Sex differences in kidney metabolism may reflect sex-dependent outcomes in human diabetic kidney disease.

Diabetic kidney disease (DKD) is the main cause of chronic kidney disease (CKD) and progresses faster in males than in females. We identify sex-based differences in kidney metabolism and in the blood metabolome of male and female individuals with diabetes. Primary human proximal tubular epithelial cells (PTECs) from healthy males displayed increased mitochondrial respiration, oxidative stress, apoptosis, and greater injury when exposed to high glucose compared with PTECs from healthy females. Male human PTECs showed increased glucose and glutamine fluxes to the TCA cycle, whereas female human PTECs showed increased pyruvate content. The male human PTEC phenotype was enhanced by dihydrotestosterone and mediated by the transcription factor HNF4A and histone demethylase KDM6A. In mice where sex chromosomes either matched or did not match gonadal sex, male gonadal sex contributed to the kidney metabolism differences between males and females. A blood metabolomics analysis in a cohort of adolescents with or without diabetes showed increased TCA cycle metabolites in males. In a second cohort of adults with diabetes, females without DKD had higher serum pyruvate concentrations than did males with or without DKD. Serum pyruvate concentrations positively correlated with the estimated glomerular filtration rate, a measure of kidney function, and negatively correlated with all-cause mortality in this cohort. In a third cohort of adults with CKD, male sex and diabetes were associated with increased plasma TCA cycle metabolites, which correlated with all-cause mortality. These findings suggest that differences in male and female kidney metabolism may contribute to sex-dependent outcomes in DKD.

Liver-restricted Type I IFN Signature Precedes Liver Damage in Chronic Hepatitis B Patients Stopping Antiviral Therapy.

Immune-mediated liver damage is the driver of disease progression in patients with chronic hepatitis B virus (HBV) infection. Liver damage is an Ag-independent process caused by bystander activation of CD8 T cells and NK cells. How bystander lymphocyte activation is initiated in chronic hepatitis B patients remains unclear. Periods of liver damage, called hepatic flares, occur unpredictably, making early events difficult to capture. To address this obstacle, we longitudinally sampled the liver of chronic hepatitis B patients stopping antiviral therapy and analyzed immune composition and activation using flow cytometry and single-cell RNA sequencing. At 4 wk after stopping therapy, HBV replication rebounded but no liver damage was detectable. There were no changes in cell frequencies at viral rebound. Single-cell RNA sequencing revealed upregulation of IFN-stimulated genes (ISGs) and proinflammatory cytokine migration inhibitory factor (MIF) at viral rebound in patients that go on to develop hepatic flares 6-18 wk after stopping therapy. The type I IFN signature was only detectable within the liver, and neither IFN-α/ß or ISG induction could be detected in the peripheral blood. In vitro experiments confirmed the type I IFN-dependent ISG profile whereas MIF was induced primarily by IL-12. MIF exposure further amplified inflammatory cytokine production by myeloid cells. Our data show that innate immune activation is detectable in the liver before clinically significant liver damage is evident. The combination of type I IFN and enhanced cytokine production upon MIF exposure represent the earliest immunological triggers of lymphocyte bystander activation observed in hepatic flares associated with chronic HBV infection.

IL-2 produced by HBV-specific T cells as a biomarker of viral control and predictor of response to PD-1 therapy across clinical phases of chronic hepatitis B.

BACKGROUND: There are no immunological biomarkers that predict control of chronic hepatitis B (CHB). The lack of immune biomarkers raises concerns for therapies targeting PD-1/PD-L1 because they have the potential for immune-related adverse events. Defining specific immune functions associated with control of HBV replication could identify patients likely to respond to anti-PD-1/PD-L1 therapies and achieve a durable functional cure. METHODS: We enrolled immunotolerant, HBeAg+ immune-active (IA+), HBeAg- immune-active (IA-), inactive carriers, and functionally cured patients to test ex vivo PD-1 blockade on HBV-specific T cell functionality. Peripheral blood mononuclear cells were stimulated with overlapping peptides covering HBV proteins +/-α-PD-1 blockade. Functional T cells were measured using a 2-color FluoroSpot assay for interferon-γ and IL-2. Ex vivo functional restoration was compared to the interferon response capacity assay, which predicts overall survival in cancer patients receiving checkpoint inhibitors. RESULTS: Ex vivo interferon-γ+ responses did not differ across clinical phases. IL-2+ responses were significantly higher in patients with better viral control and preferentially restored with PD-1 blockade. Inactive carrier patients displayed the greatest increase in IL-2 production, which was dominated by CD4 T cell and response to the HBcAg. The interferon response capacity assay significantly correlated with the degree of HBV-specific T cell restoration. CONCLUSIONS: IL-2 production was associated with better HBV control and superior to interferon-γ as a marker of T cell restoration following ex vivo PD-1 blockade. Our study suggests that responsiveness to ex vivo PD-1 blockade, or the interferon response capacity assay, may support stratification for α-PD-1 therapies.

