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Antibody-mediated protection against pathogens is crucial to a healthy life. However, the recent SARS-CoV-2 pandemic has shown that pre-existing comorbid conditions including kidney disease account for compromised humoral immunity to infections. Individuals with kidney disease are not only susceptible to infections but also exhibit poor vaccine-induced antibody response. Using multiple mouse models of kidney disease, we demonstrate that renal dysfunction inhibits germinal center (GC) response against T-dependent antigens. GC B cells exhibit increased apoptosis in kidney disease. Uremic toxin hippuric acid drives loss of mitochondrial membrane potential, leading to increased apoptosis of GC B cells in a G-protein-coupled receptor 109A dependent manner. Finally, GC B cells and antibody titer are diminished in mice with kidney disease following influenza virus infection, a major cause of mortality in individuals with renal disorders. These results provide a mechanistic understanding of how renal dysfunction suppresses humoral immunity in patients with kidney disease., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Affinity-matured plasma cells (PCs) of varying lifespans are generated through a germinal center (GC) response. The developmental dynamics and genomic programs of antigen-specific PC precursors remain to be elucidated. Here, using a model antigen in mice, we demonstrate biphasic generation of PC precursors, with those generating long-lived bone marrow PCs preferentially produced in the late phase of GC response. Clonal tracing using single-cell RNA sequencing and B cell antigen receptor sequencing in spleen and bone marrow compartments, coupled with adoptive transfer experiments, reveals a new PC transition state that gives rise to functionally competent PC precursors. The latter undergo clonal expansion, dependent on inducible expression of TIGIT. We propose a model for the proliferation and programming of precursors of long-lived PCs, based on extended antigen encounters in the GC., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background & Aims: Rifaximin-α is efficacious for the prevention of recurrent hepatic encephalopathy (HE), but its mechanism of action remains unclear. We postulated that rifaximin-α reduces gut microbiota-derived endotoxemia and systemic inflammation, a known driver of HE., Methods: In a placebo-controlled, double-blind, mechanistic study, 38 patients with cirrhosis and HE were randomised 1:1 to receive either rifaximin-α (550 mg BID) or placebo for 90 days., Primary Outcome: 50% reduction in neutrophil oxidative burst (OB) at 30 days., Secondary Outcomes: changes in psychometric hepatic encephalopathy score (PHES) and neurocognitive functioning, shotgun metagenomic sequencing of saliva and faeces, plasma and faecal metabolic profiling, whole blood bacterial DNA quantification, neutrophil toll-like receptor (TLR)-2/4/9 expression and plasma/faecal cytokine analysis., Results: Patients were well-matched: median MELD (11 rifaximin-α vs. 10 placebo). Rifaximin-α did not lead to a 50% reduction in spontaneous neutrophil OB at 30 days compared to baseline (p = 0.48). However, HE grade normalised (p = 0.014) and PHES improved (p = 0.009) after 30 days on rifaximin-α. Rifaximin-α reduced circulating neutrophil TLR-4 expression on day 30 (p = 0.021) and plasma tumour necrosis factor-α (TNF-α) (p <0.001). Rifaximin-α suppressed oralisation of the gut, reducing levels of mucin-degrading sialidase-rich species, Streptococcus spp, Veillonella atypica and parvula, Akkermansia and Hungatella. Rifaximin-α promoted a TNF-α- and interleukin-17E-enriched intestinal microenvironment, augmenting antibacterial responses to invading pathobionts and promoting gut barrier repair. Those on rifaximin-α were less likely to develop infection (odds ratio 0.21; 95% CI 0.05-0.96)., Conclusion: Rifaximin-α led to resolution of overt and covert HE, reduced the likelihood of infection, reduced oralisation of the gut and attenuated systemic inflammation. Rifaximin-α plays a role in gut barrier repair, which could be the mechanism by which it ameliorates bacterial translocation and systemic endotoxemia in cirrhosis., Clinical Trial Number: ClinicalTrials.gov NCT02019784., Lay Summary: In this clinical trial, we examined the underlying mechanism of action of an antibiotic called rifaximin-α which has been shown to be an effective treatment for a complication of chronic liver disease which effects the brain (termed encephalopathy). We show that rifaximin-α suppresses gut bacteria that translocate from the mouth to the intestine and cause the intestinal wall to become leaky by breaking down the protective mucus barrier. This suppression resolves encephalopathy and reduces inflammation in the blood, preventing the development of infection., Competing Interests: Conflict of interest Dr V Patel and Ms A Zamalloa have delivered paid lectures for Norgine Pharmaceuticals Ltd. Professor Shawcross has participated in advisory boards for Norgine Pharmaceuticals Ltd, EnteroBiotix, Kaleido Biosciences, Mallinckrodt and Shionogi and has delivered paid lectures for Norgine Pharmaceuticals Ltd, Falk Pharma and Alfa Sigma. