Tumour inflammasome-derived IL-1β recruits neutrophils and improves local recurrence-free survival in EBV-induced nasopharyngeal carcinoma

Abstract

Inflammasomes sense infection and cellular damage and are critical for triggering inflammation through IL-1β production. In carcinogenesis, inflammasomes may have contradictory roles through facilitating antitumour immunity and inducing oncogenic factors. Their function in cancer remains poorly characterized. Here we show that the NLRP3, AIM2 and RIG-I inflammasomes are overexpressed in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), and expression levels correlate with patient survival. In tumour cells, AIM2 and RIG-I are required for IL-1β induction by EBV genomic DNA and EBV-encoded small RNAs, respectively, while NLRP3 responds to extracellular ATP and reactive oxygen species. Irradiation and chemotherapy can further activate AIM2 and NLRP3, respectively. In mice, tumour-derived IL-1β inhibits tumour growth and enhances survival through host responses. Mechanistically, IL-1β-mediated anti-tumour effects depend on infiltrated immunostimulatory neutrophils. We show further that presence of tumour-associated neutrophils is significantly associated with better survival in NPC patients. Thus, tumour inflammasomes play a key role in tumour control by recruiting neutrophils, and their expression levels are favourable prognostic markers and promising therapeutic targets in patients.

Figure 1. Association of AIM2, RIG-I and NLRP3 inflammasomes with better survival in NPC patients

1. Overexpression of ASC, caspase-1 and IL-1β proteins as well as the inflammasome-receptor proteins in NPC tumour cells. Consecutive NPC tissue sections containing tumour (T) and adjacent non-tumour (N) cells were immunohistochemically stained with protein-specific antibodies. The results are shown at 100× magnification (upper panel), and the ‘N’ areas are shown at 400× magnification. The expression of these inflammasome receptors was relatively weak in the adjacent normal nasopharyngeal epithelial tissues. Bar, 100 µm.

2. Kaplan–Meier survival analysis of LRFS as a function of elevated inflammasome component expression in NPC patients.

3. Multivariant analysis of the association of inflammasome components with LRFS.

4. Kaplan–Meier survival analysis of LRFS as a function of each AIM2, RIG-I and NLRP3 inflammasome in NPC patients. AIM2 inflammasome high means the levels of all four AIM2 inflammasome components including AIM2, as well as three common components ASC, caspase-1 and IL-1β in NPC biopsy tissues are scored as high levels by immunohistochemistry analyses. Similarly, RIG-I inflammasome high or NLRP3 inflammasome high means high levels of RIG-I or NLRP3 combined with high levels of ASC, caspase-1 and IL-1β. *, statistically significant as indicated.

Figure 2. LMP1-mediated pro-IL1β induction through activation of the NF-κB and MAPK signalling pathways

1. Induction of pro-IL-1β expression by LMP1 in NPC cell lines. NPC-TW01, -TW02, -TW04 and HK1 cells were transiently transfected with LMP1-expressing plasmid (Flag-LMP1), and pro-IL-1β mRNA and protein levels were determined at 24 h post-transfection by quantitative RT-PCR and Western blotting. All results are presented as the mean ± SD of three independent experiments.

2. Mapping the domain of LMP1 responsible for pro-IL-1β induction. LMP1 and its domain-specific mutants were transfected into NPC-TW02 cells, and pro-IL-1β mRNA and protein levels were determined at 24 h post-transfection by quantitative RT-PCR and Western blotting. All results are presented as the mean ± SD of three independent experiments.

3. The effect of inhibitors on pro-IL-1β induction. NPC-TW02 cells transfected with Flag-LMP1 expressing plasmid were treated with the specific inhibitors of NF-κB (BAY11-7082), JNK (SP600125), p38 MAPK (SB203580) and ERK1/2 (PD98059). The levels of pro-IL-1β protein were determined by Western blotting.

4. The effect of p65 NF-κB, c-Jun, p38α/β MAPK and ERK1/2 knockdown on pro-IL-1β induction. NPC-TW02 cells transfected with a siRNA targeting either p65 NF-κB, c-Jun, p38α/β MAPK or ERK1/2 were further transfected with an LMP1-expressing plasmid. The levels of pro-IL-1β protein were determined by Western blotting.

