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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: Cancer Immunol Res. 2019 Apr 24;7(6):874–885. doi: 10.1158/2326-6066.CIR-18-0698

IL-1R8 Deficiency Drives Autoimmunity-Associated Lymphoma Development

Federica Riva 1,2, Maurilio Ponzoni 3, Domenico Supino 2, Maria Teresa Sabrina Bertilaccio 4,8, Nadia Polentarutti 2, Matteo Massara 2, Fabio Pasqualini 2, Roberta Carriero 2, Anna Innocenzi 3, Achille Anselmo 2, Tania Veliz-Rodriguez 4, Giorgia Simonetti 4,9, Hans-Joachim Anders 5, Federico Caligaris-Cappio 4,10, Alberto Mantovani 2,6,7, Marta Muzio 4,*, Cecilia Garlanda 2,6,*
PMCID: PMC7176492  EMSID: EMS86230  PMID: 31018956

Abstract

Chronic inflammation, including that driven by autoimmunity, is associated with development of B-cell lymphomas. IL-1R8 is a regulatory receptor belonging to the IL-1R family, which negatively regulates NF-κB activation following stimulation of IL-1R or Toll Like Receptor (TLR) family members. IL-1R8-deficiency is associated with the development of severe autoimmune lupus-like disease in lpr mice. We herein asked if concomitant exacerbated inflammation and autoimmunity caused by the deficiency of IL-1R8 could recapitulate autoimmunity-associated lymphomagenesis. We thus monitored B-cell lymphoma development during aging of IL-1R8-deficient lpr mice, observing increased lymphoid cell expansion that evolved to Diffuse Large B-cell Lymphoma (DLBCL). Molecular and gene expression analysis showed that the NF-κB pathway was constitutively activated in Il1r8-/-/lpr B-splenocytes. In human DLBCL, IL-1R8 was expressed at low levels compared to normal B cells and higher IL-1R8 expression was associated with better outcome.

Thus, IL-1R8 silencing is associated with increased lymphoproliferation and transformation in the pathogenesis of B cell lymphomas associated to autoimmunity.

Keywords: IL-1R8, autoimmunity, diffuse large B cell lymphoma, NF-kB, TLRs

Introduction

The association between chronic inflammation and promotion of malignancy was first perceived in the nineteenth century (1) and is supported by epidemiological and mechanistic data (2,3). In particular, patients suffering from selected autoimmune or inflammatory conditions, such as systemic lupus erythematosus (SLE), rheumatoid arthritis and Sjogren’s syndrome are prone to develop lymphomas, namely B-cell Non Hodgkin’s Lymphomas (B-NHL) (4–7). The mechanisms triggering the transition from benign B-cell proliferation to malignancy are still defined only in part. Chronic inflammation, antigen stimulation and B-cell receptor signaling, associated with the inherent genetic instability of lymphocytes are known to play a central role in lymphoma development (8,9). More specifically, gain of function mutations of MyD88 and constitutive activation of NF-κB have recently emerged among the most frequently recurring mutations in B cell lymphoproliferative diseases (10).

Mice homozygous for the lymphoproliferation spontaneous inactivating mutation (Faslpr) show systemic autoimmunity, massive lymphadenopathy associated with proliferation of aberrant T cells, arthritis, and immune complex glomerulonephrosis (11). In humans, germline mutations in the Fas gene have been associated with autoimmune lymphoproliferative syndrome (ALPS) (12), and somatic Fas mutations have been found in multiple myeloma and B-NHL (4).

IL-1R8 (also known as TIR8 or Single Ig IL-1 related receptor, SIGIRR) is a member of the interleukin-1 receptor (IL-1R) family acting as a negative regulatory receptor (13). IL-1R8 inhibits NF-κB and JNK activation following stimulation of IL-1R or TLR family members by interfering with the recruitment of TIR domain-containing adaptor molecules (14–17). In combination with IL-18Rα, IL-1R8 also serves as one of the receptor chains for the anti-inflammatory cytokine IL-37, activating anti-inflammatory responses (18).

IL-1R8-deficiency leads to unleashed activation of IL-1R or TLR family members and is associated with exacerbated inflammatory responses (14,19), and autoimmunity (16,20–22). Accordingly, downregulation of IL-1R8 was observed in psoriasis (23). Depending on the context, IL-1R8 is involved in modulating either inflammation-associated tumorigenesis and tumor progression, including colorectal cancer (19,24,25) and chronic lymphocytic leukemia (CLL) (26), or NK cell-mediated anti-tumor immune responses in mouse models (27,28).