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

This review is focused on the cellular dynamics and genomic programming of plasma cell (PC) precursors that arise during germinal center (GC) B cell responses in secondary lymphoid organs (SLOs) and give rise to PCs in the bone marrow. Considerable progress has been made in the phenotypic characterization of circulating and bone marrow PC precursors as well as their differentiated short-lived (SLPC) and long-lived (LLPC) counterparts, in the context of model antigen and vaccine responses. Importantly, it has been possible to infer the temporal dynamics of generation of PC precursors during a GC response. However, the nature of the PC precursors at their site of generation in SLOs, and their signaling and genomic states, remain to be elucidated. Our synthesis draws upon experimental studies conducted in murine models as well as in humans, the latter complemented with cell culture manipulations of PCs and their precursors. By integration of the studies in murine and human systems, which are being accelerated by new genomic methodologies, we highlight insights and hypotheses concerning the generation of PCs. This framework can be extended and explored from both fundamental and translational standpoints., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Antigen-activated B cells diversify variable regions of B cell antigen receptors by somatic hypermutation in germinal centers (GCs). The positive selection of GC B cells that acquire high-affinity mutations enables antibody affinity maturation. In spite of considerable progress, the genomic states underlying this process remain to be elucidated. Single-cell RNA sequencing and topic modeling revealed increased expression of the oxidative phosphorylation (OXPHOS) module in GC B cells undergoing mitoses. Coupled analysis of somatic hypermutation in immunoglobulin heavy chain (Igh) variable gene regions showed that GC B cells acquiring higher-affinity mutations had further elevated expression of OXPHOS genes. Deletion of mitochondrial Cox10 in GC B cells resulted in reduced cell division and impaired positive selection. Correspondingly, augmentation of OXPHOS activity with oltipraz promoted affinity maturation. We propose that elevated OXPHOS activity promotes B cell clonal expansion and also positive selection by tuning cell division times.

Single cell RNA sequencing (scRNAseq) can be used to infer a temporal ordering of cellular states. Current methods for the inference of cellular trajectories rely on unbiased dimensionality reduction techniques. However, such biologically agnostic ordering can prove difficult for modeling complex developmental or differentiation processes. The cellular heterogeneity of dynamic biological compartments can result in sparse sampling of key intermediate cell states. To overcome these limitations, we develop a supervised machine learning framework, called Pseudocell Tracer, which infers trajectories in pseudospace rather than in pseudotime. The method uses a supervised encoder, trained with adjacent biological information, to project scRNAseq data into a low-dimensional manifold that maps the transcriptional states a cell can occupy. Then a generative adversarial network (GAN) is used to simulate pesudocells at regular intervals along a virtual cell-state axis. We demonstrate the utility of Pseudocell Tracer by modeling B cells undergoing immunoglobulin class switch recombination (CSR) during a prototypic antigen-induced antibody response. Our results revealed an ordering of key transcription factors regulating CSR to the IgG1 isotype, including the concomitant expression of Nfkb1 and Stat6 prior to the upregulation of Bach2 expression. Furthermore, the expression dynamics of genes encoding cytokine receptors suggest a poised IL-4 signaling state that preceeds CSR to the IgG1 isotype., Competing Interests: The authors have declared that no competing interests exist.