5. Correlation of LMP1 and pro-IL-1β mRNA expression in NPC biopsies. The relative fold change of LMP1 and pro-IL-1β mRNA expression between NPC and adjacent normal tissues was determined by quantitative RT-PCR. The 20 tumour tissues were divided into two groups by their relative expression levels of LMP1: high LMP1 (n = 10) and low LMP1 (n = 10). Normal tissues, n = 7. The results are presented as the mean ± SD and analysed by Student's t test. *p = 0.045.

Figure 3. Activation of inflammasomes by EBV-associated factors and microenvironmental stressors

1. A. Expression of inflammasome components in NPC cell lines. The protein levels of individual components and actin (loading control) were determined by Western blotting of cell lysates. The THP-1 cells were used as a positive control.

2. B,C. IL-1β induction by EBV gDNA via AIM2. HK1 cells were transfected with fragmented EBV gDNA, pEGFP or poly (dA:dT) for 12 h, with or without a pretreatment with Z-VAD-FMK (10 µM) for 30 min (B), or pre-transfection with AIM2-targeting or control siRNA for 48 h (C). **p = 0.001, 0.004 and 0.002 for EBV gDNA, pEGFP and poly (dA:dT), respectively (B); *p = 0.006, 0.002 and 0.003 for EBV gDNA, pEGFP and poly (dA:dT), respectively (C). All results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

3. D,E. IL-1β induction by EBERs via RIG-I. HK1 cells were transfected with in vitro-transcribed EBER1 and EBER2 for 12 h, with or without a pretreatment with Z-VAD-FMK (D), or pre-transfection with RIG-I-targeting or control siRNA for 48 h (E). **p = 0.006 and 0.002 for EBER1 and EBER2, respectively (D); **p = 0.009 and 0.009 for EBER1 and EBER2, respectively (E). All results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

4. F. RIG-I activation by endogenous EBERs. HK1 cells were transfected with vector control (pHEBo), EBER1- or EBER2-expressing plasmid with pre-transfection with RIG-I-targeting or control siRNA for 48 h. Expression of EBERs was confirmed by RT-PCR. GAPDH was used as a positive control. The results are presented as the mean ± SD of three independent experiments.

5. G,H. IL-1β induction by ATP and H₂O₂ via NLRP3. HK1 cells were treated with ATP (5 mM) for 4 h or with H₂O₂ (10 µM) for 24 h, with or without a pretreatment with Z-VAD-FMK (G), or pre-transfection with NLRP3-targeting or control siRNA for 48 h (H). IL-1β production was used to measure inflammasome activity. **p = 0.0001 and 0.0004 for ATP and H₂O₂, respectively (G); *p = 0.022 and **p = 0.007 for ATP and H₂O₂, respectively (H). All results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

Figure 4. Enhanced activation of inflammasomes by therapeutic treatments

1. Dose-dependent induction of IL-1β by irradiation. HK1 cells were treated with various doses of irradiation for 24 h. **p = 0.001; ***p = 2.7E−05, 9.8E−06 and 9.7E−08 for 20, 30 and 40 Gy, respectively. All results are presented as the mean ± SD of six independent experiments and analysed by Student's t test.

2. ROS production is required for the induction of IL-1β by irradiation. HK-1 cells were irradiated (30 Gy) with or without a 30 min pretreatment with oATP (100 µM), apyrase (2.5 unit/ml), DPI (10 µM) or Z-VAD-FMK. *p = 0.02 and 0.035 for DPI and Z-VAD-FMK, respectively. All results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

3. Requirement of AIM2 for irradiation-induced IL-1β production. HK-1 cells transfected with NLRP3-targeting, AIM2-targeting, RIG-I-targeting, and control siRNA for 48 h were irradiated. ***p = 0.0003, all results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

4. Quantitative PCR analysis of cytosolic DNA in irradiation-treated HK1 cells. HK1 cells were treated with irradiation (30 Gy) for 24 h or ATP (5 mM) for 4 h. Nuclear DNA and mitochondrial DNA were indicated as nuDNA and mtDNA, respectively. *p = 0.005 (nuDNA analysis); *p = 0.016 and 0.017 for irradiation and ATP, respectively (mtDNA analysis). All results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