IL-1R8-deficiency in lpr mice was associated with severe lymphoproliferation and autoimmune lupus-like disease (16), due to increased dendritic cell (DC) activation and B cell proliferation in response to TLR7- and TLR9-activating autoantigens or nucleosomes (29,30).

The involvement of IL-1R8 in autoimmunity, and the critical role of constitutive activation of MyD88-dependent NF-κB activation in B cell transformation raised the hypothesis that IL-1R8 might be involved in the autoimmunity-associated risk of developing lymphoma. Here we show that IL-1R8-deficiency was associated with significantly earlier death and increased susceptibility to lymphoproliferation, which evolved in transplantable Diffuse Large B-cell Lymphoma (DLBCL). Analysis of clonality showed that multiple independent transformation events occurred in the same host. In humans, IL-1R8 was poorly expressed in DLBCL cell lines and primary lesions when compared to peripheral blood or germinal center B cells, and was associated with better outcome in terms of overall survival, suggesting that IL-1R8 downregulation is a driver of lymphomagenesis.

Material and Methods

Animals and samples

IL-1R8-deficient (Il1r8-/-) mice were generated as described (14) and backcrossed to the C57Bl/6J background (Charles River Laboratories, Calco, Italy) up to F11 generation. Il1r8-/- and B6lpr/lpr (Charles River Laboratories) were crossed to generate Il1r8-/-/lpr mice. Mice were housed in the SPF animal facility of Humanitas Research Hospital in individually ventilated cages. Mice were sacrificed at 12-18 months of age, unless they reached the established endpoints and organs were collected for histological and molecular analysis. Procedures involving animals have been conducted in accordance with, and with the approval of the Institutional Animal Care and Use Committee (IACUC) of Humanitas Research Hospital and Italian Health Ministry (authorizations 43/2012-B released on 08/02/2012 and 828/2015-PR released on 07/08/2015), in compliance with national (D.L. n.116, G.U., suppl. 40, February 18, 1992; D.L. n.26, March 4, 2014) and international law and policies (EEC Council Directive 86/609, OJ L 358,1,12-12-1987; EEC Council Directive 2010/63/UE; National Institutes of Health Guide for the Care and Use of Laboratory Animals, US National Research Council, 2011). All efforts were made to minimize the number of animals used and their suffering.

Histopathology and immunohistochemistry

5μm thick sections of formalin-fixed, paraffin embedded mouse tissues were stained with H&E. Based on lymphoid follicle morphology, a pathological score was attributed to the spleen and lymph nodes of each 10-12-month-old mouse analyzed (normal=0; reactive=1; reactive>atypical=2; atypical>reactive=3; atypical=4; atypical>lymphomatous=5; lymphomatous>atypical=6; lymphomatous=7). Slides were analyzed in blind by a certified hematopathologist (MP) and two investigators. The following antibodies were used: anti-B220 (RA3-6B2, Serotec), anti-Ki67 (SP6, Neo Markers), anti-CD3 (1F4, Biorad), anti-BCL6 (Rabbit polyclonal, Santa Cruz), anti-BCL2 (C21, Santa Cruz), anti-Multiple Myeloma 1/Interferon Regulatory Factor 4 protein (MUM1/IRF4) (3E4, Biolegend) (31).

Tumor transplantation

107 cells (5x106 splenocytes plus 5x106 lymph node cells) from 10-12-month-old Il1r8-/-/lpr (n=8) or Il1r8+/+/lpr (n=7) mice were injected ip, sc or iv into C57Bl/6J, nude or SCID mice. Recipient animals were sacrificed when clinical signs (enlargement of mandibular lymph nodes or abdomen) were evident or 12-20 months after transplantation and organs were collected for histological and molecular analysis. The genotype of the cells from the lesions developed in recipient mice was analyzed for lpr and Il1r8 mutations by PCR (14).

Western blot analysis of purified B-cell lysates (30 μg total proteins) was performed with the following antibodies: anti-p100/p52 (CS4882), anti-Phospho-p65 (CS3036), anti-p65 (CS8242) (Cell signaling); anti-beta-actin-HRP (SIGMA A3852), using precast gels.

