Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 3 (1)

Expression of AXL Receptor Tyrosine Kinase Relates to Monocyte Dysfunction and Severity of Cirrhosis

Affiliations

Expression of AXL Receptor Tyrosine Kinase Relates to Monocyte Dysfunction and Severity of Cirrhosis

Robert Brenig et al. Life Sci Alliance.

Abstract

Infectious complications in patients with cirrhosis frequently initiate episodes of decompensation and substantially contribute to the high mortality. Mechanisms of the underlying immuneparesis remain underexplored. TAM receptors (TYRO3/AXL/MERTK) are important inhibitors of innate immune responses. To understand the pathophysiology of immuneparesis in cirrhosis, we detailed TAM receptor expression in relation to monocyte function and disease severity prior to the onset of acute decompensation. TNF-α/IL-6 responses to lipopolysaccharide were attenuated in monocytes from patients with cirrhosis (n = 96) compared with controls (n = 27) and decreased in parallel with disease severity. Concurrently, an AXL-expressing (AXL+) monocyte population expanded. AXL+ cells (CD14+CD16highHLA-DRhigh) were characterised by attenuated TNF-α/IL-6 responses and T cell activation but enhanced efferocytosis and preserved phagocytosis of Escherichia coli Their expansion correlated with disease severity, complications, infection, and 1-yr mortality. AXL+ monocytes were generated in response to microbial products and efferocytosis in vitro. AXL kinase inhibition and down-regulation reversed attenuated monocyte inflammatory responses in cirrhosis ex vivo. AXL may thus serve as prognostic marker and deserves evaluation as immunotherapeutic target in cirrhosis.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1.
Figure 1.. TAM (TYRO3, AXL, and MERTK) receptor expression and functional characterisation of circulating monocytes in cirrhosis.
(A) FACS gating strategy used to identify circulating monocytes in whole blood or PBMCs (left panel). Side scatter (SSC), forward scatter (FSC). Monocyte counts (differential leucocyte count, right panel). (B) TNF-α– and IL-6–producing monocytes (%) in response to LPS ex vivo at different stages of cirrhosis and representative FACS histograms (HC, Child C, and isotype). (C, D, E) TAM receptor expression on circulating monocytes (%) at different stages of cirrhosis and representative FACS histograms for AXL expression (HC, Child C, and fluorescence minus one). (F) GAS6 levels (pg/ml) in HC and cirrhosis (upper panel) and in correlation with AXL expression (% of monocytes, lower panel). HC n = 27, CLD without (w/o) cirrhosis n = 8, Child A n = 36, Child B n = 28, Child C n = 17, and acute decompensation (AD) of cirrhosis n = 8. Data are presented as box plots showing median with 10–90 percentile. *P < 0.05/**P < 0.01 (Mann–Whitney tests, Spearman correlation coefficient).
Figure S1.
Figure S1.. Numbers of TAM receptor-expressing monocytes in patients with cirrhosis, underlying aetiologies, cohorts of patients, and follow-up data of AXL-expressing monocytes.
(A) Counts of TYRO3-, AXL-, and MERTK-expressing monocytes (G/L) in HCs and patients with cirrhosis (CLD without [w/o] cirrhosis, n = 5; Child A, n = 5; B, n = 11; C, n = 7; AD, n = 8). Median/10–90 percentile (Mann–Whitney tests). (B, C) Percentage of AXL-expressing monocytes and plasma ligand GAS6 levels (pg/ml) in different underlying aetiologies of cirrhosis. Alcoholic liver disease (AXL n = 37/ GAS6 n = 18); nonalcoholic fatty liver disease (n = 14/n = 8); hepatitis B virus (n = 7/n = 5); hepatitis C virus (n = 17/n = 10); primary biliary cholangitis (PBC; n = 2/n = 1); autoimmune hepatitis & PBC (AIH & PBC; n = 2/n = 1); alpha-1 antitrypsin deficiency (n = 1/n = 1); Wilson’s disease (n = 1/n = 1); hemochromatosis (n = 1/n = 1); and cryptogenic cirrhosis (n = 1/n = 1). Median with IQR. Statistical significance levels compared with HC and between aetiologies (Mann–Whitney tests). (D) AXL-expressing monocytes after the exclusion of distinct cohorts of patients. Median/10–90 percentile (Mann–Whitney tests). (E, F) Follow-up assessment of AXL-expressing monocytes of individual patients (E; re-compensation after AD [n = 6; n = 2 died during AD], F; 1 yr after inclusion showing Child-Pugh and MELD scores in parallel). *P < 0.05, **P < 0.01 (Wilcoxon test).
Figure S2.
