Clonal haematopoiesis and risk of chronic liver disease

Abstract

Chronic liver disease is a major public health burden worldwide¹. Although different aetiologies and mechanisms of liver injury exist, progression of chronic liver disease follows a common pathway of liver inflammation, injury and fibrosis². Here we examined the association between clonal haematopoiesis of indeterminate potential (CHIP) and chronic liver disease in 214,563 individuals from 4 independent cohorts with whole-exome sequencing data (Framingham Heart Study, Atherosclerosis Risk in Communities Study, UK Biobank and Mass General Brigham Biobank). CHIP was associated with an increased risk of prevalent and incident chronic liver disease (odds ratio = 2.01, 95% confidence interval (95% CI) [1.46, 2.79]; P < 0.001). Individuals with CHIP were more likely to demonstrate liver inflammation and fibrosis detectable by magnetic resonance imaging compared to those without CHIP (odds ratio = 1.74, 95% CI [1.16, 2.60]; P = 0.007). To assess potential causality, Mendelian randomization analyses showed that genetic predisposition to CHIP was associated with a greater risk of chronic liver disease (odds ratio = 2.37, 95% CI [1.57, 3.6]; P < 0.001). In a dietary model of non-alcoholic steatohepatitis, mice transplanted with Tet2-deficient haematopoietic cells demonstrated more severe liver inflammation and fibrosis. These effects were mediated by the NLRP3 inflammasome and increased levels of expression of downstream inflammatory cytokines in Tet2-deficient macrophages. In summary, clonal haematopoiesis is associated with an elevated risk of liver inflammation and chronic liver disease progression through an aberrant inflammatory response.

Extended Data Fig. 1. CHIP ascertainment.

a, Proportion of CHIP by mutated gene among 11,783 individuals with CHIP. b, Prevalence of CHIP by age.

Extended Data Fig. 2. Association of CHIP with chronic liver disease.

a, Association of CHIP with prevalent or incident chronic liver disease by variant allele fraction. b, Association of CHIP with chronic liver disease by mutated gene. c, Cumulative risk of chronic liver disease by clonal hematopoiesis status in the UK Biobank. d, Cumulative risk of chronic liver disease by clonal hematopoiesis status in the UK Biobank by age 80 years. Estimates derived using logistic regression with adjustment for age and sex in the UK Biobank, Framingham Heart Study and Atherosclerosis Risk in Communities study and pooled using inverse variance weighted fixed effects meta-analysis. Cumulative risk of chronic liver disease by age was modeled using Cox proportional hazards model with age as the underlying time variable and adjustment for sex. CHIP, clonal hematopoiesis of indeterminate potential; CI, 95% confidence interval; OR, odds ratio; VAF, variant allele fraction.

Extended Data Fig. 3. Mendelian randomization analysis of CHIP association with chronic liver disease.

a, Effect of genetic variants against exposure (CHIP) and outcome (cirrhosis). Effect estimates are oriented to CHIP-increasing alleles. b, Phenome-wide mendelian randomization analysis of CHIP with 22 phenotypes. MR analysis was performed using MR Base platform. Estimates were derived using inverse variance weighted meta-analysis using 90 independent variants associated with CHIP with p < 5 x 10⁻⁵.

Extended Data Fig. 4. Association of CHIP with serum biomarkers.

a, Association of CHIP with serum biomarkers in the UK Biobank. b, Association of CHIP with serum biomarkers in the UK Biobank excluding JAK2-mutant CHIP. c, Association of CHIP with serum biomarkers in the UK Biobank excluding TET2-mutant CHIP. ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; CHIP, clonal hematopoiesis of indeterminate potential; CRP, C-reactive protein; GGT, gamma-glutamyl transferase; TBili, total bilirubin; WBC, white blood cell count. A P value of 0.006 after Bonferroni adjustment (0.05/9 = 0.006) was considered significant.

Extended Data Fig. 5. Metabolic phenotype of Tet2^−/− bone marrow transplanted mice fed CDAHFD.

Lethally irradiated C57BL/6J mice were transplanted with Tet2^−/− (n = 30) or control vavCre⁺ (WT; n = 25) bone marrow cells. After hematopoietic reconstitution, mice were fed CDAHFD and body weight (a) and food intake (b) were measured over 30 days. Mice were sacrificed and terminal liver weight (c-d) and serum biomarkers (e) were measured. Control mice were transplanted with Tet2^−/− (n = 6) or control vavCre⁺ (WT; n = 6) bone marrow cells and fed standard chow for the same duration. Data from one (a-d) or two independent experiments (e) are shown. AST, aspartate transaminase; ALT, alanine transaminase; TBILI, total bilirubin; ALB, albumin; TRIG, triglycerides; GLUC, glucose; CHOL, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NEFA, non-essential fatty acids.