Real-Time PCR and Real-Time PCR array

Total RNA from mouse spleen purified B cells, DLBCL cell lines and B cells from healthy donor buffy coats was isolated with a column-based kit followed by DNAse treatment (Promega) (for PCR array) or TRI Reagent (Sigma-Aldrich) (for PCR).

RNA was retrotranscribed and cDNA used for gene expression analysis by Real-Time PCR and Real-Time PCR array (Biorad Prime PCR ARRAY code:10034381).

Real-time PCR was performed in QuantStudium 7 Flex (Applied Biosystems, Thermo Fisher) or 7900 Sequence Detection System (Applied Biosystem), in duplicate using Power Sybr Green PCR Master Mix (Applied Biosystem) and primers (300 nM) in MicroAmp optical 96-well plates (25μl). The following primer pairs were purchased from Invitrogen: Nfkbiz for 5’-GCGCTCTCGTATGTCC-3’; Nfkbiz rev 5’-AGACTGCCGATTCCTC-3’; GAPDH for 5’-GCAAAGTGGAGATTGTTGCCAT-3’; GAPDH rev 5’-CCTTGA CTGTGCCGTTGAATTT-3’ (28); human IL-1R8 For: 5’-CCGACCTTTTGG TGAACCTGA-3’; human IL-1R8 Rev: 5’-TGGCCCTCAAAGGTGATGAAG-3’; Universal actin For: 5’-CCCAAGGCCAACCGCGAGAAGAT-3’; Universal actin Rev: 5’-GTCCCGGCCAGCCAGGTCCAG-3’. Experiments were repeated at least twice. The expression of the target gene was normalized using GAPDH or β-actin cDNA expression of the same sample and run, and reported as 2^(-deltaCT).

For Real-Time PCR array, the analysis of 84 NF-κB signaling target genes was performed as described (32). Data were reported as 2^(-deltaCT), relative to the average of 6 housekeeping genes. To note, the specific assay for Fas mRNA expression is designed within the exons 1 and 2, and it recognizes both wild type and mutant Fas (33).

IgH gene rearrangement analysis

IgH gene rearrangement was investigated by Southern blot analysis of genomic DNA from different organs of a 20-month-old wild type mouse transplanted sc with total lymph node and spleen cells collected form a 11-month-old Il1r8-/-/lpr mouse. DNA (5 μg) was extracted from spleen, lymph nodes and solid lesions (100 mg each), digested with EcoRI or StuI and subsequently hybridized with a 32P-labeled DNA probe PJ3 representing the JH4 region of the IgH locus, as described (34).

Cell culture

B cells were isolated from 100x106 splenocytes using a B cell isolation kit (Miltenyi Biotec), plated in 48 well plate at 1x106/ml and cultured overnight. Human DLBCL cell lines SU-DHL-2 (ATCC CRL-2956), SU-DHL-4 (ATCC CRL-2957), SU-DHL-6 (ATCC CRL-2959), SU-DHL-8 (ATCC CRL-2961), RC-K8 (DSMZ ACC-561), and RIVA (RI-1; DSMZ ACC-585) were grown in RPMI or IMDM (RIVA, RC-K8) medium supplemented with 10-20% FCS, 2mM L-glutamine and 100U/ml streptomycin.

Flow cytometric analysis

Mouse spleen B cells overnight cultured in medium or in presence of LPS (100 ng-1 μg/ml, Sigma-Aldrich) were incubated with anti-mouse CD86 (GL1, eBioscience) and anti-mouse CD19 (1D3, BD Bioscience) antibodies and analyzed by FACS canto II (Becton Dickinson, Franklin Lakes, NJ, USA).

IL-1R8 cell surface staining on human cells was performed with biotinylated goat anti-human IL-1R8/SIGIRR (R&D Systems), followed by Alexa-647 conjugated streptavidin (Molecular Probes, Invitrogen), and analyzed with FACS Canto I flow cytometer (BD Bioscience). Diva software (BD Pharmingen) and Flow-jo (Tree Star) were used for data acquisition and analysis, respectively.

