Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump.

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.

Activated eosinophils in early life impair lung development and promote long-term lung damage.

Exaggeration of type 2 immune responses promotes lung inflammation and altered lung development; however, eosinophils, despite expansion in the postnatal lung, have not been specifically assessed in the context of neonatal lung disease. Furthermore, early life factors including prematurity and respiratory infection predispose infants to chronic obstructive pulmonary disease later in life. To assess eosinophils in the developing lung and how they may contribute to chronic lung disease, we generated mice harboring eosinophil-specific deletion of the negative regulatory enzyme SH2 domain-containing inositol 5' phosphatase-1. This increased the activity and number of pulmonary eosinophils in the developing lung, which was associated with impaired lung development, expansion of activated alveolar macrophages (AMφ), multinucleated giant cell formation, enlargement of airspaces, and fibrosis. Despite regression of eosinophils following completion of lung development, AMφ-dominated inflammation persisted, alongside lung damage. Bone marrow chimera studies showed that SH2 domain-containing inositol 5' phosphatase-1-deficient eosinophils were not sufficient to drive inflammatory lung disease in adult steady-state mice but once inflammation and damage were present, it could not be resolved. Depletion of eosinophils during alveolarization alleviated pulmonary inflammation and lung pathology, demonstrating an eosinophil-intrinsic effect. These results show that the presence of activated eosinophils during alveolarization aggravates AMφs and promotes sustained inflammation and long-lasting lung pathology.

A new superhero, the intraepithelial mast cell, joins the "Guardians of the Gut".

The intestinal epithelium interacts with immune cells to support tissue homeostasis and coordinate responses against pathogens. In this issue of Immunity, Yang et al. unveil a central role for mast cell-epithelial cell interactions in orchestrating protective type 2 immune responses following intestinal helminth infection.

Multi-omics integration reveals a nonlinear signature that precedes progression of lung fibrosis.

Objectives: Idiopathic pulmonary fibrosis (IPF) is a devastating progressive interstitial lung disease with poor outcomes. While decades of research have shed light on pathophysiological mechanisms associated with the disease, our understanding of the early molecular events driving IPF and its progression is limited. With this study, we aimed to model the leading edge of fibrosis using a data-driven approach. Methods: Multiple omics modalities (transcriptomics, metabolomics and lipidomics) of healthy and IPF lung explants representing different stages of fibrosis were combined using an unbiased approach. Multi-Omics Factor Analysis of datasets revealed latent factors specifically linked with established fibrotic disease (Factor1) and disease progression (Factor2). Results: Features characterising Factor1 comprised well-established hallmarks of fibrotic disease such as defects in surfactant, epithelial-mesenchymal transition, extracellular matrix deposition, mitochondrial dysfunction and purine metabolism. Comparatively, Factor2 identified a signature revealing a nonlinear trajectory towards disease progression. Molecular features characterising Factor2 included genes related to transcriptional regulation of cell differentiation, ciliogenesis and a subset of lipids from the endocannabinoid class. Machine learning models, trained upon the top transcriptomics features of each factor, accurately predicted disease status and progression when tested on two independent datasets. Conclusion: This multi-omics integrative approach has revealed a unique signature which may represent the inflection point in disease progression, representing a promising avenue for the identification of therapeutic targets aimed at addressing the progressive nature of the disease.

Neuroimmune Interactions in the Lung.

Barrier tissues are highly innervated by sensory and autonomic nerves that are positioned in close proximity to both stromal and immune cell populations. Together with a growing awareness of the far-reaching consequences of neuroimmune interactions, recent studies have uncovered key mechanisms through which they contribute to organ homeostasis and immunity. It has also become clear that dysregulation of such interactions is implicated in the development of chronic lung diseases. This review describes the characteristics of the lung nervous system and discusses the molecular mechanisms that underlie lung neuroimmune interactions in infection and disease. We have contextualized the current literature and identified opportune areas for further investigation. Indeed, both the lung-brain axis and local neuroimmune interactions hold enormous potential for the exploration and development of novel therapeutic strategies targeting lung diseases. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Serological response and breakthrough infection after COVID-19 vaccination in patients with cirrhosis and post-liver transplant.