Extended Data Fig. 6. Liver and hematological parameters of Tet2^−/− bone marrow transplanted mice.

a, Ldlr^−/− mice were transplanted with Tet2^−/− (n = 25) or control vavCre⁺ (WT; n = 20) bone marrow cells and fed Western diet for 10 weeks. b, B6.SJL mice were transplanted with Tet2^−/− (n = 19) or control vavCre⁺ (WT; n = 12) bone marrow cells and fed CDAHFD for 11 weeks. c, C57BL/6J mice were transplanted with Tet2^−/− (n = 12) or control vavCre⁺ (WT; n = 13) bone marrow cells and fed CDAHFD for 19 weeks. After the prescribed dietary periods, mice were sacrificed and terminal liver weight and peripheral blood chimerism and hematological parameters were measured. Data from one (c) or two independent experiments (a, b) are shown. WBC, white blood cell count; Hgb, hemoglobin; Hct, hematocrit; RBC, red blood cell count; Plt, platelet count.

Extended Data Fig. 7. Steatohepatitis and liver fibrosis in Tet2^−/− and Dnmt3a^−/− transplanted mice.

a, Selected gene signatures enriched in bulk liver transcripts from Tet2^−/− (n = 4) transplanted mice fed CDAHFD relative to control vavCre⁺ (WT; n = 4) transplanted mice. b, Histologic features of steatohepatitis in Ldlr^−/− mice transplanted with Tet2^−/− (n = 27) and control vavCre⁺ (WT; n = 30) bone marrow and fed Western diet for 10 weeks were graded on a semiquantitative scale and aggregated into a NASH activity score (NAS) using CRN histologic scoring criteria. c-f, Graded histologic features included steatosis (c), inflammatory foci (d), hepatocyte ballooning (e, arrowhead) and apoptosis (f, arrow). g, Masson’s trichrome staining demonstrates absence of perivenular fibrosis in control and Tet2^−/− transplanted mice. h, B6.SJL mice were transplanted with Dnmt3a^−/− (n = 24) or control vavCre⁺ (WT; n = 20) bone marrow cells and fed CDAHFD for 11 weeks. Steatohepatitis was assessed histologically for steatosis, inflammation, and hepatocyte ballooning. Collagen fibrosis was measured by Masson’s trichrome staining. i, B6.SJL mice were transplanted with Tet2^−/− (n = 20) or control vavCre⁺ (WT; n = 15) bone marrow cells and fed CDAHFD for 11 weeks, then reverted to standard chow for 10 days. Compared to control animals, Tet2^−/− transplanted mice show similar resolution of liver fat but show persistently greater inflammation and more hepatocyte ballooning. Collagen fibrosis, as measured by Masson’s trichrome staining, was not significantly different. Data from one (a) or two independent experiments (b, h, i) are shown. NES, normalized enrichment score; FDR, false discovery rate.

Extended Data Fig. 8. Fibrogenic response in hepatic stellate cells co-cultured with Tet2^−/− hematopoietic cell populations.

Hepatic stellate cells were isolated from wild type livers (n = 5) and co-cultured with CD19⁺ B cells, CD3⁺ T cells, or CD11b⁺ hepatic macrophages isolated from Tet2^−/− (n = 5) or control vavCre⁺ (WT; n = 5) mice for 2 days. Co-cultured hepatic stellate cells were harvested for RNA sequencing and selected differentially expressed genes (relative to hepatic stellate cell mono-culture) are shown (a). Gene expression profiles of co-cultured hepatic stellate cells were compared to published gene signatures of activated hepatic stellate cells from Zhang DY et al. (b) and Wang H et al. (c). Data from one experiment are shown. NES, normalized enrichment score; FWER, family-wise error rate.

Extended Data Fig. 9. Donor-derived Kupffer cells and hepatic macrophages after bone marrow transplantation.

a, C57BL/6J mice were transplanted with CD45.1⁺Tet2^−/− (n = 2) bone marrow cells and fed CDAHFD or standard chow. After 4 weeks, mice were sacrificed and dissociated liver cells were subjected to flow cytometric analysis of CD45.1 (donor) and CD45.2 (recipient) expression in F4/80^hiCD11b^mod Kupffer cells and CD11b^hiF4/80^mod hepatic macrophages. b, B6.SJL mice were transplanted with CD45.2⁺vavCre⁺ (n = 2) bone marrow cells and fed CDAHFD or standard chow for 19 weeks. Immunohistochemical stains demonstrate the presence of CD45.2⁺CD45.1⁻F4/80⁺ macrophages (arrowheads) in livers of bone marrow transplanted mice fed CDAHFD. Representative data from one mouse per condition are shown. c, Selected gene signatures enriched in sorted liver macrophages from Tet2^−/− (n = 4) transplanted mice fed CDAHFD relative to control vavCre⁺ (WT; n = 4) transplanted mice. Data shown are from one experiment. NES, normalized enrichment score; FWER, family-wise error rate.

