Ductular Reaction Cells Display an Inflammatory Profile and Recruit Neutrophils in Alcoholic Hepatitis

Abstract

Chronic liver diseases are characterized by the expansion of ductular reaction (DR) cells and the expression of liver progenitor cell (LPC) markers. In alcoholic hepatitis (AH), the degree of DR expansion correlates with disease progression and short-term survival. However, little is known about the biological properties of DR cells, their impact on the pathogenesis of human liver disease, and their contribution to tissue repair. In this study, we have evaluated the transcriptomic profile of DR cells by laser capture microdissection in patients with AH and assessed its association with disease progression. The transcriptome analysis of cytokeratin 7-positive (KRT7⁺ ) DR cells uncovered intrinsic gene pathways expressed in DR and genes associated with alcoholic liver disease progression. Importantly, DR presented a proinflammatory profile with expression of neutrophil recruiting C-X-C motif chemokine ligand (CXC) and C-C motif chemokine ligand chemokines. Moreover, LPC markers correlated with liver expression and circulating levels of inflammatory mediators such as CXCL5. Histologically, DR was associated with neutrophil infiltration at the periportal area. In order to model the DR and to assess its functional role, we generated LPC organoids derived from patients with cirrhosis. Liver organoids mimicked the transcriptomic and proinflammatory profile of DR cells. Conditioned medium from organoids induced neutrophil migration and enhanced cytokine expression in neutrophils. Likewise, neutrophils promoted the proinflammatory profile and the expression of chemokines of liver organoids. Conclusion: Transcriptomic and functional analysis of KRT7⁺ cells indicate that DR has a proinflammatory profile and promote neutrophil recruitment. These results indicate that DR may be involved in the liver inflammatory response in AH, and suggest that therapeutic strategies targeting DR cells may be useful to mitigate the inflammatory cell recruitment in AH.

FIG. 1.

Gene expression profile of ductular reaction in AH. (A) KRT7 staining with and without counterstaining of paraffin-embedded liver sections of patients with AH. Scheme of populations selected by laser capture microdissection: KRT7-positive (n = 6), -negative (n = 6), and total (n = 4) areas. (B) Principal component analysis showing the three different fractions that underwent RNA sequencing. Each sample is placed in the two-dimensional space according to its RNA expression. The KRT7-positive fraction (POS) is represented in blue, the KRT7-negative fraction (NEG) in red, and the total fraction in green. (C) Heat map of the top 50 differentially expressed genes in KRT7-positive fraction compared with the negative fraction. The red color indicates an enrichment of gene expression, whereas the blue color shows decreased gene expression. (D) Immunohistochemistry of liver progenitor cell markers HNF1β, KRT19, EpCAM, KRT7, SOX9, and KRT23 in hepatic sections of patients with AH. Abbreviation: HNF1β, hepatocyte nuclear factor 1β.

FIG. 2.

Expression of DR genes in ALD progression. (A) Expression of DR genes that positively correlate with MELD and Child-Pugh score along ALD progression (data set containing healthy controls [n = 6], patients with precirrhosis [n = 4], and patients with cirrhosis without AH [n = 7] and with AH [n = 11]). Two differentiated group of genes are represented according to the pattern of expression along ALD progression. (B) Correlation between expression of LPC markers and proinflammatory mediators in the ALD data set. Correlations are shown as a heat map. Patient cohort characteristics are shown in Supporting Table S1. Abbreviation: FC, fold change.

FIG. 3.

Correlation of ductular reaction with inflammatory mediators in patients with AH. (A) Correlation of real-time quantitative PCR gene expression (Fc vs. healthy individuals) of KRT7 with inflammatory cytokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8) in patients with AH (n = 40). The regression coefficient (r) and P value of each correlation are indicated. (B) Serum levels (pg/mL) of CXCL5 and TROP2 in patients with AH (n = 20 and n = 14, respectively) compared with healthy individuals (n = 6 and n = 3, respectively); *, P < 0.05 compared with healthy individuals. (C) Correlation of CXCL5 and TROP2 serum levels (pg/mL) in patients with AH (n = 14). r value and P value is indicated. (D) Representative immunohistochemistry of CXCL5 and TROP2 in liver sections of patients with AH. Abbreviation: Fc, fold change.

FIG. 4.

Transcriptomic and functional analysis of liver organoids from patients with cirrhosis. (A) Immunofluorescence of LPC markers, EpCAM and KRT7, in liver organoids. (B) Gene set enrichment analysis between liver organoid transcriptome and genes expressed in KRT7⁺ microdissected fraction. The gene expression profile of human organoids (n = 4) versus liver tissue from healthy patients (n = 4) was used as a data set. NES and significance is shown. (C) Heat map illustrating gene expression of LPC markers and inflammatory cytokines in liver organoids compared with liver tissue from healthy patients. (D) Real-time PCR gene expression of LPC markers (KRT7, KRT19, EpCAM, and SOX9) and inflammatory mediators (CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, and CCL28) in human organoids. mRNA levels are shown as ΔCt values compared with glyceraldehyde 3-phosphate dehydrogenase Ct values (n = 4). (E) Expression of inflammatory cytokines in organoids after IL-1β stimulation and NF-κB inhibition. Organoids were incubated with VEH or NF-κB INH and stimulated with IL-1β for 6 hours. Organoid gene expression of CXCL1, CXCL5, CXCL6, and CXCL8 is shown as fold change versus VEH. n = 3; *, P < 0.05 compared with VEH; #, P < 0.05 compared with IL-1β. Abbreviations: NE S, normalized enrichment score; NF-κB INH, NF-κB inhibitior; VEH, vehicle.

FIG. 5.

DR recruits neutrophils. (A) Representative double staining of KRT7 (green) and MPO (red) in alcohol-induced cirrhotic liver sections from 2 different patients. (B) Neutrophil migration assay in response to CM LPCs. Conditioned medium from three different organoid lines was used in three independent neutrophil isolations. (C) Neutrophil response to organoid-conditioned medium. Freshly isolated human neutrophils were incubated with CM LPCs from 3 different patients with cirrhosis for 18 hours. Gene expression is shown as fold change versus VEH after incubation with CM LPCs. n = 3; *, P < 0.05 compared with VEH. (D) Organoid response to neutrophil-conditioned medium. Conditioned media from neutrophils stimulated with vehicle or LPS for 6 hours was used for organoid stimulation. Results are shown as fold change versus VEH after incubation with CM of neutrophils. n = 5; *, P < 0.05 compared with VEH. #, P < 0.05 compared with CM. Abbreviations: CM LPCs, liver progenitor cells organoid conditioned medium; VEH, vehicle.