Background & Aim: Ammonia is central in the pathogenesis of brain edema in acute liver failure (ALF) with infection and systemic inflammation expediting development of intracranial hypertension (ICH). Patients with acetaminophen-induced ALF have increased neutrophil TLR9 expression which can be induced by ammonia. We determined whether ammonia-induced brain edema and immune dysfunction are mediated by TLR9 and if this could be prevented in a TLR9-deficient mouse model., Methods: Ammonium acetate (NH 4 -Ac; 4mmol/kg) was injected intraperitoneally in wild type (WT), Tlr9 -/- and Lysm-Cre Tlr9 fl/fl mice (TLR9 absent in neutrophils and macrophages including Kupffer cells) and compared to controls. Six hours after NH 4 -Ac injection, intracellular cytokine production was determined in splenic macrophages, CD4 + and CD8 + T cells. Brain water (BW) and total plasma DNA (tDNA) were also measured. The impact of the TLR9 antagonist ODN2088 (50μg/mouse) was evaluated., Results: Following NH 4 -Ac injection, BW, macrophage and T cell cytokine production increased (P < .0001) in WT but not Tlr9 -/- mice (P < .001). ODN2088 inhibited macrophage and T cell cytokine production (P < .05) and prevented an increase in BW (P < .0001). Following NH 4 -Ac injection, macrophage cytokine production and BW were ameliorated in Lysm-Cre Tlr9 fl/fl mice compared to WT mice (P < .05) but there was no difference compared to Tlr9 -/- mice. Following NH 4 -Ac injection, plasma tDNA levels increased in WT and Tlr9 -/- mice (P < .05) suggesting that TLR9 may be activated by DNA released from ammonia-stimulated cells., Conclusion: Ammonia-induced brain edema requires macrophage and T cell expression of TLR9. Amelioration of brain edema and lymphocyte cytokine production by ODN2088 supports exploration of TLR9 antagonism in early ALF to prevent progression to ICH., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Thrombocytopenia and circulating dysfunctional immune cells are commonly observed in patients with cirrhosis. Platelets may form complexes with neutrophils, monocytes and T cells modulating their function. We recently reported increased frequencies of platelet-complexed neutrophils in cirrhosis with evidence of neutrophil activation upon contact with healthy platelets in vitro. Whether this occurs in vivo following platelet transfusion and contributes to systemic inflammation and endothelial activation is unknown., Aims: To characterise platelet-leucocyte aggregation in cirrhosis and to determine whether elective platelet transfusion results in perturbations associated with changes in markers of haemostasis, inflammation or endothelial activation., Methods: We collected blood from cirrhotics (n = 19) before and following elective platelet transfusion. We measured platelet-leucocyte aggregation, activation and function, and markers of platelet activation, systemic inflammation and endothelial activation by flow cytometry. Haemostasis was assessed by thromboelastometry and plasma haemostatic proteins., Results: We observed a 2.5-fold increase in platelet-complexed neutrophils in patients with cirrhosis compared with healthy subjects and twofold more platelets attached per monocyte and T cell. All platelet-complexed leucocytes expressed higher levels of activation markers and platelet-complexed neutrophils had higher resting oxidative burst and phagocytic capacity than their nonplatelet-complexed counterparts (P < 0.001); most pronounced in patients with cirrhosis. Paradoxically, platelet-complexed leucocyte frequency decreased with increasing MELD score. Platelet transfusion increased soluble CD40 ligand (platelet activation marker), the frequency of platelet-complexed monocytes (P < 0.05) and improved haemostatic status., Conclusion: Cirrhotic patients have activated circulating platelet-complexed leucocytes with increased platelet-monocyte aggregation following elective platelet transfusion. Elective platelet transfusion might therefore exacerbate immune dysfunction in cirrhosis., (© 2018 John Wiley & Sons Ltd.)