Analysis of IL-1R8 and IL-37 expression in human DLBCL

Public gene expression data of DLBCL were retrieved from GEO. In the first study (GSE43677) samples of naïve B cells (n=8), germinal center (GC) B cells (n=13), postGC B cells (n=9), tonsils (n=10), and DLBCL (n=12) were analyzed. In the second study (GSE32018) gene expression profiling was conducted in a series of B-cell non-Hodgkin lymphoma patients (17 CLL, 22 DLBCL, 23 Follicular Lymphoma (FL), 24 Mantle Cell Lymphoma (MCL), 15 marginal zone lymphoma-MALT type (MALT), 13 Nodal Marginal Zone Lymphoma (NMZL)) and 7 freshly frozen lymph nodes. Differential expression analysis was performed using limma (version 3.26.8) (35). For prognosis evaluation, expression and clinical data of 98 DLBCL cases selected among 220 lymphoma samples (GSE4475) were used. DLBCL patients treated with radiotherapy were excluded.

The Gene Set Enrichment Analysis (GSEA) software was used to perform the over-representation analysis with gene sets coming from the Molecular Signature Database (36). The entire datamatrix containing normalized gene expression values (log-scale) was used and the expression profile of IL-1R8 gene was tested as continuous phenotype label. The analysis was performed choosing the Pearson correlation as the metric to investigate gene sets enriched by genes correlated with the expression profile of IL-1R8. The Reactome database, belonging to the C2 collection (c2.cp.reactome.v6.1) was used. Resulting gene sets were considered significantly enriched according to the False Discovery Rate (FDR) threshold of 5%.

Statistical analysis

Statistical differences in mouse mortality and lymphoma incidence rates were analyzed with Mantel-Cox test and Fisher test, respectively. Mann Whitney test or Student's T test with Welch's correction were performed as specified. Survival analysis of human DLBCL was performed using Kaplan-Meier and Mantel-Cox tests. The median gene expression value was used to classify patients into IL-1R8low and IL-1R8high or IL-37low and IL-37high gene expression groups. A P value < 0.05 was considered statistically significant. Statistical analysis was performed using Graph Pad Prism software (Graph Pad Software, San Diego, CA).

Results

IL-1R8-deficiency is associated with severe lymphadenopathy and lymphoma in lpr mice

We previously observed that 6-month-old Il1r8-/-/lpr mice were affected by enhanced lymphoproliferation and lymph follicle hyperplasia compared to Il1r8+/+/lpr mice (16). In order to address whether this benign lymphoproliferation eventually evolved to malignancy, we analyzed survival and followed the evolution of lymphoid organs in older animals. As shown by survival curves reported in Fig. 1A, Il1r8-/-/lpr mice reached the endpoints earlier than Il1r8+/+/lpr mice, and mortality was 100% (23/23) at 15 months of age in Il1r8-/-/lpr mice compared to 22% (6/27) in Il1r8+/+/lpr mice (P=0.0001, Mantel-Cox test). Splenomegaly and lymphadenomegaly were dramatic in 10-12-month-old Il1r8-/-/lpr mice compared to Il1r8+/+/lpr mice of the same age (Fig.1B). The spleen weight was significantly increased in both groups compared to wild type or Il1r8-/- mice, and in Il1r8-/-/lpr mice it was significantly higher than in Il1r8+/+/lpr mice (Fig. 1C).

Figure 1. IL-1R8 deficiency increases the severity of the lymphoproliferative disorder of lpr mice.

Figure 1

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A) Mortality rate at 15 months of age in Il1r8−/−/lpr mice (100%; n=23) and in Il1r8+/+/lpr mice (22%; n=27) (Mantel-Cox test P<0.0001). B) Spleen (lower panel) and mandibular lymph nodes (upper panel) from 10-12-month-old Il1r8+/+, Il1r8−/−, Il1r8+/+/lpr, Il1r8−/−/lpr mice. C) Spleen weight of 10-12-month-old Il1r8+/+ (n=6), Il1r8−/− (n=6), Il1r8+/+/lpr (n=30), Il1r8−/−/lpr (n=33) mice (unpaired Student’s t-test with Welch’s correction; mean and SD are indicated).

