Fluorogenic Chemical Probes for Wash-free Imaging of Cell Membrane Damage in Ferroptosis, Necrosis, and Axon Injury.

Selectively labeling cells with damaged membranes is needed not only for identifying dead cells in culture, but also for imaging membrane barrier dysfunction in pathologies in vivo. Most membrane permeability stains are permanently colored or fluorescent dyes that need washing to remove their non-uptaken extracellular background and reach good image contrast. Others are DNA-binding environment-dependent fluorophores, which lack design modularity, have potential toxicity, and can only detect permeabilization of cell volumes containing a nucleus (i.e., cannot delineate damaged volumes in vivo nor image non-nucleated cell types or compartments). Here, we develop modular fluorogenic probes that reveal the whole cytosolic volume of damaged cells, with near-zero background fluorescence so that no washing is needed. We identify a specific disulfonated fluorogenic probe type that only enters cells with damaged membranes, then is enzymatically activated and marks them. The esterase probe MDG1 is a reliable tool to reveal live cells that have been permeabilized by biological, biochemical, or physical membrane damage, and it can be used in multicolor microscopy. We confirm the modularity of this approach by also adapting it for improved hydrolytic stability, as the redox probe MDG2. We conclude by showing the unique performance of MDG probes in revealing axonal membrane damage (which DNA fluorogens cannot achieve) and in discriminating damage on a cell-by-cell basis in embryos in vivo. The MDG design thus provides powerful modular tools for wash-free in vivo imaging of membrane damage, and indicates how designs may be adapted for selective delivery of drug cargoes to these damaged cells: offering an outlook from selective diagnosis toward therapy of membrane-compromised cells in disease.

Retinoic acid drives intestine-specific adaptation of effector ILC2s originating from distant sites.

Adaptation of immune cells to tissue-specific microenvironments is a crucial process in homeostasis and inflammation. Here, we show that murine effector type 2 innate lymphoid cells (ILC2s) from various organs are equally effective in repopulating ILC2 niches in other anatomical locations where they adapt tissue-specific phenotypes of target organs. Single-cell transcriptomics of ILC2 populations revealed upregulation of retinoic acid (RA) signaling in ILC2s during adaptation to the small intestinal microenvironment, and RA signaling mediated reprogramming of kidney effector ILC2s toward the small intestinal phenotype in vitro and in vivo. Inhibition of intestinal ILC2 adaptation by blocking RA signaling impaired worm expulsion during Strongyloides ratti infection, indicating functional importance of ILC2 tissue imprinting. In conclusion, this study highlights that effector ILC2s retain the ability to adapt to changing tissue-specific microenvironments, enabling them to exert tissue-specific functions, such as promoting control of intestinal helminth infections.

Increased Alveolar Epithelial Damage Markers and Inflammasome-Regulated Cytokines Are Associated with Pulmonary Superinfection in ARDS.

Acute respiratory distress syndrome (ARDS) is a life-threatening form of respiratory failure defined by dysregulated immune homeostasis and alveolar epithelial and endothelial damage. Up to 40% of ARDS patients develop pulmonary superinfections, contributing to poor prognosis and increasing mortality. Understanding what renders ARDS patients highly susceptible to pulmonary superinfections is therefore essential. We hypothesized that ARDS patients who develop pulmonary superinfections display a distinct pulmonary injury and pro-inflammatory response pattern. Serum and BALF samples from 52 patients were collected simultaneously within 24 h of ARDS onset. The incidence of pulmonary superinfections was determined retrospectively, and the patients were classified accordingly. Serum concentrations of the epithelial markers soluble receptor for advanced glycation end-products (sRAGE) and surfactant protein D (SP-D) and the endothelial markers vascular endothelial growth factor (VEGF) and angiopoetin-2 (Ang-2) as well as bronchoalveolar lavage fluid concentrations of the pro-inflammatory cytokines interleukin 1ß (IL-1ß), interleukin 18 (IL-18), interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-a) were analyzed via multiplex immunoassay. Inflammasome-regulated cytokine IL-18 and the epithelial damage markers SP-D and sRAGE were significantly increased in ARDS patients who developed pulmonary superinfections. In contrast, endothelial markers and inflammasome-independent cytokines did not differ between the groups. The current findings reveal a distinct biomarker pattern that indicates inflammasome activation and alveolar epithelial injury. This pattern may potentially be used in future studies to identify high-risk patients, enabling targeted preventive strategies and personalized treatment approaches.