Single-cell RNA sequencing of liver fine-needle aspirates captures immune diversity in the blood and liver in chronic hepatitis B patients.

BACKGROUND AND AIMS: HBV infection is restricted to the liver, where it drives exhaustion of virus-specific T and B cells and pathogenesis through dysregulation of intrahepatic immunity. Our understanding of liver-specific events related to viral control and liver damage has relied almost solely on animal models, and we lack useable peripheral biomarkers to quantify intrahepatic immune activation beyond cytokine measurement. Our objective was to overcome the practical obstacles of liver sampling using fine-needle aspiration and develop an optimized workflow to comprehensively compare the blood and liver compartments within patients with chronic hepatitis B using single-cell RNA sequencing. APPROACH AND RESULTS: We developed a workflow that enabled multi-site international studies and centralized single-cell RNA sequencing. Blood and liver fine-needle aspirations were collected, and cellular and molecular captures were compared between the Seq-Well S 3 picowell-based and the 10× Chromium reverse-emulsion droplet-based single-cell RNA sequencing technologies. Both technologies captured the cellular diversity of the liver, but Seq-Well S 3 effectively captured neutrophils, which were absent in the 10× dataset. CD8 T cells and neutrophils displayed distinct transcriptional profiles between blood and liver. In addition, liver fine-needle aspirations captured a heterogeneous liver macrophage population. Comparison between untreated patients with chronic hepatitis B and patients treated with nucleoside analogs showed that myeloid cells were highly sensitive to environmental changes while lymphocytes displayed minimal differences. CONCLUSIONS: The ability to electively sample and intensively profile the immune landscape of the liver, and generate high-resolution data, will enable multi-site clinical studies to identify biomarkers for intrahepatic immune activity in HBV and beyond.

Longitudinal liver sampling in patients with chronic hepatitis B starting antiviral therapy reveals hepatotoxic CD8+ T cells.

Accumulation of activated immune cells results in nonspecific hepatocyte killing in chronic hepatitis B (CHB), leading to fibrosis and cirrhosis. This study aims to understand the underlying mechanisms in humans and to define whether these are driven by widespread activation or a subpopulation of immune cells. We enrolled CHB patients with active liver damage to receive antiviral therapy and performed longitudinal liver sampling using fine-needle aspiration to investigate mechanisms of CHB pathogenesis in the human liver. Single-cell sequencing of total liver cells revealed a distinct liver-resident, polyclonal CD8+ T cell population that was enriched at baseline and displayed a highly activated immune signature during liver damage. Cytokine combinations, identified by in silico prediction of ligand-receptor interaction, induced the activated phenotype in healthy liver CD8+ T cells, resulting in nonspecific Fas ligand-mediated killing of target cells. These results define a CD8+ T cell population in the human liver that can drive pathogenesis and a key pathway involved in their function in CHB patients.

The Human Male Liver Is Predisposed to Inflammation Via Enhanced Myeloid Responses to Inflammatory Triggers.

Background & Aim: Men have a higher prevalence of liver disease. Liver myeloid cells can regulate tissue inflammation, which drives progression of liver disease. We hypothesized that sex alters the responsiveness of liver myeloid cells, predisposing men to severe liver inflammation. Methods: Luminex was done on plasma from Hepatitis B Virus infected patients undergoing nucleoside analogue cessation in 45 male and female patients. We collected immune cells from the sinusoids of uninfected livers of 53 male and female donors. Multiparametric flow cytometry was used to phenotype and characterize immune composition. Isolated monocytes were stimulated with TLR ligands to measure the inflammatory potential and the expression of regulators of TLR signaling. Results: We confirmed that men experienced more frequent and severe liver damage upon Hepatitis B Virus reactivation, which was associated with inflammatory markers of myeloid activation. No differences were observed in the frequency or phenotype of sinusoidal myeloid cells between male and female livers. However, monocytes from male livers produced more inflammatory cytokines and chemokines in response to TLR stimulation than female monocytes. We investigated negative regulators of TLR signaling and found that TOLLIP was elevated in female liver-derived monocytes. Conclusions: Our data show that enhanced responsiveness of myeloid cells from the male liver predisposes men to inflammation, which was associated with altered expression of negative regulators of TLR signaling.

Immunomodulation and RNA interference alter hepatitis B virus-specific CD8 T-cell recognition of infected HepG2-NTCP.