Figure S2.. AXL expression levels on circulatory monocyte subsets and other leukocytes.
(A) AXL expression on monocytes illustrated by a representative flow cytometry histogram, flow cytometry viSNE (visualization tool for high-dimensional single-cell data based on the t-Distributed Stochastic Neighbor Embedding [t-SNE] algorithm) , analysis of cirrhotic monocytes illustrating AXL expression on classical (CD14+CD16), intermediate (CD14++CD16+), and nonclassical (CD14lowCD16+) subsets, and its corresponding quantification shown in percentage and MFI. (B) Representative flow cytometry viSNE analyses and quantification (% of monocytes and MFI) for HCs, patients with CLD without (w/o) cirrhosis, and patients with cirrhosis Child A, B, and C showing AXL expression on different leukocytes such as monocytes, lymphocytes, and granulocytes. Leukocyte count (G/L). Side scatter (SSC); forward scatter (FSC). Median/10–90 percentile. *P < 0.05, **P < 0.01 (Mann–Whitney test).
Figure 2.
Figure 2.. The AXL-expressing monocyte population in patients with cirrhosis in relation to disease severity, complications and prognosis.
(A) Correlations of AXL-expressing monocytes (%) with Child-Pugh (n = 78) and MELD (n = 73) scores and the classification by D’Amico et al (21). HC, CLD without (w/o) cirrhosis. Box plots showing median/10–90 percentile. (B) AXL expression predicted 1-yr mortality (criterion MFI > 440, sensitivity 80%, specificity 79.2%), development of further episodes of AD of cirrhosis within 4 mo (criterion MFI > 362, sensitivity 66.7%, specificity 67.1%), and development of infection over 4 wk (criterion MFI > 389, sensitivity 60%, specificity 65.5%). Median/interquartile range (IQR). (C, D) AXL-expressing monocytes in relation to portal hypertension (C: ascites, hepatic venous pressure [HVPG, n = 14], varices, hepatic encephalopathy; D: bilirubin, n = 72; INR, n = 74; albumin, n = 72; and creatinine, n = 75). Median/10–90 percentile. *P < 0.05/**P < 0.01 (Mann–Whitney tests, Spearman correlation coefficient).
Figure S3.
Figure S3.. sAXL plasma levels in cirrhosis related to disease severity and acute decompensated cirrhosis.
(A) Correlation of sAXL levels with AXL-expressing monocytes (n = 64) and absolute numbers (G/L; n = 27). Spearman correlation coefficient. (B) sAXL plasma levels (ng/ml) in patients with cirrhosis compared with HCs. Median/10–90 percentile (Mann–Whitney tests). (C) sAXL plasma levels in different underlying aetiologies of cirrhosis. Alcoholic liver disease (n = 20); nonalcoholic fatty liver disease (n = 8); hepatitis B virus (n = 7); hepatitis C virus (n = 10); (PBC; n = 1); autoimmune hepatitis & PBC (AIH & PBC; n = 1); alpha-1 antitrypsin deficiency (n = 1); Wilson’s disease (n = 1); hemochromatosis (n = 1); and cryptogenic cirrhosis (n = 1). Median with IQR. Statistical significance levels compared with HC (Mann–Whitney test). (D) Correlation of sAXL levels with Child-Pugh (n = 47) and MELD scores (n = 47). Spearman correlation coefficient. (E) sAXL predicted the onset of acute decompensated cirrhosis (AD) episodes within 4 mo following inclusion for the criterion sAXL > 88.1 ng/ml (sensitivity 83.3%, specificity 82.2%). Median with IQR. *P < 0.05, **P < 0.01 (Mann–Whitney test).
Figure S4.
Figure S4.. AXL is associated with 1-yr mortality, development of acute decompensated cirrhosis, and inflammation and possibly predicts need for transplantation, transplantation-free 1-yr survival, and development of HCC.
(A) AXL predicts (A) 1-yr mortality (sensitivity 77.8%, specificity 77.9%, and criterion AXL [%] > 21.6; sensitivity 87.5%, specificity 88.14%, and criterion AXL [G/L] > 0.17), onset of acute decompensated cirrhosis (AD) within 4 mo following inclusion (sensitivity 77.8%, specificity 77.8%, and criterion AXL [%] > 15.8; sensitivity 74.5%, specificity 75%, and criterion AXL [G/L] > 0.097), and development of infection within 4 wk (sensitivity 80%, specificity 79.8%, and criterion AXL [%] > 19.4; sensitivity 100%, specificity 94.6%, and criterion AXL [G/L] > 0.2). Median with IQR (Mann–Whitney tests). (B) Correlation of AXL-expressing monocytes with CRP (n = 64). Spearman correlation coefficient. (C, D, E) AXL expression on monocytes is associated with the need for transplantation within 1 yr (sensitivity 75%, specificity 85.4%, and criterion AXL MFI > 490; sensitivity 75%, specificity 74.8%, and criterion AXL [%] > 13.6; sensitivity 66.7%, specificity 66.1%, and criterion AXL [G/L] > 0.067), with (D) transplantation-free 1-yr survival (sensitivity 85.7%, specificity 85.4%, and criterion AXL [MFI] > 463.5; sensitivity 76.9%, specificity 76.1%, and criterion AXL [%] > 13.1; sensitivity 75%, specificity 74.5%, and criterion AXL [G/L] > 0.081), and with (E) the development of HCC within 1 yr (sensitivity 75%, specificity 80.1%, and criterion AXL [MFI] > 444; sensitivity 75%, specificity 74.4%, and criterion AXL [%] > 14.5). Median with IQR. *P < 0.05, **P < 0.01 (Mann–Whitney tests).
