Proinflammatory macrophages release CXCL5 to regulate T cell function and limit effects of αPD-1 in steatosis-driven liver cancer

Abstract

Background & aims: Steatosis is a comorbid factor for cancer development. Patients with steatosis do not respond well to current immune checkpoint therapy (CPI) treatment. We explored the roles of neutrophil-activating chemokines (NACs) in the response of steatosis/liver cancer to CPI.

Methods: We used a steatosis-driven liver cancer model induced by the deletion of Pten in the liver (LiPten) and a high-fat diet + carbon tetrachloride (CCl₄) fibrosis model to study the effects of targeting CXCL5. We also studied the role of CXCL5 in the liver immune microenvironment in vitro and in vivo. ANOVA/t tests were used for data analysis.

Results: Using LiPten steatosis-tumor mice, we identified CXCL5 as the NAC most robustly upregulated as steatosis progresses to cancer (>100 fold, n = 6-11). We also validated this observation in patient samples. When used together with αPD-1, inhibiting the NAC receptor CXCR2 promoted (100% vs. 80% in untreated LiPten mice), whereas anti-CXCL5 suppressed (25%), tumor progression (n = 4-6) suggesting unique functions of CXCL5 independent of CXCR2. Similar effects were observed for anti-CXCL5 (0/4 with fibrosis) vs. CXCR2 inhibition (4/4 with fibrosis) of fibrosis in the HFD + CCl₄ model. Using a Transwell assay, we identified a novel inhibitory function of CXCL5 in the recruitment of CD4⁺ T cells (p <0.02, n = 4) and potentiation of CD8⁺ T cell cytotoxicity (p <0.001, n = 4). In vivo, we showed that neutralizing CXCL5 increased the CD8/CD4 ratio (p = 0.03 and 0.07) and synergized with αPD-1 for its anti-tumor and anti-fibrosis activity (n = 4-6).

Conclusions: Our discovery of the novel inhibitory role of CXCL5 in T cells suggests that NACs have additional functions in modulating the immune system beyond neutrophil chemotaxis. The discovery of this novel CXCL5 role presents additional therapeutical targets alongside current immune checkpoint therapy.

Impact and implications: In this study, we investigated the role of CXCL5 in the progression from steatosis to liver cancer. We uncovered a novel inhibitory role of CXCL5 in T cell recruitment, with implications for NAC-targeted therapy and immune checkpoint synergy in liver cancer. We believe our findings will be of interest to physicians, researchers, and patients interested in therapeutic development and translational research in liver disease.

Keywords: Fibrosis; HCC; NAFLD/NASH; PTEN.

Graphical abstract

Fig. 1

Identification of CXCL5 as a steatosis-induced NAC. (A) Progression of steatosis to liver cancer in LiPten livers. Microscopic images showing steatosis (fatty liver) in mice at 1 and 3 months of age, inflammation/fibrosis at 9 months, and tumor after 12 months. Percentage indicates percentage of mice that were tumor bearing at the indicated age. (B, C) Induction of CXCL5 (and CXCL2) among the NACs in LiPten livers. qPCR analysis of bulk liver tissues from the Con and LiPten mice at the indicated age. (D) Reducing liver steatosis via deletion of Akt2 (A2-LiPten) inhibits the expression of CXCL5 compared with LiPten mice. Left: H&E image of liver tissues from indicated genotype group. Right: qPCR analysis of bulk liver tissues. Each data point is one mouse. Data are presented as mean ± SEM, analyzed with unpaired Student t test; statistical significance at p <0.05. Scale bars: 50 µm. Con, controls (Pten^loxP/loxP; Alb-Cre^–); CXCL5, C-X-C ligand 5; LiPten, Pten^LoxP/LoxP; Alb-Cre⁺; NAC, neutrophil-activating chemokine; PV, portal vein.