Impact of digital health interventions for adolescents with type 1 diabetes mellitus on health literacy: a systematic review.

BACKGROUND: Evidence shows that living with diabetes mellitus type 1 (T1DM) in adolescent age is particularly challenging and difficult to manage. A high level of health literacy is important to prevent and avoid debilitating complications. Despite the increasing prevalence and incidence of T1DM by adolescent and the large use of digital health interventions, little is known about the association between this use and health literacy. This systematic review provides an overview on the impact of digital health interventions for adolescents with type 1 diabetes on health literacy and derive recommendations for further research. METHODS: Electronic searches were performed in five databases in Medline (Medline, PubMed + via PubMed), The Cochrane Library, EMBASE (via Ovid), Web of Science and PsycINFO from 2011 to 2021. In addition, grey literature searches were conducted in Google Scholar, OAlster and Trip. Relevant studies that have been missed by electronic and hand-searching strategies were searched in the reference lists of all included studies. The review followed PRISMA guidelines. Two researchers independently screened abstracts for initial eligibility and applied the inclusion and exclusion criteria to the relevant full-text articles. Quality was assessed using the tools RoB2 Cochrane, ROBINS I, NOS (Newcastle-Ottawa Scale), CASP (Critical Appraisal Skills Programme) for primary studies and Amstar-2 for secondary studies. RESULTS: Out of 981 studies, 22 were included in the final review. Most primary studies included in this review were judged as moderate overall risk of bias or with some concerns and most of the secondary studies as critically low quality reviews. Our findings suggest that the interplay of health care providers (HCP) and patients through social media helps the management of the disease. This corroborates Bröder et al.' (2017) dimension of 'communication and interactions' in their concept of health literacy. CONCLUSIONS: For adolescents with T1DM, social media may be a specific and beneficial intervention for an improved communication and interaction with their HCP. Further research should investigate what specific form of social media suits best for which adolescents. TRIAL REGISTRATION: The study protocol was registered on the 15th of November 2021 on Prospero (reg. NR: CRD42021282199).

Fight INflammation to Improve outcome after aneurysmal Subarachnoid HEmorRhage (FINISHER) trial: Study protocol for a randomized controlled trial.

RATIONALE: Aneurysmal subarachnoid hemorrhage (SAH) has high morbidity and mortality. While the primary injury results from the initial bleeding cannot currently be influenced, secondary injury through vasospasm and delayed cerebral ischemia worsens outcome and might be a target for interventions to improve outcome. To date, beside the aneurysm treatment to prevent re-bleeding and the administration of oral nimodipine, there is no therapy available, so novel treatment concepts are needed. Evidence suggests that inflammation contributes to delayed cerebral ischemia and poor outcome in SAH. Some studies suggest a beneficial effect of anti-inflammatory glucocorticoids, but there are no data from randomized controlled trials examining the efficacy of glucocorticoids. Therefore, current guidelines do not recommend the use of glucocorticoids in SAH. AIM: The Fight INflammation to Improve outcome after aneurysmal Subarachnoid HEmorRhage (FINISHER) trial aims to determine whether dexamethasone improves outcome in a clinically relevant endpoint in SAH patients. METHODS AND DESIGN: FINISHER is a multicenter, prospective, randomized, double-blinded, placebo-controlled clinical phase III trial which is testing the outcome and safety of anti-inflammatory treatment with dexamethasone in SAH patients. SAMPLE SIZE ESTIMATES: In all, 334 patients will be randomized to either dexamethasone or placebo within 48 h after SAH. The dexamethasone dose is 8 mg tds for days 1-7 and then 8 mg od for days 8-21. STUDY OUTCOME: The primary outcome is the modified Rankin Scale (mRS) at 6 months, which is dichotomized to favorable (mRS 0-3) versus unfavorable (mRS 4-6). DISCUSSION: The results of this study will provide the first phase III evidence as to whether dexamethasone improves outcome in SAH.

Food for thought - ILC metabolism in the context of helminth infections.

Helminths are multicellular ancient organisms residing as parasites at mucosal surfaces of their host. Through adaptation and co-evolution with their hosts, helminths have been able to develop tolerance mechanisms to limit inflammation and avoid expulsion. The study of helminth infections as an integral part of tissue immunology allowed us to understand fundamental aspects of mucosal and barrier immunology, which led to the discovery of a new group of tissue-resident immune cells, innate lymphoid cells (ILC), over a decade ago. Here, we review the intricate interplay between helminth infections and type 2 ILC (ILC2) biology, discuss the host metabolic adaptation to helminth infections and the metabolic pathways fueling ILC2 responses. We hypothesize that nutrient competition between host and helminths may have prevented chronic inflammation in the past and argue that a detailed understanding of the metabolic restraints imposed by helminth infections may offer new therapeutic avenues in the future.

