Temporal dynamics and genomic programming of plasma cell fates.

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.

Rifaximin-α reduces gut-derived inflammation and mucin degradation in cirrhosis and encephalopathy: RIFSYS randomised controlled trial.

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.

Pseudocell Tracer-A method for inferring dynamic trajectories using scRNAseq and its application to B cells undergoing immunoglobulin class switch recombination.

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.

Ammonia-Induced Brain Edema Requires Macrophage and T Cell Expression of Toll-Like Receptor 9.

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 (NH4-Ac; 4mmol/kg) was injected intraperitoneally in wild type (WT), Tlr9-/- and Lysm-Cre Tlr9fl/fl mice (TLR9 absent in neutrophils and macrophages including Kupffer cells) and compared to controls. Six hours after NH4-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 NH4-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 NH4-Ac injection, macrophage cytokine production and BW were ameliorated in Lysm-Cre Tlr9fl/fl mice compared to WT mice (P < .05) but there was no difference compared to Tlr9-/- mice. Following NH4-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.