BACKGROUND: Vaccine hesitancy and lack of access remain major issues in disseminating COVID-19 vaccination to liver patients globally. Factors predicting poor response to vaccination and risk of breakthrough infection are important data to target booster vaccine programs. The primary aim of the current study was to measure humoral responses to 2 doses of COVID-19 vaccine. Secondary aims included the determination of factors predicting breakthrough infection. METHODS: COVID-19 vaccination and Biomarkers in cirrhosis And post-Liver Transplantation is a prospective, multicenter, observational case-control study. Participants were recruited at 4-10 weeks following first and second vaccine doses in cirrhosis [n = 325; 94% messenger RNA (mRNA) and 6% viral vaccine], autoimmune liver disease (AILD) (n = 120; 77% mRNA and 23% viral vaccine), post-liver transplant (LT) (n = 146; 96% mRNA and 3% viral vaccine), and healthy controls (n = 51; 72% mRNA, 24% viral and 4% heterologous combination). Serological end points were measured, and data regarding breakthrough SARS-CoV-2 infection were collected. RESULTS: After adjusting by age, sex, and time of sample collection, anti-Spike IgG levels were the lowest in post-LT patients compared to cirrhosis (p < 0.0001), AILD (p < 0.0001), and control (p = 0.002). Factors predicting reduced responses included older age, Child-Turcotte-Pugh B/C, and elevated IL-6 in cirrhosis; non-mRNA vaccine in AILD; and coronary artery disease, use of mycophenolate and dysregulated B-call activating factor, and lymphotoxin-α levels in LT. Incident infection occurred in 6.6%, 10.6%, 7.4%, and 15.6% of cirrhosis, AILD, post-LT, and control, respectively. The only independent factor predicting infection in cirrhosis was low albumin level. CONCLUSIONS: LT patients present the lowest response to the SARS-CoV-2 vaccine. In cirrhosis, the reduced response is associated with older age, stage of liver disease and systemic inflammation, and breakthrough infection with low albumin level.

Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing.

BACKGROUND: The exploitation of anti-tumour immunity, harnessed through immunomodulatory therapies, has fundamentally changed the treatment of primary liver cancer (PLC). However, this has posed significant challenges in preclinical research. Novel immunologically relevant models for PLC are urgently required to improve the translation from bench to bedside and back, explore and predict effective combinatorial therapies, aid novel drug discovery and develop personalised treatment modalities. METHODS: We used human precision-cut tissue slices (PCTS) derived from resected tumours to create a patient-specific immunocompetent disease model that captures the multifaceted and intricate heterogeneity of the tumour and the tumour microenvironment. Tissue architecture, tumour viability and treatment response to single agent and combination therapies were assessed longitudinally over 8 days of ex vivo culture by histological analysis, detection of proliferation/cell death markers, ATP content via HPLC. Immune cell infiltrate was assessed using PCR and immunofluorescence. Checkpoint receptor expression was quantified via Quantigene RNA assay. FINDINGS: After optimising the culture conditions, PCTS maintained the original tissue architecture, including tumour morphology, stroma and tumour-infiltrated leukocytes. Moreover, PCTS retained the tumour-specific immunophenotype over time, suggesting the utility of PCTS to investigate immunotherapeutic drug efficacy and identify non-responsiveness. INTERPRETATION: Here we have characterised the PCTS model and demonstrated its effectiveness as a robust preclinical tool that will significantly support the development of successful (immuno)therapeutic strategies for PLC. FUNDING: Foundation for Liver Research, London.

Development of severe colitis is associated with lung inflammation and pathology.

Inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis are chronic relapsing diseases that affect the gastrointestinal tract, most commonly the colon. A link between the gut and the lung is suggested since patients with IBD have an increased susceptibility for chronic inflammatory lung disease. Furthermore, in the absence of overt lung disease, IBD patients have worsened lung function and more leukocytes in sputum than healthy individuals, highlighting a conduit between the gut and lung in disease. To study the gut-lung axis in the context of IBD, we used TCRδ-/- mice, which are highly susceptible to dextran sulfate sodium (DSS) due to the importance of γδ T cells in maintenance of barrier integrity. After induction of experimental colitis using DSS, the lungs of TCRδ-/- mice exhibited signs of inflammation and mild emphysema, which was not observed in DSS-treated C57BL/6 mice. Damage to the lung tissue was accompanied by a large expansion of neutrophils in the lung parenchyma and an increase in alveolar macrophages in the lung wash. Gene expression analyses showed a significant increase in Csf3, Cxcl2, Tnfa, and Il17a in lung tissue in keeping with neutrophil infiltration. Expression of genes encoding reactive oxygen species enzymes and elastolytic enzymes were enhanced in the lungs of both C57BL/6 and TCRδ-/- mice with colitis. Similarly, surfactant gene expression was also enhanced, which may represent a protective mechanism. These data demonstrate that severe colitis in a susceptible genetic background is sufficient to induce lung inflammation and tissue damage, providing the research community with an important tool for the development of novel therapeutics aimed at reducing co-morbidities in IBD patients.