Figure 1.. CHIP is associated with chronic liver disease.

a, Association of CHIP with prevalent and incident chronic liver disease. WES, whole-exome sequencing. b, Association of clonal haematopoiesis with subtypes of incident chronic liver disease in the UK Biobank. ALD, alcohol-related liver disease. c, Association of clonal haematopoiesis with biopsy-proven NASH in MGB Biobank. Estimates in prevalent analyses were derived using logistic regression, with adjustment for age, sex, type 2 diabetes and smoking. d, Mendelian randomization estimates of the association of CHIP with chronic liver disease. Estimates were derived using MR-RAPS with 184 independent genetic variants with significance of P < 0.0001. Estimates in incident analyses were derived using Cox proportional hazards regression, with adjustment for age, sex, type 2 diabetes and smoking. MGB Biobank cohorts were matched for age, sex, type 2 diabetes, body mass index and smoking. NASH was defined as chronic liver disease among individuals with a body mass index of 30 kg m⁻² or more who consumed 21 drinks or fewer per week for men and 14 drinks or fewer per week for women. Alcohol-related liver disease was defined as chronic liver disease among individuals who consumed 21 drinks or more per week for men or 14 drinks or more per week for women.

Figure 2.. CHIP is associated with steatohepatitis.

a, Prevalence of liver inflammation and hepatic steatosis on magnetic resonance imaging among 8,251 individuals in the UK Biobank. Liver inflammation and fibrosis was defined as a cT1 signal ≥ 795 ms. Fatty liver was defined as a proton density fat fraction ≥ 5%. Logistic regression, with adjustment for age and sex, was used to test the association between CHIP status and the presence of fatty liver and liver inflammation. b, Perspectum MultiScan cT1 image (left) and proton density fat fraction (right) of a patient with CHIP. Image reproduced with permission from the UK Biobank. c–g, B6.SJL mice were transplanted with Tet2^−/− (n = 20), Tet2^−/−Nlrp3^−/− (n = 10) or control vavCre⁺ (wild type (WT); n = 13) bone marrow cells and fed CDAHFD for 11 weeks. Steatohepatitis was graded using modified NASH CRN (Clinical Research Network) histologic criteria. Compared to vavCre⁺ and Tet2^−/−Nlrp3^−/− animals, Tet2^−/−-transplanted mice show similar accumulation of liver fat (c), increased inflammation (d), macrophage crown structures (e, arrows) and hepatocyte ballooning (f, arrowhead), resulting in a higher aggregate NAS (g). h, B6.SJL mice were transplanted with Tet2^−/− (n = 24) or vavCre⁺ wild-type control (n = 21) bone marrow cells and fed CDAHFD for 19 weeks. Collagen fibrosis, highlighted by Picrosirius red staining (left and middle panels), was quantified as the percentage of positive area using ImageJ (right panel). Statistical analysis was performed using two-tailed unpaired t-test (a), chi-square test (g) and Mann–Whitney test (h). For a and h, error bars represent mean ± s.d.

Figure 3.. Proinflammatory signaling in CHIP.

a, Association of the IL6R germline mutation resulting in p.Asp358Ala with chronic liver disease in individuals with CHIP (variant allele fraction ≥ 10%) versus individuals without CHIP. b, Unsupervised hierarchical clustering of differentially regulated genes in sorted liver macrophages from B6.SJL mice transplanted with Tet2^−/− (n = 4) or control vavCre⁺ (WT; n = 4) bone marrow cells and fed CDAHFD for 11 weeks. c, After 19 weeks of CDAHFD, Tet2^−/−-transplanted (n = 12) or control vavCre⁺-transplanted (WT; n = 14) mice were bled and serum was obtained for cytokine measurements. Statistical analysis was performed using two-tailed unpaired t-test (IL-6, CXCL1) or Mann–Whitney test (CCL22, CCL17) with Bonferroni correction for multiple hypothesis testing. d, Bone marrow-derived macrophages from Tet2^−/− (n = 6), Tet2^−/−Nlrp3^−/− (n = 4) or control vavCre⁺ (WT, n = 7) mice were primed with low-dose lipopolysaccharide (LPS) for 2 h and stimulated with palmitic acid or cholesterol monohydrate crystals as indicated for 6 h. Statistical analysis was performed using two-way ANOVA. For c and d, individual measurements, mean and standard deviation from two independent experiments are shown.