma (PDAC), whose lack of recognizable symptoms and early detection assays make this type of cancer highly lethal. The wild-type p53 protein, frequently mutated in PDAC, prevents tumorigenesis by regulating a plethora of signaling pathways. The importance of the p53 tumor suppressive activity is not only primarily involved within cells to limit tumor cell proliferation but also in the extracellular space. Thus, loss of p53 has a profound impact on the secretome composition of cancer cells and marks the transition to invasiveness. Here, we demonstrate the tumor suppressive role of wild-type p53 on cancer cell secretome, showing the anti-proliferative, apoptotic and chemosensitivity effects of wild-type p53 driven conditioned medium. By using high-resolution SWATH-MS technology, we characterized the secretomes of p53-deficient and p53-expressing PDAC cells. We found a great number of secreted proteins that have known roles in cancer-related processes, 30 of which showed enhanced and 17 reduced secretion in response to p53 silencing. These results are important to advance our understanding on the link between wt-p53 and cancer microenvironment. In conclusion, this approach may detect a secreted signature specifically driven by wild-type p53 in PDAC.

Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors owing to its robust desmoplasia, low immunogenicity, and recruitment of cancer-conditioned, immunoregulatory myeloid cells. These features strongly limit the success of immunotherapy as a single agent, thereby suggesting the need for the development of a multitargeted approach. The goal is to foster T lymphocyte infiltration within the tumor landscape and neutralize cancer-triggered immune suppression, to enhance the therapeutic effectiveness of immune-based treatments, such as anticancer adoptive cell therapy (ACT). METHODS: We examined the contribution of immunosuppressive myeloid cells expressing arginase 1 and nitric oxide synthase 2 in building up a reactive nitrogen species (RNS)-dependent chemical barrier and shaping the PDAC immune landscape. We examined the impact of pharmacological RNS interference on overcoming the recruitment and immunosuppressive activity of tumor-expanded myeloid cells, which render pancreatic cancers resistant to immunotherapy. RESULTS: PDAC progression is marked by a stepwise infiltration of myeloid cells, which enforces a highly immunosuppressive microenvironment through the uncontrolled metabolism of L-arginine by arginase 1 and inducible nitric oxide synthase activity, resulting in the production of large amounts of reactive oxygen and nitrogen species. The extensive accumulation of myeloid suppressing cells and nitrated tyrosines (nitrotyrosine, N-Ty) establishes an RNS-dependent chemical barrier that impairs tumor infiltration by T lymphocytes and restricts the efficacy of adoptive immunotherapy. A pharmacological treatment with AT38 ([3-(aminocarbonyl)furoxan-4-yl]methyl salicylate) reprograms the tumor microenvironment from protumoral to antitumoral, which supports T lymphocyte entrance within the tumor core and aids the efficacy of ACT with telomerase-specific cytotoxic T lymphocytes. CONCLUSIONS: Tumor microenvironment reprogramming by ablating aberrant RNS production bypasses the current limits of immunotherapy in PDAC by overcoming immune resistance.

Fatal cytokine release syndrome by an aberrant FLIP/STAT3 axis.

Inflammatory responses rapidly detect pathogen invasion and mount a regulated reaction. However, dysregulated anti-pathogen immune responses can provoke life-threatening inflammatory pathologies collectively known as cytokine release syndrome (CRS), exemplified by key clinical phenotypes unearthed during the SARS-CoV-2 pandemic. The underlying pathophysiology of CRS remains elusive. We found that FLIP, a protein that controls caspase-8 death pathways, was highly expressed in myeloid cells of COVID-19 lungs. FLIP controlled CRS by fueling a STAT3-dependent inflammatory program. Indeed, constitutive expression of a viral FLIP homolog in myeloid cells triggered a STAT3-linked, progressive, and fatal inflammatory syndrome in mice, characterized by elevated cytokine output, lymphopenia, lung injury, and multiple organ dysfunctions that mimicked human CRS. As STAT3-targeting approaches relieved inflammation, immune disorders, and organ failures in these mice, targeted intervention towards this pathway could suppress the lethal CRS inflammatory state.

Tryptophan and indole metabolism in immune regulation.

L-tryptophan is an essential amino acid that undergoes complex metabolic routes, resulting in production of many types of signaling molecules that fall into two types: retaining the indole ring such as serotonin, melatonin and indole-pyruvate or breaking the indole ring to form kynurenine. Kynurenines are the precursor of signaling molecules and are the first step in de novo NAD+ synthesis. In mammalian cells, the kynurenine pathway is initiated by the rate-limiting enzymes tryptophan-2,3-dioxygenase (TDO) and interferon responsive indoleamine 2,3-dioxygenase (IDO1) and is the major route for tryptophan catabolism. IDO1 regulates immune cell function through the kynurenine pathway but also by depleting tryptophan in microenvironments, and especially in tumors, which led to the development of IDO1 inhibitors for cancer therapy. However, the connections between tryptophan depletion versus product supply remain an ongoing challenge in cellular biochemistry and metabolism. Here, we highlight current knowledge about the physiological and pathological roles of tryptophan signaling network with a focus on the immune system.

Anti-ferroptotic mechanism of IL4i1-mediated amino acid metabolism.

Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologs in snake venoms (L-amino acid oxidases [LAAO]), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function. Using mammalian expressed recombinant proteins, we found that venom LAAO kills cells via hydrogen peroxide generation. By contrast, mammalian IL4i1 is non-cytotoxic and instead elicits a cell protective gene expression program inhibiting ferroptotic redox death by generating indole-3-pyruvate (I3P) from tryptophan. I3P suppresses ferroptosis by direct free radical scavenging and through the activation of an anti-oxidative gene expression program. Thus, the pro-tumor effects of IL4i1 are likely mediated by local anti-ferroptotic pathways via aromatic amino acid metabolism, arguing that an IL4i1 inhibitor may modulate tumor cell death pathways.

The immune modulatory effects of umbilical cord-derived mesenchymal stromal cells in severe COVID-19 pneumonia.

Coronavirus disease 2019 (COVID-19) may result in a life-threatening condition due to a hyperactive immune reaction to severe acute respiratory syndrome-coronavirus-2 infection, for which no effective treatment is available. Based on the potent immunomodulatory properties of mesenchymal stromal cells (MSCs), a growing number of trials are ongoing. This prompted us to carry out a thorough immunological study in a patient treated with umbilical cord-derived MSCs and admitted to the Intensive Care Unit for COVID-19-related pneumonia. The exploratory analyses were assessed on both peripheral blood and bronchoalveolar fluid lavage samples at baseline and after cellular infusion by means of single-cell RNA sequencing, flow cytometry, ELISA, and functional assays. Remarkably, a normalization of circulating T lymphocytes count paralleled by a reduction of inflammatory myeloid cells, and a decrease in serum levels of pro-inflammatory cytokines, mostly of interleukin-6 and tumor necrosis factor-α, were observed. In addition, a drop of plasma levels of those chemokines essential for neutrophil recruitment became evident that paralleled the decrease of lung-infiltrating inflammatory neutrophils. Finally, circulating monocytes and low-density gradient neutrophils acquired immunosuppressive function. This scenario was accompanied by an amelioration of respiratory, renal, inflammatory, and pro-thrombotic indexes. Our results provide the first immunological data possibly related to the use of umbilical cord-derived MSCs in severe COVID-19 context.