Impaired ketogenesis ties metabolism to T cell dysfunction in COVID-19.

Anorexia and fasting are host adaptations to acute infection, and induce a metabolic switch towards ketogenesis and the production of ketone bodies, including ß-hydroxybutyrate (BHB)1-6. However, whether ketogenesis metabolically influences the immune response in pulmonary infections remains unclear. Here we show that the production of BHB is impaired in individuals with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) but not in those with  influenza-induced ARDS. We found that BHB promotes both the survival of and the production of interferon-γ by CD4+ T cells. Applying a metabolic-tracing analysis, we established that BHB provides an alternative carbon source to fuel oxidative phosphorylation (OXPHOS) and the production of bioenergetic amino acids and glutathione, which is important for maintaining the redox balance. T cells from patients with SARS-CoV-2-induced ARDS were exhausted and skewed towards glycolysis, but could be metabolically reprogrammed by BHB to perform OXPHOS, thereby increasing their functionality. Finally, we show in mice that a ketogenic diet and the delivery of BHB as a ketone ester drink restores CD4+ T cell metabolism and function in severe respiratory infections, ultimately reducing the mortality of mice infected with SARS-CoV-2. Altogether, our data reveal that BHB is an alternative source of carbon that promotes T cell responses in pulmonary viral infections, and highlight impaired ketogenesis as a potential confounding factor in severe COVID-19.

The neuropeptide VIP potentiates intestinal innate type 2 and type 3 immunity in response to feeding.

The nervous system and the immune system both rely on an extensive set of modalities to perceive and act on perturbations in the internal and external environments. During feeding, the intestine is exposed to nutrients that may contain noxious substances and pathogens. Here we show that Vasoactive Intestinal Peptide (VIP), produced by the nervous system in response to feeding, potentiates the production of effector cytokines by intestinal type 2 and type 3 innate lymphoid cells (ILC2s and ILC3s). Exposure to VIP alone leads to modest activation of ILCs, but strongly potentiates ILCs to concomitant or subsequent activation by the inducer cytokines IL-33 or IL-23, via mobilization of cAMP and energy by glycolysis. Consequently, VIP increases resistance to intestinal infection by the helminth Trichuris muris and the enterobacteria Citrobacter rodentium. These findings uncover a functional neuro-immune crosstalk unfolding during feeding that increases the reactivity of innate immunity necessary to face potential threats associated with food intake.

Mitochondrial Dysfunction Contributes to Impaired Cytokine Production of CD56bright Natural Killer Cells From Human Immunodeficiency Virus-Infected Individuals Under Effective Antiretroviral Therapy.

Human immunodeficiency virus (HIV) infection is associated with impaired natural killer (NK) cell activity, which is only incompletely restored under antiretroviral therapy. Analyzing the bioenergetics profiles of oxygen consumption, we observed that several parameters were significantly reduced in HIV+ NK cells, indicating a mitochondrial defect. Accordingly, we found HIV+ CD56bright NK cells to display a decreased mitochondrial membrane potential and mitochondrial mass. Both parameters were positively correlated with interferon gamma (IFN-γ) production of NK cells. Finally, we demonstrated that stimulation of HIV+ NK cells with MitoTEMPO, a mitochondria-targeting antioxidant, significantly improved IFN-γ production. We identified mitochondrial dysfunction as a mechanism that contributes to impaired NK cell function.

Tetracycline ameliorates silica-induced pulmonary inflammation and fibrosis via inhibition of caspase-1.