BACKGROUND AND AIMS: CD8 T cells are essential in controlling HBV infection. Viral control is dependent on efficient recognition of HBV-infected hepatocytes by CD8 T cells, which can induce direct lysis of infected hepatocytes. In addition, CD8 T cells produce interferon (IFN)-γ, which mediates noncytopathic viral clearance. Innate immunomodulators and HBV-targeted RNA interference (RNAi) are being developed to treat chronic hepatitis B (CHB), but may modify HBV antigen presentation and impact CD8 T-cell recognition, in addition to their primary mechanisms of action. APPROACH AND RESULTS: HBV-infected HepG2-NTCP cells were treated with tenofovir disoproxil fumarate (TDF), Toll-like receptor (TLR) 7/8 agonists, TLR7/8 conditioned media (CM) collected from immune cells, or RNAi using short interfering RNAs. The effect of these treatments on antigen presentation was measured through coculture with CD8 T cells recognizing human leukocyte antigen-A0201 restricted epitopes, HBc18-27 or HBs183-191. Cytokine profiles of TLR7/8 CM were measured using a cytometric bead array. TDF reduced viral replication, but not CD8 T-cell recognition, of infected cells. Direct exposure of infected HepG2-NTCP to TLR7/8 agonists had no impact on T-cell recognition. Exposure of infected HepG2-NTCP to TLR7/8 CM enhanced HBV-specific CD8 T-cell recognition through type 1 interferon (IFN) and IFN-γ-dependent mechanisms. RNAi rapidly suppressed HBV-DNA, HBcAg, and HBsAg expression, impairing recognition by HBV-specific CD8 T cells. CONCLUSIONS: Immunomodulation and RNAi, but not nucleos(t)ide analogues, alter the recognition of infected HepG2-NTCP by HBV-specific CD8 T cells. Understanding these changes will inform combination treatments for CHB.

Optimized ex vivo stimulation identifies multi-functional HBV-specific T cells in a majority of chronic hepatitis B patients.

High antigen burden during chronic hepatitis B (CHB) results in a low frequency HBV-specific T cell response with restricted functionality. However, this observation is based on limited data because low T cell frequencies have hindered effective ex vivo analysis. We adapted the ELISpot assay to overcome this obstacle to measure ex vivo T cell responses in CHB patients. We modified the key variables of cell number and the peptide pulsing method to improve ex vivo detection of HBV-specific T cells. We detected IFN-γ responses in 10/15 vaccinated controls and 20/30 CHB patients, averaging 195 and 84 SFUs/2 × 106 PBMCs respectively. Multi-analyte FluoroSpots improved functional characterization of T cells. We detected IFN-γ responses in all tested vaccinated controls (n = 10) and CHB patients (n = 13). IL-2 responses were detectable in 9/10 controls and 10/13 patients. TNF-α displayed less sensitivity, detectable in only 7/10 controls and 7/13 patients. Antigen-specific analysis demonstrated that IFN-γ responses were dominated by polymerase and core, with weak responses to envelope and X. IL-2 responses were found in 3/5 patients and equally directed towards polymerase and core. While their ex vivo frequency is extremely low, a fraction of HBV-specific T cells are detectable and display multi-functionality ex vivo.

IFN-α Suppresses Myeloid Cytokine Production, Impairing IL-12 Production and the Ability to Support T-Cell Proliferation.

BACKGROUND: Interferon-α (IFN-α) can suppress production of T-cell polarizing cytokines or induce inhibitory antigen-presenting cells that suppress T-cell activation. Previous studies showed that IFN-α therapy fails to boost virus-specific T-cell immunity in patients with chronic hepatitis B virus infection. Our aim was to determine whether IFN-α exposure alters human antigen-presenting cell function in vivo. METHODS: We investigated the immunomodulatory effects using peripheral blood mononuclear cells from healthy donors exposed to IFN-α and chronic hepatitis B (CHB) patients starting IFN-α therapy. RESULTS: IFN-α increased HLA-DR, CD80, CD86, and PD-L1 expression on healthy donor monocytes. In contrast to the activated phenotype, IFN-α inhibited Toll-like receptor-induced cytokine production and monocyte-induced T-cell proliferation. In CHB patients, peg-IFN treatment induced an interferon-stimulated gene signature in monocytes and increased HLA-DR, CD80, CD86, and PD-L1 expression. As early as 3 days after CHB patients started treatment, IFN-α inhibited monocyte cytokine production and T-cell stimulation ex vivo. IFN-α-mediated inhibition of IL-12 production, rather than inhibitory receptor expression, was responsible for inhibition of T-cell proliferation. Addition of IL-12 restored T-cell proliferation to baseline levels. CONCLUSIONS: Understanding how professional antigen-presenting cells respond to immunomodulation is important for both new innate and adaptive-targeted immunotherapies. CLINICAL TRIALS REGISTRATION: NCT00962871.