Figure S5.
Figure S5.. Phenotypic characterisation of circulating monocytes.
(A) Immunophenotyping of monocytes in HCs, CLD without (w/o) cirrhosis, and cirrhosis: Fcγ-receptors (CD16, CD32, and CD64), MHC class II receptor HLA-DR, chemokine receptors (CX3CR1, CCR5, and CCR7), TLR4, 3, 9, and 2, scavenger receptor CD163, and IFN-α/β receptor IFNAR. Median/10–90 percentile. *P < 0.05, **P < 0.01 (Mann–Whitney tests).
Figure 3.
Figure 3.. Phenotypic characterisation of the AXL-expressing monocyte subset.
(A) Gating strategy with representative FACS scatter plots and histograms for AXL expression used to distinguish AXL-expressing (AXL+) from AXL-negative (AXL) monocytes. Side scatter (SSC), forward scatter (FSC), fluorescence minus one. (B) Immunophenotyping of AXL+ and AXL monocytes in cirrhosis. Glycoprotein CD14, MHC class II receptor HLA-DR, Fcγ-receptors (CD16 and CD32), TAM receptors (MERTK and TYRO3), chemokine receptors (CX3CR1, CCR5, and CCR7), and TLR4. Box plots showing median/10–90 percentile. (C) Viability (7-AADAnnexinV-cells) of AXL+-/AXL-monocytes. Median/10–90 percentile. *P < 0.05/**P < 0.01 (Wilcoxon tests).
Figure S6.
Figure S6.. Expansion of immune-regulatory AXL-expressing monocytes and immunosuppressive M-MDSCs in progressing cirrhosis.
(A) M-MDSCs in patients with stable and advanced cirrhosis compared with patients with CLD without (w/o) cirrhosis and HCs. Median/10–90 percentile (Mann–Whitney tests). (B) Distinct monocytic subsets (M-MDSCs, CD14+HLA-DR+AXL+) and “functionally intact” CD14+HLA-DR+AXL monocytes (% in pie charts and absolute cell counts in table) with progressing disease severity from Child-Pugh A to C compared with HC represented. Mean with SD. (C) AXL expression in M-MDSCs and CD14+HLA-DR+ monocytic subsets. Median/10–90 percentile. *P < 0.05, **P < 0.01 (Wilcoxon tests).
Figure 4.
Figure 4.. Functional characterisation of AXL-expressing circulating monocytes ex vivo.
(A) TNF-α and IL-6 production upon LPS treatment of CD14+HLA-DR+AXL+, CD14+HLA-DR+AXL monocytes and M-MDSCs from HC and patients with cirrhosis (% of monocytes). (B) SOCS1/3 mRNA-expression of monocytes from HC and cirrhosis. (C) T cell proliferation in co-culture with monocytes at day 2 in a mixed lymphocyte reaction (HC versus cirrhosis; AXLlow versus AXLhigh). Data shown as MFI−1 of carboxyfluorescein succinimidyl ester. (D) Phagocytosis of E. coli bioparticles of the entire monocyte population from different patient groups (HC, CLD without [w/o] cirrhosis, cirrhosis, left panel) and CD14+HLA-DR+AXL+, CD14+HLA-DR+AXL subsets, and M-MDSCs from HC and patients with cirrhosis. Box plots showing median/10–90 percentile. *P < 0.05/**P < 0.01 (Mann–Whitney, Wilcoxon tests).
Figure S7.
Figure S7.. Pro-inflammatory cytokine production in response to LPS, phagocytic capacity of monocytes, and correlations of AXL expression with monocyte function.
(A, B) TNF-α and IL-6 production (% of monocytes/MFI) upon LPS treatment of monocyte subgroups (AXL+-/AXL-monocytes [A]/monocytic myeloid-derived suppressor cells [M-MDSCs; B]) from HCs and patients with cirrhosis at different stages of disease with representative FACS histograms (A). Median/10–90 percentile (Wilcoxon tests). (C) Proportion of phagocyting (E. coli) monocytes (AXL+-/AXL-monocytes/M-MDSCs) from HC and patients with cirrhosis. Median/10–90 percentile (Wilcoxon, Mann–Whitney tests). (D, E) Correlation of phagocytosis capacity of E. coli bioparticles with (D) AXL expression (n = 57) and with (E) TNF-α production upon LPS treatment (n = 25). *P < 0.05, **P < 0.01 (Spearman correlation coefficient).
Figure 5.
Figure 5.. AXL overexpression in THP-1 cells and LPS-induced inflammatory cytokine production in vitro.