Networking between helminths, microbes, and mammals.

Just as mammals have coevolved with the intestinal bacterial communities that are part of the microbiota, intestinal helminths represent an important selective force on their mammalian host. The complex interaction between helminths, microbes, and their mammalian host is likely an important determinant of mutual fitness. The host immune system in particular is a critical interface with both helminths and the microbiota, and this crosstalk often determines the balance between tolerance and resistance against these widespread parasites. Hence, there are many examples of how both helminths and the microbiota can influence tissue homeostasis and homeostatic immunity. Understanding these processes at a cellular and molecular level is an exciting area of research that we seek to highlight in this review and that will potentially guide future treatment approaches.

The skin microbiome in the first year of life and its association with atopic dermatitis.

BACKGROUND: Early-life microbial colonization of the skin may modulate the immune system and impact the development of atopic dermatitis (AD) and allergic diseases later in life. To address this question, we assessed the association between the skin microbiome and AD, skin barrier integrity and allergic diseases in the first year of life. We further explored the evolution of the skin microbiome with age and its possible determinants, including delivery mode. METHODS: Skin microbiome was sampled from the lateral upper arm on the first day of life, and at 3, 6, and 12 months of age. Bacterial communities were assessed by 16S rRNA gene amplicon sequencing in 346 infants from the PreventADALL population-based birth cohort study, representing 970 samples. Clinical investigations included skin examination and skin barrier function measured as trans-epidermal water loss (TEWL) at the site and time of microbiome sampling at 3, 6, and 12 months. Parental background information was recorded in electronic questionnaires, and delivery mode (including vaginal delivery (VD), VD in water, elective caesarean section (CS) and emergency CS) was obtained from maternal hospital charts. RESULTS: Strong temporal variations in skin bacterial community composition were found in the first year of life, with distinct patterns associated with different ages. Confirming our hypothesis, skin bacterial community composition in the first year of life was associated with skin barrier integrity and later onsets of AD. Delivery mode had a strong impact on the microbiome composition at birth, with each mode leading to distinct patterns of colonization. Other possible determinants of the skin microbiome were identified, including environmental and parental factors as well as breastfeeding. CONCLUSION: Skin microbiome composition during infancy is defined by age, transiently influenced by delivery mode as well as environmental, parental factors and breastfeeding. The microbiome is also associated with skin barrier integrity and the onset of AD.

Helminth Infection-Induced Increase in Virtual Memory CD8 T Cells Is Transient, Driven by IL-15, and Absent in Aged Mice.

CD8 virtual memory T (TVM) cells are Ag-naive CD8 T cells that have undergone partial differentiation in response to common γ-chain cytokines, particularly IL-15 and IL-4. TVM cells from young individuals are highly proliferative in response to TCR and cytokine stimulation but, with age, they lose TCR-mediated proliferative capacity and exhibit hallmarks of senescence. Helminth infection can drive an increase in TVM cells, which is associated with improved pathogen clearance during subsequent infectious challenge in young mice. Given the cytokine-dependent profile of TVM cells and their age-associated dysfunction, we traced proliferative and functional changes in TVM cells, compared with true naive CD8 T cells, after helminth infection of young and aged C57BL/6 mice. We show that IL-15 is essential for the helminth-induced increase in TVM cells, which is driven only by proliferation of existing TVM cells, with negligible contribution from true naive cell differentiation. Additionally, TVM cells showed the greatest proliferation in response to helminth infection and IL-15 compared with other CD8 T cells. Furthermore, TVM cells from aged mice did not undergo expansion after helminth infection due to both TVM cell-intrinsic and -extrinsic changes associated with aging.

Human Precision-Cut Liver Slices: A Potential Platform to Study Alcohol-Related Liver Disease.

Alcohol-related liver disease (ALD) encompasses a range of pathological conditions that are complex to study at the clinical and preclinical levels. Despite the global burden of ALD, there is a lack of effective treatments, and mortality is high. One of the reasons for the unsuccessful development of novel therapies is that experimental studies are hindered by the challenge of recapitulating this multifactorial disorder in vitro, including the contributions of hepatotoxicity, impaired lipid metabolism, fibrosis and inflammatory cytokine storm, which are critical drivers in the pathogenesis of ALD in patients and primary targets for drug development. Here, we present the unique characteristics of the culture of human precision-cut liver slices (PCLS) to replicate key disease processes in ALD. PCLS were prepared from human liver specimens and treated with ethanol alone or in combination with fatty acids and lipopolysaccharide (FA + LPS) for up to 5 days to induce hepatotoxic, inflammatory and fibrotic events associated with ALD. Alcohol insult induced hepatocyte death which was more pronounced with the addition of FA + LPS. This mixture showed a significant increase in the cytokines conventionally associated with the prototypical inflammatory response observed in severe ALD, and interestingly, alcohol alone exhibited a different effect. Profibrogenic activation was also observed in the slices and investigated in the context of slice preparation. These results support the versatility of this organotypic model to study different pathways involved in alcohol-induced liver damage and ALD progression and highlight the applicability of the PCLS for drug discovery, confirming their relevance as a bridge between preclinical and clinical studies.