Objectives: There is a marked propensity for patients with acetaminophen-induced acute liver failure to develop sepsis, which may culminate in multiple organ failure and death. Toll-like receptors sense pathogens and induce inflammatory responses, but whether this is protective or detrimental in acetaminophen-induced acute liver failure remains unknown., Design, Setting, and Patients: We assessed Toll-like receptor expression on circulating neutrophils and their function in 24 patients with acetaminophen-induced acute liver failure and compared with 10 healthy controls., Interventions: Neutrophil Toll-like receptor 2, -4, and -9 expression and cytokine production and function were studied ex vivo at baseline and following stimulation with lipopolysaccharide, oligodeoxynucleotides, ammonium chloride, and interleukin-8. To examine the influence of acetaminophen-induced acute liver failure plasma and endogenous DNA on Toll-like receptors-9 expression, healthy neutrophils were incubated with acetaminophen-induced acute liver failure plasma with and without deoxyribonuclease-I., Measurements and Main Results: Circulating neutrophil Toll-like receptor 9 expression was increased in acetaminophen-induced acute liver failure on day 1 compared with healthy controls (p = 0.0002), whereas Toll-like receptor 4 expression was decreased compared with healthy controls (p < 0.0001). Toll-like receptor 2 expression was unchanged. Neutrophil phagocytic activity was decreased, and spontaneous oxidative burst increased in all patients with acetaminophen-induced acute liver failure compared with healthy controls (p < 0.0001). Neutrophil Toll-like receptor 9 expression correlated with plasma interleukin-8 and peak ammonia concentration (r = 0.6; p < 0.05) and increased with severity of hepatic encephalopathy (grade 0-2 vs 3/4) and systemic inflammatory response syndrome score (0-1 vs 2-4) (p < 0.05). Those patients with advanced hepatic encephalopathy (grade 3/4) or high systemic inflammatory response syndrome score (2-4) on day 1 had higher neutrophil Toll-like receptor 9 expression, arterial ammonia concentration, and plasma interleukin-8 associated with neutrophil exhaustion. Healthy neutrophil Toll-like receptor 9 expression increased upon stimulation with acetaminophen-induced acute liver failure plasma, which was abrogated by preincubation with deoxyribonuclease-I. Intracellular Toll-like receptor 9 was induced by costimulation with interleukin-8 and ammonia., Conclusion: These data point to neutrophil Toll-like receptor 9 expression in acetaminophen-induced acute liver failure being mediated both by circulating endogenous DNA as well as ammonia and interleukin-8 in a synergistic manner inducing systemic inflammation, neutrophil exhaustion, and exacerbating hepatic encephalopathy.

Background & Aims: Susceptibility to bacterial infection is a feature of alcohol-related liver disease. Programmed cell death 1 (PD1), the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3, also known as hepatitis A virus cellular receptor 2), and their respective ligands-CD274 (also known as PD ligand 1 [PDL1]) and galectin-9-are inhibitory receptors that regulate the balance between protective immunity and host immune-mediated damage. However, their sustained hyperexpression promotes immune exhaustion and paralysis. We investigated the role of these immune inhibitory receptors in driving immune impairments in patients with alcoholic liver disease., Methods: In a prospective study, we collected blood samples from 20 patients with acute alcoholic hepatitis (AAH), 16 patients with stable advanced alcohol-related cirrhosis, and 12 healthy individuals (controls). Whole blood or peripheral blood mononuclear cells were assessed for expression of PD1, PDL1, TIM3, galectin-9, and Toll-like receptors on subsets of innate and adaptive immune effector cells. We measured antibacterial immune responses to lipopolysaccharide (endotoxin) using ELISpot assays, and used flow cytometry to quantify cytokine production, phagocytosis, and oxidative burst in the presence or absence of blocking antibodies against PD1 or TIM3., Results: Antibacterial innate and adaptive immune responses were greatly reduced in patients with AAH, compared with controls, and patients with alcohol-related cirrhosis had less severe dysfunctions in innate immune effector cells and preserved functional T-cell responses. Fewer T cells from patients with AAH produced interferon gamma in response to lipopolysaccharide, compared with controls. In addition, patients with AAH had greater numbers of interleukin 10-producing T cells, and reduced levels of neutrophil phagocytosis and oxidative burst in response to Escherichia coli stimulation, compared with controls. T cells from patients with AAH, but not alcohol-related cirrhosis, expressed higher levels of PD1 and PDL1, or TIM3 and