(A) Schematic model of retroviral transduction of THP-1 cells and representative FACS histogram of AXL expression in THP-1-AXL+ cells. Side scatter (SSC), forward scatter (FSC). (B) AXL expression in THP-1 cells and AXL-expressing THP-1 cells (%). (C) TNF-α and IL-6 secretion (pg/ml) in response to LPS in THP-1-AXL+ and THP-1 cells. Bar plots showing mean/SD. *P < 0.05/**P < 0.01 (t tests).
Figure S8.
Figure S8.. Phenotypic characterisation of monocytic THP-1 and transduced THP-1-AXL+ cells.
(A) Relative AXL mRNA expression in THP-1 compared with THP-1-AXL+ cells. (B) Difference of protein surface receptors (Fcγ-receptors [CD16, CD32, and CD64], MHC class II receptor HLA-DR, chemokine receptors [CX3CR1, CCR5, and CCR7], TLR4, and scavenger receptor CD163) on THP-1 and THP-1-AXL+ cells. Mean % of THP-1/THP-1-AXL+ cells with SD. *P < 0.05, **P < 0.01 (t tests).
Figure 6.
Figure 6.. AXL expression on monocytes in response to bacterial and inflammatory stimuli and following phagocytosis and efferocytosis.
(A) AXL expression after incubation with bacterial/inflammatory stimuli as indicated in vitro for 18 h. Time-dependent effect of LPS on AXL expression. Delta AXL (ΔAXL) MFI shows difference to untreated cells. Bar plots showing mean/SD (t tests). (B) AXL expression after monocyte incubation in 25% plasma of HCs and patients with cirrhosis for 24 h. (C) Representative FACS scatter plots for monocyte phagocytosis of microbial products ex vivo. Forward scatter (FSC). AXL expression after E. coli and S. aureus bioparticle uptake (15 min) and live GFP-E. coli ingestion (60 min) on monocytes from HC and patients with cirrhosis. Box plots showing median/10–90 percentile (Mann–Whitney tests). (D) Representative FACS scatter plots for resting (CellTracker) and efferocytosing (CellTracker+) monocytes after co-culture with apoptotic cells for 8 h. Apoptosis of neutrophils and HepG2 cells after 24 h. AXL expression after efferocytosis, AXL expression of resting and efferocytosing monocytes, and efferocytosis capacity for neutrophils of AXL+-/AXL-monocytes. Bar plots showing mean/SD. *P < 0.05/**P < 0.01 (unpaired/paired t tests).
Figure S9.
Figure S9.. Cytokine responses following up-regulation of AXL.
(A, B) TNF-α and IL-6 production of healthy monocytes upon LPS treatment (100 ng/ml, 5 h) after 24-h pre-incubation with LPS (which resulted in AXL up-regulation; Fig 6A) compared with monocytes not previously exposed to LPS (A) and after 8-h co-culture with apoptotic neutrophils (which also resulted in AXL up-regulation; Fig 6D; AXL+ versus AXL monocytes) (B). Mean % of monocytes with SD. *P < 0.05, **P < 0.01 (t tests).
Figure 7.
Figure 7.. Innate immune responses and phagocytosis capacity of monocytes from patients with cirrhosis after AXL inhibition and down-regulation ex vivo.
(A, B, C) AXL expression (% of monocytes), (B) TNF-α and IL-6 production in response to LPS (total monocyte population and AXL+/AXL-cells), and (C) monocyte phagocytosis capacity of E. coli bioparticles (%CD14+ cells) after small molecule inhibitor BGB324 and metformin treatment compared with untreated cells (w/o) in HCs and patients with cirrhosis. Box plots showing median/10–90 percentile. *P < 0.05/**P < 0.01 (Mann–Whitney, Wilcoxon tests).
Figure S10.
Figure S10.. AXL expression, viability, and cytokine expression after BGB324 and metformin treatment.
(A) AXL expression (MFI) after BGB324 and metformin treatment compared with untreated (w/o) cells in HCs and patients with cirrhosis. Median/10–90 percentile (Mann–Whitney tests). (B, C) Healthy monocytes were treated with 10 mM of metformin (24 h) (B), 0.25, 0.5, 1, and 2 μM of BGB324 (24 h) (C) and proportion of viable (7-AAD-Annexin V), apoptotic (7-AAD-Annexin V+), and dead (7-AAD+-Annexin V) monocytes was assessed. (D) TNF-α/IL-6 production was assessed in response to LPS (100 ng/ml, 5 h) after different doses of BGB324. DMSO. Mean % of monocytes with SD. *P < 0.05, **P < 0.01 (t tests).
Figure S11.
Figure S11.. Effect of GAS6 on cytokine production upon LPS treatment.
(A) Differences of TNF-α/IL-6 production of healthy monocytes and monocytes from patients (Child C) in response to LPS (100 ng/ml, 5 h) after the addition of GAS6 (20 nM) and other complements (10% FCS, 25% autologous plasma) to X-VIVO culture medium.