Histopathological analysis of the spleen of 12-14-month-old Il1r8-/-/lpr mice showed an enlargement of the white pulp and a complete loss of the normal architecture of the organ in most animals (Fig. 2A). In the spleen of 12-14-month-old Il1r8+/+/lpr mice, we observed a moderate enlargement of the white pulp, but the architecture of the organ remained recognizable despite the presence of enlarged germinal centers (Fig. 2A). Similarly, most (62.5%; 20/32) lymph nodes from 10-12-month-old Il1r8-/-/lpr mice presented abnormal histological architecture, without any evident follicle (Fig. 2B). In contrast, lymph nodes from 10-12-month-old Il1r8+/+/lpr mice were enlarged, but generally retained a preserved normal morphology of the follicles (Fig. 2B). As shown in Fig. 2C and 2D, the pathological score of lymphoid follicles (based on the presence of normal, reactive, atypical, or lymphomatous follicles) was significantly higher in 10-12-month-old Il1r8-/-/lpr mice compared to 12-14-month-old Il1r8+/+/lpr mice (p=0.0001 in spleen and p=0.0022 in lymph nodes), and the diagnosis of lymphoma was mostly limited to Il1r8-/-/lpr mice. Splenic and lymph nodal plasmacytosis occurred in spleen and lymph nodes of both Il1r8+/+/lpr and Il1r8-/-/lpr mice, in agreement with the role of TLR ligands and autoantigens in inducing cellular differentiation into mature plasma cells and plasma cell expansion (37,38).

Figure 2. IL-1R8 deficiency is associated with increased susceptibility to lymphoma development in lpr mice.

Figure 2

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A, B) Histopathological analysis of the spleen (A) and lymph nodes (B) of 10-12-month-old Il1r8+/+, Il1r8−/−, Il1r8+/+/lpr, Il1r8−/−/lpr mice stained with H&E (400x; Axioskop 40 microscope equipped with AxioCam MRc camera and AxioVision Rel. 4.8 acquisition software; Zeiss). C, D) Pathological score of the spleen (C) and lymph nodes (D) of 10-12-month-old Il1r8+/+ (n=2), Il1r8-/- (n=2), Il1r8+/+/lpr (n=20), Il1r8-/-/lpr (n=26) mice (unpaired Student’s t-test; mean and SD are indicated). E) Incidence of DLBCL in Il1r8+/+/lpr (3/23) and Il1r8-/-/lpr (13/26) mice (Fisher test).

Development of Diffuse Large B Cell Lymphoma in Il1r8-/-/lpr mice

Histopathological analysis showed that a diffuse organ replacement by large cells in spleen and lymph nodes consistent with a diagnosis of lymphoma occurred in 13/26 Il1r8-/-/lpr mice and 3/23 Il1r8+/+/lpr mice (P= 0.0073, Fisher test) (Fig. 2E). In 8 out of 13 mice carrying lymphoma (61.5%), large cells were observed in liver, lung, kidney, and gut, indicating the development of Diffuse Large B Cell Lymphoma (DLBCL). Immunostaining for B220 highlighted a diffuse infiltration by large neoplastic B-cells (Fig. 3A). CD3-positive T cell occurred haphazardly without the classical distribution within and around follicles of the splenic white pulp in lymphoma-bearing Il1r8-/-/lpr mice (Fig. 3B). Neoplastic B lymphocytes were relatively monomorphic, with large nuclei and abundant cytoplasm; within this population, high mitotic rate as well as diffusely elevated Ki67 immunoreactivity confirmed the dramatically increased proliferation rate of these lymphomas (Fig. 3C). In addition, neoplastic B cells were immunoreactive for Bcl-2, suggesting an activation of an anti-apoptotic machinery, and negative for Bcl-6 and MUM1 (Fig. 3D, 3E and 3F, respectively). In Il1r8-/-/lpr lymph nodes, we observed lesions with similar features and in few cases (3/13, 23%), bone marrow of Il1r8-/-/lpr mice showed small and focal areas of DLBCL.

Figure 3. Il1r8-/-/lpr mice develop DLBCL lesions.

Figure 3

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Immunohistochemical analysis of: B220 (A), CD3 (B) and Ki67 (C) in the spleen of 10-12-month-old Il1r8+/+, Il1r8-/-, Il1r8+/+/lpr, Il1r8-/-/lpr mice (40x). Immunohistochemical analysis of: bcl-2 (D), bcl-6 (E) and MUM-1 (F) in the spleen of 10-12-month-old Il1r8-/-/lpr mice (400x; Axioskop 40 microscope equipped with AxioCam MRc camera and AxioVision Rel. 4.8 acquisition software; Zeiss).

Rarely, in Il1r8+/+/lpr mice older than 12 months, DLBCL was diagnosed as well (3/23, 13 %). The vast majority of Il1r8+/+/lpr old mice actually showed an irregular enlargement of germinal centers with predominance of intermediate cells, without fulfilling the required criteria for follicular lymphoma; in addition, the presence of few, scattered large B220+ cells was consistent with atypical, lymphoproliferative, potentially pre-neoplastic disorder (Fig. 3A, 3B and 3C).