Adding data to gut feelings.

Pain-sensing neurons detect environmental insults and tissue injury, driving avoidance behavior and the local release of neuropeptides. Two related papers in this issue of Cell report that gut-innervating pain neurons sense bacterial presence to both shape the constituents of the gut microbiome and protect against excessive inflammation.

Human Immunocompetent Model of Neuroendocrine Liver Metastases Recapitulates Patient-Specific Tumour Microenvironment.

Neuroendocrine liver metastases (LM-NEN) develop in a considerable proportion of patients with gastroenteropancreatic neuroendocrine neoplasms. There is a paucity of experimental models that accurately recapitulate this complex metastatic human liver microenvironment precluding scientific and clinical advancements. Here, we describe the development of a novel personalised immunocompetent precision cut tumour slice (PCTS) model for LM-NEN using resected human liver tissue. The histological assessment throughout the culture demonstrated that slices maintain viability for at least 7 days and retain the cellular heterogeneity of the original tumour. Essential clinical features, such as patient-specific histoarchitecture, tumour grade, neuroendocrine differentiation and metabolic capacity, are preserved in the slices. The PCTS also replicate the tumor-specific immunological profile as shown by the innate and adaptive immunity markers analysis. Furthermore, the study of soluble immune checkpoint receptors in the culture supernatants proves that these immunomodulators are actively produced by LM-NEN and suggests that this process is epithelium-dependent. This model can be employed to investigate these pathways and provides a powerful platform for mechanistic, immunological and pre-clinical studies.

Tetracycline-induced mitohormesis mediates disease tolerance against influenza.

Mitohormesis defines the increase in fitness mediated by adaptive responses to mild mitochondrial stress. Tetracyclines inhibit not only bacterial but also mitochondrial translation, thus imposing a low level of mitochondrial stress on eukaryotic cells. We demonstrate in cell and germ-free mouse models that tetracyclines induce a mild adaptive mitochondrial stress response (MSR), involving both the ATF4-mediated integrative stress response and type I interferon (IFN) signaling. To overcome the interferences of tetracyclines with the host microbiome, we identify tetracycline derivatives that have minimal antimicrobial activity, yet retain full capacity to induce the MSR, such as the lead compound, 9-tert-butyl doxycycline (9-TB). The MSR induced by doxycycline (Dox) and 9-TB improves survival and disease tolerance against lethal influenza virus (IFV) infection when given preventively. 9-TB, unlike Dox, did not affect the gut microbiome and also showed encouraging results against IFV when given in a therapeutic setting. Tolerance to IFV infection is associated with the induction of genes involved in lung epithelial cell and cilia function, and with downregulation of inflammatory and immune gene sets in lungs, liver, and kidneys. Mitohormesis induced by non-antimicrobial tetracyclines and the ensuing IFN response may dampen excessive inflammation and tissue damage during viral infections, opening innovative therapeutic avenues.

Methods to study folding of alpha-helical membrane proteins in lipids.

How alpha-helical membrane proteins fold correctly in the highly hydrophobic membrane interior is not well understood. Their folding is known to be highly influenced by the lipids within the surrounding bilayer, but the majority of folding studies have focused on detergent-solubilized protein rather than protein in a lipid environment. There are different ways to study folding in lipid bilayers, and each method has its own advantages and disadvantages. This review will discuss folding methods which can be used to study alpha-helical membrane proteins in bicelles, liposomes, nanodiscs or native membranes. These folding methods include in vitro folding methods in liposomes such as denaturant unfolding studies, and single-molecule force spectroscopy studies in bicelles, liposomes and native membranes. This review will also discuss recent advances in co-translational folding studies, which use cell-free expression with liposomes or nanodiscs or are performed in vivo with native membranes.

Acetate, a metabolic product of Heligmosomoides polygyrus, facilitates intestinal epithelial barrier breakdown in a FFAR2-dependent manner.