5. Dose-dependent induction of IL-1β by cisplatin. HK1 cells were treated with various doses of cisplatin for 24 h. *p = 0.012. **p = 0.007 and 0.003 for 40 and 50 µM, respectively. All results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

6. Requirement of cathepsin B activity for the induction of IL-1β by cisplatin. HK-1 cells were incubated with 40 µM cisplatin with or without a 30 min pretreatment with oATP, apyrase, DPI, CA-074 Me (10 µM) or Z-VAD-FMK. **p = 0.0017 and 0.0005 for CA-074 Me and Z-VAD-FMK, respectively. All results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

7. Requirement of cathepsin B for cisplatin-induced IL-1β production. HK-1 cells transfected with cathepsin B-targeting and control siRNA for 48 h were treated with cisplatin. *p = 0.037. The results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

8. Requirement of NLRP3 for cisplatin-induced IL-1β production. HK-1 cells transfected with NLRP3-targeting, AIM2-targeting, RIG-I-targeting and control siRNA for 48 h were treated with cisplatin. *p = 0.029. The results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

9. Enhanced induction of tumour microenvironmental factor-stimulated IL-1β by therapeutic treatments. HK1 cells were treated with tumour microenvironmental factors, EBV gDNA/EBERs/ATP/H₂O₂ as previously described with or without therapies, irradiation and cisplatin. IL-1β production was used to measure inflammasome activity. *p = 0.00004, the results are presented as the mean ± SD of four independent experiments and analysed by Student's t test.

Figure 5. Inhibition of growth by tumour-derived IL-1β in vivo

1. Tumour growth of IL-1β-expressing HK1 cells in a xenograft model. Nude mice were injected with HK1-vector or HK1-IL-1β cells (n = 9 per group). *p = 4.6E−04, 6.2E−06, 4.0E−06, 4.1E−07, 5.4E−09, 5.0E−08, 1.0E−06, 5.3E−07 and 4.3E−07 at day 15, 18, 22, 25, 29, 32, 36, 39 and 43 post-inoculation, respectively. All results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

2. Tumour growth of IL-1β- and pro-IL-1β-expressing B16F10 cells in a syngeneic mouse model. Wild-type mice were injected with B16F10-vector, B16F10-pro-IL-1β and B16F10-IL-1β cells (n = 12 per group). *p = 1.1E−06, 1.5E−06, 6.9E−03, 9.7E−04 and 5.7E−03 at day 29, 32, 36, 39 and 43 post-inoculation, respectively. All results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

3. Tumour growth of B16F10-vector and B16F10-IL-1β cells in wild-type and Il1r1^−/− mice (n = 4–6 per group). All results are presented as the mean ± SD of three independent experiments.

4. Dose effect of IL-1β secretion on tumour growth inhibition. IL-1β in supernatants from B16F10-IL-1β cells premixed with B16F10-vector cells by indicated ratio (left panel). Tumour growth of B16F10-IL-1β mixed cells in a syngeneic mouse model (middle panel, n = 8 per group). Correlation between the tumour sizes at day 43 and IL-1β secretion of B16F10-IL-1β premixed cells (right panel). *p = 0.022 (1/16 IL-1β vs. vector); **p = 8.5E−04 (1/4 IL-1β vs. vector) and 1.2E−05 (IL-1β vs. vector). All results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

5. Survival rate in mice bearing IL-1β- and pro-IL-1β-expressing B16F10 cells. Syngeneic mice were injected with B16F10-vector, B16F10-pro-IL-1β or B16F10-IL-1β cells (n = 12 per group) and survival curves were plotted using the Kaplan–Meier method and compared using the log-rank test. *p = 0.009. The results were obtained from three independent experiments.

6. Survival rate in mice with surgical removal of primary tumours. B16F10-vector, B16F10-pro-IL-1β and B16F10-IL-1β tumours were established and then surgically removed after 14 days (n = 12 per group) and survival curves were plotted using the Kaplan–Meier method and compared using the log-rank test. *p = 0.036. The results were obtained from three independent experiments.