BACKGROUND: Inhalation of dust containing silica particles is associated with severe pulmonary inflammation and lung injury leading to chronic silicosis including fibrotic remodeling of the lung. Silicosis represents a major global health problem causing more than 45.000 deaths per year. The inflammasome-caspase-1 pathway contributes to the development of silica-induced inflammation and fibrosis via IL-1ß and IL-18 production. Recent studies indicate that tetracycline can be used to treat inflammatory diseases mediated by IL-1ß and IL-18. Therefore, we hypothesized that tetracycline reduces silica-induced lung injury and lung fibrosis resulting from chronic silicosis via limiting IL-1ß and IL-18 driven inflammation. METHODS: To investigate whether tetracycline is a therapeutic option to block inflammasome-caspase-1 driven inflammation in silicosis, we incubated macrophages with silica alone or combined with tetracycline. The in vivo effect of tetracycline was determined after intratracheal administration of silica into the mouse lung. RESULTS: Tetracycline selectively blocks IL-1ß production and pyroptotic cell death via inhibition of caspase-1 in macrophages exposed to silica particles. Consistent, treatment of silica-instilled mice with tetracycline significantly reduced pulmonary caspase-1 activation as well as IL-1ß and IL-18 production, thereby ameliorating pulmonary inflammation and lung injury. Furthermore, prolonged tetracycline administration in a model of chronic silicosis reduced lung damage and fibrotic remodeling. CONCLUSIONS: These findings suggest that tetracycline inhibits caspase-1-dependent production of IL-1ß in response to silica in vitro and in vivo. The results were consistent with tetracycline reducing silica-induced pulmonary inflammation and chronic silicosis in terms of lung injury and fibrosis. Thus, tetracycline could be effective in the treatment of patients with silicosis as well as other diseases involving silicotic inflammation.

Enemy or ally? Fasting as an essential regulator of immune responses.

Nutrition is essential for supplying an organism with sufficient energy to maintain its bodily functions. Apart from serving as an energy supply, the immunomodulatory effects of diet are emerging as a central aspect of human health. The latest evidence suggests that dietary restriction may play an important regulatory role by influencing the activation and effector functions of immune cells. However, depending on the context, nutrient restriction may have both pathogenic and beneficial effects. Here, we discuss the diverse roles of fasting programs, including ketogenesis in infection and chronic inflammation, aiming to clarify their detrimental and/or beneficial effects. Understanding these differences may help identify conditions under which dietary interventions might serve as putative effective approaches to treat various diseases.

Inhibition of Caspase-1 with Tetracycline Ameliorates Acute Lung Injury.

Rationale: Acute respiratory distress syndrome (ARDS) is a heterogeneous syndrome with a mortality of up to 40%. Precision medicine approaches targeting patients on the basis of their molecular phenotypes of ARDS might help to identify effective pharmacotherapies. The inflammasome-caspase-1 pathway contributes to the development of ARDS via IL-1ß and IL-18 production. Recent studies indicate that tetracycline can be used to treat inflammatory diseases mediated by IL-1ß and IL-18, although the molecular mechanism by which tetracycline inhibits inflammasome-caspase-1 signaling remains unknown. Objectives: To identify patients with ARDS characterized by IL-1ß and IL-18 expression and investigate the ability of tetracycline to inhibit inflammasome-caspase-1 signaling in ARDS. Methods: IL-1ß and IL-18 concentrations were quantified in BAL fluid from patients with ARDS. Tetracycline's effects on lung injury and inflammation were assessed in two mouse models of direct (pulmonary) acute lung injury, and its effects on IL-1ß and IL-18 production were assessed by alveolar leukocytes from patients with direct ARDS ex vivo. Murine macrophages were used to further characterize the effect of tetracycline on the inflammasome-caspase-1 pathway. Measurements and Main Results: BAL fluid concentrations of IL-1ß and IL-18 are significantly higher in patients with direct ARDS than those with indirect (nonpulmonary) ARDS. In experimental acute lung injury, tetracycline significantly diminished lung injury and pulmonary inflammation by selectively inhibiting caspase-1-dependent IL-1ß and IL-18 production, leading to improved survival. Tetracycline also reduced the production of IL-1ß and IL-18 by alveolar leukocytes from patients with direct ARDS. Conclusions: Tetracycline may be effective in the treatment of direct ARDS in patients with elevated caspase-1 activity. Clinical Trial registered with www.clinicaltrials.gov (NCT04079426).

SP-D Serum Levels Reveal Distinct Epithelial Damage in Direct Human ARDS.