Approximately 2 billion people worldwide and a significant part of the domestic livestock are infected with soil-transmitted helminths, of which many establish chronic infections causing substantial economic and welfare burdens. Beside intensive research on helminth-triggered mucosal and systemic immune responses, the local mechanism that enables infective larvae to cross the intestinal epithelial barrier and invade mucosal tissue remains poorly addressed. Here, we show that Heligmosomoides polygyrus infective L3s secrete acetate and that acetate potentially facilitates paracellular epithelial tissue invasion by changed epithelial tight junction claudin expression. In vitro, impedance-based real-time epithelial cell line barrier measurements together with ex vivo functional permeability assays in intestinal organoid cultures revealed that acetate decreased intercellular barrier function via the G-protein coupled free fatty acid receptor 2 (FFAR2, GPR43). In vivo validation experiments in FFAR2-/- mice showed lower H. polygyrus burdens, whereas oral acetate-treated C57BL/6 wild type mice showed higher burdens. These data suggest that locally secreted acetate - as a metabolic product of the energy metabolism of H. polygyrus L3s - provides a significant advantage to the parasite in crossing the intestinal epithelial barrier and invading mucosal tissues. This is the first and a rate-limiting step for helminths to establish chronic infections in their hosts and if modulated could have profound consequences for their life cycle.

Novel High-Throughput Fluorescence-Based Assay for the Identification of Nematocidal Compounds That Target the Blood-Feeding Pathway.

Hookworm infections cause a neglected tropical disease (NTD) affecting ~740 million people worldwide, principally those living in disadvantaged communities. Infections can cause high morbidity due to their impact on nutrient uptake and their need to feed on host blood, resulting in a loss of iron and protein, which can lead to severe anaemia and impaired cognitive development in children. Currently, only one drug, albendazole is efficient to treat hookworm infection and the scientific community fears the rise of resistant strains. As part of on-going efforts to control hookworm infections and its associated morbidities, new drugs are urgently needed. We focused on targeting the blood-feeding pathway, which is essential to the parasite survival and reproduction, using the laboratory hookworm model Nippostrongylus brasiliensis (a nematode of rodents with a similar life cycle to hookworms). We established an in vitro-drug screening assay based on a fluorescent-based measurement of parasite viability during blood-feeding to identify novel therapeutic targets. A first screen of a library of 2654 natural compounds identified four that caused decreased worm viability in a blood-feeding-dependent manner. This new screening assay has significant potential to accelerate the discovery of new drugs against hookworms.

Small intestinal resident eosinophils maintain gut homeostasis following microbial colonization.

The intestine harbors a large population of resident eosinophils, yet the function of intestinal eosinophils has not been explored. Flow cytometry and whole-mount imaging identified eosinophils residing in the lamina propria along the length of the intestine prior to postnatal microbial colonization. Microscopy, transcriptomic analysis, and mass spectrometry of intestinal tissue revealed villus blunting, altered extracellular matrix, decreased epithelial cell turnover, increased gastrointestinal motility, and decreased lipid absorption in eosinophil-deficient mice. Mechanistically, intestinal epithelial cells released IL-33 in a microbiota-dependent manner, which led to eosinophil activation. The colonization of germ-free mice demonstrated that eosinophil activation in response to microbes regulated villous size alterations, macrophage maturation, epithelial barrier integrity, and intestinal transit. Collectively, our findings demonstrate a critical role for eosinophils in facilitating the mutualistic interactions between the host and microbiota and provide a rationale for the functional significance of their early life recruitment in the small intestine.

Microbial regulation of intestinal motility provides resistance against helminth infection.

Soil-transmitted helminths cause widespread disease, infecting ~1.5 billion people living within poverty-stricken regions of tropical and subtropical countries. As adult worms inhabit the intestine alongside bacterial communities, we determined whether the bacterial microbiota impacted on host resistance against intestinal helminth infection. We infected germ-free, antibiotic-treated and specific pathogen-free mice, with the intestinal helminth Heligmosomoides polygyrus bakeri. Mice harboured increased parasite numbers in the absence of a bacterial microbiota, despite mounting a robust helminth-induced type 2 immune response. Alterations to parasite behaviour could already be observed at early time points following infection, including more proximal distribution of infective larvae along the intestinal tract and increased migration in a Baermann assay. Mice lacking a complex bacterial microbiota exhibited reduced levels of intestinal acetylcholine, a major excitatory intestinal neurotransmitter that promotes intestinal transit by activating muscarinic receptors. Both intestinal motility and host resistance against larval infection were restored by treatment with the muscarinic agonist bethanechol. These data provide evidence that a complex bacterial microbiota provides the host with resistance against intestinal helminths via its ability to regulate intestinal motility.