Figure 6. TANs as important effector cells for the IL-1β-mediated antitumour activity

1. Leukocyte subsets in tumours. Percentage of leukocytes (CD45⁺), myeloid cells (CD11b⁺), neutrophils (CD11b⁺/Ly6G⁺), macrophages (CD11b⁺/F4/80⁺), dendritic cells (CD11c⁺), T cells (CD3⁺), B cells (B220⁺) and NK cells (NK1.1⁺) in the B16F10-IL-1β-, B16F10-pro-IL-1β- or B16F10-vector-bearing tumours (n = 3 per group) was determined by flow cytometry. *p = 0.0364, 0.0222 and 0.0004 for leukocytes, myeloid cells and neutrophils, respectively. The results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

2. Induction of intratumoural neutrophils by IL-1β. Percentage of TANs (CD11b⁺/Ly6G⁺) in the B16F10-IL-1β and B16F10-vector-bearing tumours (n = 3 per group) in wild-type and Il1r1^−/− mice was determined by flow cytometry. *p = 0.003. The results are presented as the mean ± SD of three independent experiments and analysed by Student's t test.

3. Increased TANs in B16F10-IL-1β tumour. Ly6G immunohistochemistry performed on tumour tissues. Scale bars, 100 µm.

4. The morphology of TANs. Neutrophils were sorted from CD11b⁺/Ly6G⁺ cells in B16F10-vector or B16F10-IL-1β tumours. Scale bars, 10 µm.

5. Quantitative RT-PCR analysis. The fold change of gene expression in purified neutrophils (CD11b⁺/Ly6G⁺) of B16F10-IL-1β- and B16F10-vector-tumours, using the expression level in TANs from B16F10-vector-tumours as the denominator, was calculated (n = 3 per group). The results were obtained from three independent experiments and analysed by Student's t test.

6. Neutrophil depletion. Mice bearing B16F10-IL-1β- or B16F10-vector-tumours were injected with either the anti-Ly6G 1A8 or a control IgG antibody intraperitoneally twice a week (n = 4 per group). After 2 weeks, the percentages of TANs (CD11b⁺/Ly6G⁺) in whole tumour cells were determined by flow cytometry. *p = 0.005. The results are presented as the mean ± SD of four independent experiments and analysed by Student's t test.

7. Effect of neutrophil depletion on tumour growth. Mice bearing B16F10-IL-1β- or B16F10-vector-tumours were injected with antibodies described in (F) intraperitoneally (arrowheads) twice a week during the study period (n = 7–9 per group). *p = 0.006, 0.003 and 0.002 at day 39, 43 and 46 post-inoculation, respectively. All results are presented as the mean ± SD and analysed by Student's t test. Each experiment was repeated at least twice.

8. TANs in NPC tumours. The H&E results are shown at 400× magnification (upper panel) and the enlarged box areas (lower panel). Arrowhead indicates the TANs. Scale bars, 50 µm.

9. Kaplan–Meier survival analysis of LRFS.

10. Correlation of TANs and pro-IL-1β and inflammasome components in NPC biopsies. The positive rates of TANs in patients with high expression levels of AIM2, RIG-I, NLRP3, ASC, caspase-1 and IL-1β versus the positive rates of TANs in patients with low expression levels of the above-described proteins are analysed using Student's t test (Supporting Information Table S13).

Figure 7. Role of inflammasomes in tumour cell responses to microenvironmental factors and therapy

Before treatment, tumour cells are latently infected with EBV and express LMP1, which can induce pro-IL-1β production through NF-κB and MAPK signalling. In the tumour microenvironment, tumour cells encounter PAMPs (EBV gDNA and EBV-encoded RNAs, and EBER1 and EBER2) and DAMPs (intracellular ROS or H₂O₂, and extracellular ATP) produced by dying or stressed cells. The PAMPs and DAMPs can stimulate AIM2, RIG-I and NLRP3, which then form inflammasomes with ASC and caspase-1, resulting in cleavage of pro-IL-1β and low-level basal IL-1β secretion. Low levels of tumour-derived IL-1β can facilitate tumour growth. Treatment of NPC patients with irradiation leads to ROS production that leads to the release of nuclear and mitochondrial DNA into the cytosol of tumour cells, while chemotherapy (cisplatin) causes cathepsin B to be released from ruptured lysosomes due to cisplatin accumulation. Thus, AIM2 and NLRP3 in tumour cells are further activated to secret IL-1β at higher levels. Finally, high levels of tumour-derived IL-1β achieved by therapeutic treatment can inhibit tumour growth and local relapse by recruiting neutrophils, especially immunostimulatory N1 TANs.