Similar articles

See all similar articles

References

    1. Arvaniti V, D’Amico G, Fede G, Manousou P, Tsochatzis E, Pleguezuelo M, Burroughs AK. (2010) Infections in patients with cirrhosis increase mortality four-fold and should be used in determining prognosis. Gastroenterology 139: 1246–1256.e5. 10.1053/j.gastro.2010.06.019 - DOI - PubMed
    1. Tandon P, Garcia-Tsao G. (2008) Bacterial infections, sepsis, and multiorgan failure in cirrhosis. Semin Liver Dis 28: 26–42. 10.1055/s-2008-1040319 - DOI - PubMed
    1. Fernández J, Navasa M, Gómez J, Colmenero J, Vila J, Arroyo V, Rodés J. (2002) Bacterial infections in cirrhosis: Epidemiological changes with invasive procedures and norfloxacin prophylaxis. Hepatology 35: 140–148. 10.1053/jhep.2002.30082 - DOI - PubMed
    1. Borzio M, Salerno F, Piantoni L, Cazzaniga M, Angeli P, Bissoli F, Boccia S, Colloredo-Mels G, Corigliano P, Fornaciari G, et al. (2001) Bacterial infection in patients with advanced cirrhosis: A multicentre prospective study. Dig Liver Dis 33: 41–48. 10.1016/s1590-8658(01)80134-1 - DOI - PubMed
    1. Moreau R, Jalan R, Gines P, Pavesi M, Angeli P, Cordoba J, Durand F, Gustot T, Saliba F, Domenicali M, et al. (2013) Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis. Gastroenterology 144: 1426–1437.e9. 10.1053/j.gastro.2013.02.042 - DOI - PubMed
Feedback