Fig. 2

CXCL5 correlates with poor patient response to CPI and poor overall survival. (A) Kaplan–Meier survival analysis of TCGA HCC patient data. (B) Correlation analysis of CXCL5 with proliferation marker MKi67 using TCGA HCC patient data. (C) Left: qPCR analysis of CXCL5 in healthy donor (n = 5) and HCC samples (n = 29). Right: immunoblotting analysis of tumors (T1–T4) and adjacent tissues (N1–N4) for CXCL5. Numbers below show densitometry quantification of the bands displayed as CXCL5:β-Actin ratio. (D) Analysis of three GEO data sets containing healthy (H), non-tumor (NT) and tumor (T) samples. Red arrows indicate CXCL5. (E) Plasma CXCL5 levels in patients before they underwent immune checkpoint therapy. Complete response (n = 1), partial response (n = 7), stable disease (n = 27) and progressive disease (n = 8). The line connects the mean in each group, indicating the trend in CXCL5 change from one group to the next. Data are presented as mean ± SEM, analyzed with Student t test; p = 0.3 (C). CPI, immune checkpoint therapy; CXCL, C-X-C ligand; GEO, Gene Expression Omnibus; HCC, hepatocellular carcinoma; TCGA,The Cancer Genome Atlas.

Fig. 3

αCXCL5 but not CXCR2 inhibitor synergizes with αPD-1 for attenuating progression from steatosis to cancer. (A) Effects of CXCR2 inhibitor SB225002, αPD-1 in combination with SB225002 or αCXCL5 on tumor growth in LiPten mice. Macro- and microscopic images of livers and liver sections. Numbers indicate number of tumor-bearing mice/total mice in the group. Arrows indicate tumor nodules and ‘T’ indicates tumor. Scale bar: 100 µm (B) Flow cytometry analysis of liver immune cells shows that αCXCL5 + αPD-1 treatment induces T cell recruitment to tumors in LiPten liver. Each data point is one mouse. Data are presented as mean ± SEM, analyzed with ANOVA followed by Tukey post hoc test; statistical significance at p <0.05. CXCL, C-X-C ligand; LiPten, Pten^LoxP/LoxP; Alb-Cre⁺.

Fig. 4

Blocking CXCL5 reprograms the TME and suppresses T cell cytotoxicity. (A) Schematic of the Transwell T cell cytotoxicity experiment. (B) Transwell assay performed with LMac from LiPten mice, LiPten tumor hepatocytes, and liver immune cells with or without CXCL5 neutralizing antibody to determine the effects of CXCL5 on T cell-mediated cytotoxicity. All cultures treated with LPS and αPD-1 to enable T cell-mediated cytotoxicity. Crystal violet staining was performed on cells in the bottom chamber 3 days after co-culture. Each column represents one mouse. (C) Areas with crystal violet staining quantified and reported as a percentage. Each data point is one mouse. (D) Transwell assay showing that CXCL5 induces the migration of neutrophils while inhibiting the migration of T cells. Treatment with AZD5069 inhibits the migration of neutrophils (p = 0.0026) but has limited effects on the migration of T cells. Each line is one mouse. Data are presented as mean ± SEM, analyzed with ANOVA followed by Tukey post hoc test (C) or Student t test (D); statistical significance at p <0.05. Ab, antibody; CXCL, C-X-C ligand; LPS, lipopolysaccharide; LiPten, Pten^LoxP/LoxP; Alb-Cre⁺; LMac, liver macrophages; TME, tumor microenvironment.