Il1r8-/-/lpr DLBCL are transplantable and oligoclonal

In order to further prove the malignant capability of lesions developing in aged Il1r8-/-/lpr and Il1r8+/+/lpr mice, splenocytes and lymph node cells were injected through different routes in immunodeficient or immunocompetent mice. Irrespectively of the immunocompetence of the recipient mouse and route of injection, after 4-20 months of observation, cells from 4 out of 8 Il1r8-/-/lpr mice generated histologically-proven parental DLBCL in recipient mice (Fig. 4A). In contrast, mice injected with cells collected from 7 Il1r8+/+/lpr mice did never develop lymphoma. Genotyping for lpr and Il1r8 gene modifications by PCR analysis of genomic DNA of recipient’s organs affected by lymphoma (spleen, lymph nodes) and control tissues (skeletal muscle) confirmed that malignant cells originated from the Il1r8-/-/lpr donor (Fig. 4B).

Figure 4. DLBCL lesions are transplantable and oligoclonal.

Figure 4

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A) Histopathological analysis of the spleen of a 12-month-old Il1r8-/-/lpr donor mice compared to the spleen of Il1r8+/+ recipient mice stained with H&E (Axioskop 40, Zeiss, 200 and 400x). B) Genomic analysis by PCR of lpr and Il1r8 targeted genes in organs of recipient mice 6.5 months after transplantation with Il1r8-/-/lpr spleen and lymph node cells. C, D) Southern blot analysis of Ig genes shows rearrangement and oligoclonal expansion of B cells in recipient mice of Il1r8-/-/lpr spleen and lymph node cells. Genomic DNA from different organs and tissues of the recipient animal was digested with EcoRI (C) or StuI (D). Yellow arrows indicate clonal bands.

Since in Il1r8-/-/lpr mice lymphoma lesions infiltrated more than one lymphoid organ, we next assessed whether these lesions originated from a common B cell clone. Southern blot analysis was performed to detect immunoglobulin (Ig) heavy chain gene (IgH) rearrangements in DLBCL developed in different organs of a recipient wild type mouse transplanted with Il1r8-/-/lpr splenocytes and lymph node cells. The analysis revealed bands of IgH rearrangement of different size in the spleen, lymph nodes and other organs indicating that multiple B cell clones were transformed in the donor mouse (Fig. 4C-D).

These results indicate that lymphomas of Il1r8-/-/lpr mice can be transplanted in wild type recipient mice giving rise to lymphoma.

Constitutive activation of the NF-κB pathway in Il1r8-/-/lpr B cells

Hyper-activation of the NF-κB pathways and overexpression of NFKBIZ are hallmarks of a sub-type of Diffuse Large B-cell lymphoma in humans (39–41). IL-1R8 dampens NF-κB activation induced by TLR and IL-1R family members (42) and Fas mutations impact on B-cell activation (16). In addition, we previously showed that IL-1R8-deficiency significantly increased B cell proliferation upon exposure to RNA and DNA immune complexes and other TLR agonists (16). To further investigate the NF-κB pathway in B cells of Il1r8-/-/lpr old mice, we analyzed NF-κB activation and the expression of specific NF-κB regulated genes in CD19+ cells purified from the spleen of 12-month-old Il1r8+/+, Il1r8-/-, Il1r8+/+/lpr and Il1r8-/-/lpr mice, in resting conditions or after stimulation with LPS. Non-canonical and canonical NF-κB activation can be monitored by the cleavage of p100 (Nfkb2) into p52 fragment, and phosphorylation of p65 (RelA), respectively. In contrast to wild type mice, we observed activation of non-canonical NF-κB pathway in both Il1r8+/+/lpr and Il1r8-/-/lpr mice, while the canonical pathway appeared mostly activated in the Il1r8+/+/lpr mice (Fig. 5A-C). We did not observe any significant difference in Il1r8-/- mice at this time point compared to Il1r8+/+mice in the absence of ex vivo stimulation.

Figure 5. Deregulated NF-κB activation in Il1r8+/+/lpr and Il1r8-/-/lpr mice.