Acute respiratory distress syndrome (ARDS) is a heterogeneous syndrome with multiple underlying diseases. Particularly epithelial damage results from direct (e.g., pneumonia) rather than indirect lung injury (e.g., nonpulmonary sepsis), which is more likely associated with endothelial damage. Hence, targeting ARDS patients based on their molecular phenotypes is a promising approach to improve outcome. With regard to distinct inflammatory responses and subsequent lung damage in direct ARDS due to the causing pathogen, we quantified markers of epithelial and endothelial damage and pro-inflammatory cytokines in patients with ARDS triggered by bacterial, viral, and atypical pathogen pneumonia or indirect ARDS. The serum levels of interleukin-6 (IL-6) and interleukin-8 (IL-8), lung epithelial injury markers surfactant protein D (SP-D), and soluble receptor for advanced glycation end-products (sRAGE) as well as endothelial injury marker angiopoietin-2 (Ang-2) from 49 patients with distinct types of ARDS were analyzed by multiplex immunoassay. Epithelial damage marker SP-D was significantly higher in direct ARDS caused by viral and atypical pathogens in contrast to ARDS caused by typical bacterial pneumonia and nonpulmonary sepsis. In contrast, sRAGE levels did not differ due to the causing pathogen. Patients with atypical pathogen pneumonia related ARDS showed significantly lower Ang-2 levels compared to patients with viral and indirect ARDS. Patients with viral and atypical pneumonia related ARDS possessed significantly lower serum IL-6 levels compared to bacterial pneumonia related ARDS and IL-6 levels in atypical pneumonia related ARDS were significantly lower than in indirect ARDS. Current findings report a potential difference in ARDS biomarkers due to the underlying disease and pathogen.

Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells.

Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.

Lipid-Droplet Formation Drives Pathogenic Group 2 Innate Lymphoid Cells in Airway Inflammation.

Innate lymphoid cells (ILCs) play an important role in the control and maintenance of barrier immunity. However, chronic activation of ILCs results in immune-mediated pathology. Here, we show that tissue-resident type 2 ILCs (ILC2s) display a distinct metabolic signature upon chronic activation. In the context of allergen-driven airway inflammation, ILC2s increase their uptake of both external lipids and glucose. Externally acquired fatty acids are transiently stored in lipid droplets and converted into phospholipids to promote the proliferation of ILC2s. This metabolic program is imprinted by interleukin-33 (IL-33) and regulated by the genes Pparg and Dgat1, which are both controlled by glucose availability and mTOR signaling. Restricting dietary glucose by feeding mice a ketogenic diet largely ablated ILC2-mediated airway inflammation by impairing fatty acid metabolism and the formation of lipid droplets. Together, these results reveal that pathogenic ILC2 responses require lipid metabolism and identify ketogenic diet as a potent intervention strategy to treat airway inflammation.

Adiponectin Limits IFN-γ and IL-17 Producing CD4 T Cells in Obesity by Restraining Cell Intrinsic Glycolysis.

Compared to the innate immune system, the contribution of the adaptive immune response during obesity and insulin resistance is still not completely understood. Here we demonstrate that high fat diet (HFD) increases the frequencies of activated CD4+ and CD8+ T cells and frequencies of T cells positive for IFN-γ and IL-17 in the adipose tissue. The adipocyte-derived soluble factor adiponectin reduces IFN-γ and IL-17 positive CD4+ T cells from HFD mice and dampens the differentiation of naïve T cells into Th1 cells and Th17 cells. Adiponectin reduces Th17 cell differentiation and restrains glycolysis in an AMPK dependent fashion. Treatment with adult worm extracts of the rodent filarial nematode Litomosoides sigmodontis (LsAg) reduces adipose tissue Th1 and Th17 cell frequencies during HFD and increases adiponectin levels. Stimulation of T cells in the presence of adipocyte-conditioned media (ACM) from LsAg-treated mice reduces Th1 and Th17 frequencies and this effect was abolished when ACM was treated with an adiponectin neutralizing antibody. Collectively, these data reveal a novel role of adiponectin in controlling pro-inflammatory CD4+ T cells during obesity and suggest that the beneficial role of helminth infections and helminth-derived products on obesity and insulin resistance may be in part mediated by adiponectin.

Innate lymphoid cells-key immune integrators of overall body homeostasis.

The maintenance of the tissue barrier is essential to protect the host from external pathogens, thus ensuring the survival of the organism. This process requires the integration of various physiological signals originating from the digestive, immune, endocrine, and the nervous system as indicators of overall body fitness. Innate lymphoid cells (ILC) are a group of immune cells equipped for the guarding and maintenance of the tissue barrier against invading pathogens. Extensive research has focused on the regulation of ILC by cytokines derived from immune or non-immune cells, such as the epithelium. However, recent findings suggest that ILC may play an additional role in the monitoring of the overall health status of the host. This requires the combined sensing of cytokines, metabolites, hormones, and neuropeptides. ILC appear to be essential in this process functioning as hubs for the integration of different physiological signals to facilitate barrier immunity. Here, we discuss the emerging literature revealing dietary, metabolic, hormonal, and neuronal signals as important controllers and modulators of ILC function in health and disease.