Fig. 5

Macrophages are the source of CXCL5 in the liver. (A) Analysis of TCGA data shows that CXCL5 expression is associated with Mac. (B) Flowcytometry analysis shows highest expression of CXCL5 in KCs. Left: IM also express higher CXCL5 compared with other immune cells. Right: MFI of CXCL5 in the sorted cell populations. (C) Analysis of CXCL5 (left) and other NACs (right) in LiPten livers treated with or without Cld to deplete macrophages. Expression of Clec4f is also quantified to validate successful depletion. Each data point is one mouse. (D) Top: staining of CXCL5 in mouse liver (mCXCL5) shows colocalization with Clec4f, a marker of liver macrophages. Bottom: staining of CXCL5 in HCC patient liver (hCXCL5) shows CXCL5 is not localized in Ki67-positive tumor cells. Right: quantification of mCXCL5 and Clec4f staining and co-staining in mouse tissues. Scale bar: 50 µm. Each data point corresponds to an image. Data are presented as mean ± SEM, analyzed with ANOVA followed by Tukey post hoc test (B) or Student t test (C); statistical significance at p <0.05. CAF, cancer-associated fibroblasts; Cld, chlodronate; CXCL, C-X-C ligand; Endo, endothelial cells; HCC, hepatocellular carcinoma; IM, infiltrating macrophages; KP, Kupffer cells; LiPten, Pten^LoxP/LoxP; Alb-Cre⁺; Mac, macrophages; MFI, mean fluorescent intensity; NAC, neutrophil-activating chemokine; NK, natural killer cells.

Fig. 6

Stimulating proinflammatory response in macrophages induces CXCL5 release. (A) LPS treatment induces CXCL5 expression in primary LMac. Left: qPCR analysis of CXCL5 in LPS-treated primary LMac. Right: LPS induces a proinflammatory response in primary LMac as indicated by the secretion of TNFα, a proinflammatory cytokine. Galectin-3 is detected as negative control. Each data point is one mouse. (B) Analysis of TCGA data shows induction of the proinflammatory gene iNOS and inhibition of anti-inflammatory gene CD163 in HCC samples. Normal (n = 50); HCC (n = 371). (C) Left: flowcytometry analysis of CD64+ macrophages in the non-parenchymal immune cells from Con and LiPten livers. Right: qPCR analysis of CD64, a marker of proinflammatory macrophages in the same group of mice. Data are presented as mean ± SEM, analyzed with Student t test; statistical significance at p <0.05. Con, control; CXCL, C-X-C ligand; HCC, hepatocellular carcinoma; LiPten, Pten^LoxP/LoxP; Alb-Cre⁺; LMac, liver macrophages; LPS, lipopolysaccharide; TGCA, The Cancer Genome Atlas.

Fig. 7

CXCL5 inhibits T cell recruitment. (A) Left: experimental schematics of Transwell culture of splenocytes with LMac with or without LPS. Right: quantification of CD45+ immune cells in the bottom chamber of the Transwell following migration. (B) Migration index for each cell type calculated based on the number of each cell type migrated with the co-culture with LMac considered as 1. Each data point is one mouse. (C) CXCL5 inhibits T cell migration induced by CCL20. Each data point is one mouse. Data are presented as mean ± SEM, analyzed with ANOVA followed by Tukey post hoc test; statistical significance at p <0.05. Ab, antibody; CXCL, C-X-C ligand; LMac, liver macrophages; LPS, lipopolysaccharide.

Fig. 8

Blocking CXCL5 increases recruitment of T cells and attenuates fibrosis development. (A) Morphology of livers from HFD + CCl₄-induced fibrosis model treated with different drug combinations. Left: H&E; Scale bar: 50 μm. right: Sirius Red staining. Scale bar: 100 μm. (B) αCXCL5 + αPD-1 treatment increases T cells in livers of HFD + CCl4 mice. Each data point is one mouse. (C) αCXCL5 enhances CD8+/CD4+ ratio better than CXCR2 inhibition in combination with αPD-1. Each data point is one mouse. (D) Comparison of T cell subpopulations in αCXCL5 + αPD-1 vs. SB225002 + αPD-1-treated mouse livers. Each data point is one mouse. (E) Schematic of the findings showing the roles of CXCL5 in T cells in the liver TME. Data are presented as mean ± SEM, analyzed with Student t test; statistical significance at p <0.05. CCl₄, carbon tetrachloride; CXCL, C-X-C ligand; HFD, high-fat diet; LMac, liver macrophages; LPS, lipopolysaccharide; Mac, macrophages; Neu, neutrophils; TME, tumor microenvironment.