galectin-9, than T cells from controls. Antibodies against PD1 and TIM3 restored T-cell production of interferon gamma, reduced the numbers of interleukin 10-producing T cells, and increased neutrophil antimicrobial activities. Circulating levels of endotoxin in plasma from patients with AAH caused over expression of immune inhibitory receptors on T cells via Toll-like receptor 4 binding to CD14(+) monocytes., Conclusions: Antibacterial immune responses are impaired in patients with AAH. Lymphocytes from these patients express high levels of immune inhibitory receptors, produce lower levels of interferon gamma, and have increased IL10 production due to chronic endotoxin exposure. These effects can be reversed by blocking PD1 and TIM3, which increase the antimicrobial activities of T cells and neutrophils., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Patients with cirrhosis are susceptible to sepsis, pre-disposing to the development of encephalopathy, bleeding and organ dysfunction with associated high mortality., Aim: To characterise circulating neutrophil function in a cirrhotic cohort as a determinant of 90-day and 1-year mortality., Methods: Sixty-two patients with cirrhosis [49 stable (Child-Pugh A/B/C = 24%/39%/37%); 13 acute-on-chronic liver failure] were prospectively studied and compared with 11 healthy controls. Neutrophil function was evaluated at baseline and repeated at critical points during the course of the patient's illness until death/transplantation. Neutrophil phenotype was determined using fluorochrome-labelled antibodies to CD16/CD11b and assessed by flow cytometry. Neutrophil phagocytic activity (NPA) and capacity (NPC) were determined using FITC-labelled opsonised Escherichia coli. Oxidative burst (OB) was quantified by the percentage of neutrophils producing reactive oxygen species (ROS) and mean fluorescence intensity at rest, and after stimulation with E. coli. Physiological variables, biochemistry, microbiology and outcomes were collected. Plasma pro- and anti-inflammatory cytokine profiles were performed by ELISA., Results: NPA/NPC was impaired in cirrhosis with the most significant dysfunction being observed in those with advanced disease and in those treated with propranolol. NPC predicted survival in stable cirrhosis [AUROC 0.83 (95% CI 0.68-0.97); P = 0.021] and differentiated survivors from nonsurvivors (90-day P = 0.01; 1 year P < 0.001). Resting OB ≥12% predicted 90-day mortality with 80% sensitivity and 71% specificity [AUROC 0.81 (95% CI 0.64-0.97); P = 0.026 and differentiated survivors from nonsurvivors; P = 0.015]., Conclusion: Circulating neutrophils in patients with cirrhosis are dysfunctional and predict the development of infection, organ dysfunction and survival at 90 days and 1 year., (© 2014 John Wiley & Sons Ltd.)

Unlabelled: Systemic inflammation and susceptibility to developing sepsis is common in acute liver failure (ALF) resulting in tissue damage and organ failure. This study characterized the function of circulating neutrophils in 25 patients with ALF and subacute liver failure (SALF). ALF (n=15)/SALF (n=10) patients were prospectively studied and compared with 11 healthy (HC) and 6 septic controls (SC). Neutrophils were isolated on admission to intensive care and every 3-4 days until death / liver transplantation / recovery. Neutrophil phenotype was determined using fluorochrome-labeled antibodies to CD16 and CD11b and assessed by flow cytometry. Neutrophil phagocytic activity (NPA) was determined using fluorescein isothiocyanate-labeled opsonized Escherichia coli and oxidative burst (OB) was determined by the percentage of neutrophils producing reactive oxygen species (ROS) at rest and after stimulation with opsonized E. coli. Physiological variables, biochemistry, arterial ammonia, microbiology, and outcomes were collected. Plasma pro- and antiinflammatory cytokine profiles were performed by enzyme-linked immunosorbent assay. Neutrophil expression of CD16 which recognizes the FcγRIII region of immunoglobulin G was significantly reduced in the ALF cohort (P<0.001) on day 1 compared to HC. NPA was significantly impaired in the SALF cohort compared to HC (P<0.01). Impaired NPA in the ALF and SALF cohorts on admission predicted nonsurvival without liver transplantation (P=0.01). Spontaneous neutrophil production of ROS was not significantly increased in any of the cohorts. E. coli-stimulated OB was preserved in ALF/SALF cohorts but was significantly impaired in the SC group (P<0.05)., Conclusion: Circulating neutrophils in ALF/SALF have impaired bacteriocidal function similar to that seen in severe sepsis. Neutrophil function indices are important biomarkers in ALF and may be implicated in the development of organ dysfunction and the increased susceptibility to developing sepsis., (Copyright © 2012 American Association for the Study of Liver Diseases.)