Figure 5

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A) Western blot of spleen B cells with the indicated antibodies. β-actin expression was analyzed as internal control. B, C) Densitometric signal ratios of p52/p100 and phospho-p65/p65 shown in panel A (Mann Whitney test; * = P-Value<0.05). D) Real-Time PCR array of NF-κB signaling target genes. Expression data are shown only for the genes for which a Fold Difference (FD) >2 was observed in at least one comparison between two groups of mice (see supplementary Table 2 for individual data). In the graph, a two-color scale formatting scheme was used to format cells: red is the maximum level of expression; blue, minimum. Each column represents one sample (from one mouse). E) Nfkbiz mRNA expression in purified B cells (unpaired Student’s t-test with Welch’s correction). F) FACS analysis of CD86 expression in overnight cultured purified B cells in basal condition and after LPS stimulation. One representative experiment out of 2 performed is shown (unpaired Student’s t-test with Welch’s correction). B, C, E, F: mean and SD are shown.

Next, we analyzed by Real-time PCR array 84 genes known to be targets of the NF-κB signaling pathway (Supplementary Table 1). We compared the results obtained from non-stimulated B cells collected from 4 wild type, 4 Il1r8-/- mice, 5 Il1r8+/+/lpr mice and 5 Il1r8-/-/lpr mice (Fig. 5D and Supplementary Table 2). 14 genes were deregulated in at least one group, with 13 upregulated and only one downregulated as compared to wild type animals, again suggesting constitutive hyperactivation of this pathway in Il1r8+/+/lpr and Il1r8-/-/lpr B cells from aged mice. In detail, most of the NF-κB targets were upregulated in both Il1r8+/+/lpr and Il1r8-/-/lpr mice, including pro-inflammatory genes (e.g. Il1b, Ifng, Csf1, Stat1, Il12b) and genes associated with proliferation or anti-apoptosis (e.g. Ccnd1) (Supplementary Table 2). The Bcl2a1a gene coding for an antiapoptotic protein necessary for cell transformation and growth in anaplastic lymphoma (43) was specifically downregulated in lpr mice (fold difference =0.47 and p=0.04 as shown in Supplementary Table 2), while IL-1R8 deficiency did not influence its expression (fold difference =0.93; p=0.8) and restored its levels in Il1r8-/-/lpr mice (fold difference =1.08 and p=0.8 as shown in Supplementary Table 2).

We then analyzed a secondary response gene prototypically induced by TLRs and regulated by NF-κB, namely Nfkbiz. In basal conditions we observed low levels of Nfkbiz mRNA in B-cells isolated from wild type mice; notably, Nfkbiz was significantly induced in Il1r8-/- mice, and this induction was sustained in Il1r8-/-/lpr mice (Fig. 5E) suggesting that a TLR-dependent NF-κB secondary response is constitutively activated in Il1r8-/- and Il1r8-/-/lpr mice.

In agreement with deregulated activation of the NF-κB pathway, flow cytometry analysis showed that overnight-cultured spleen-purified Il1r8-/-/lpr B cells expressed higher levels of CD86, an activation marker downstream of TLR activation (44,45), compared to wild type, Il1r8−/− and Il1r8+/+/lpr B cells, both in basal conditions and after LPS stimulation (Fig. 5F).

Taken together, these results show that both Fas and IL-1R8-deficiency contribute to constitutive deregulated NF-κB signaling and increased B cell activation with few distinct differences characteristic associated with IL-1R8 deficiency (e.g. Nfkbiz). Interestingly, the double mutation rendered the cells highly responsive to TLR activation in terms of CD86 expression.

IL-1R8 expression is downmodulated in human DLBCL cells and correlates with prognosis

To assess the relevance of these results to human disease, we first studied the expression of IL-1R8 in human lymphoma cell lines compared with normal circulating mature B cells. As shown in Fig. 6A and B, all DLBCL cell lines analyzed expressed lower levels of IL-1R8 mRNA and protein, respectively, compared to peripheral blood B cells.

Figure 6. IL-1R8 is downmodulated in human lymphoma cell lines.

Figure 6

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A) Real-Time PCR analysis of IL-1R8 mRNA expression (unpaired Student’s t-test of each line vs normal B cells with Welch’s correction; mean and SD are indicated; the highest P value is reported) and B) Flow cytometric analysis of IL-1R8 protein expression in human lymphoma cell lines compared to circulating B (CD19+) cells from 3-4 healthy donors (unpaired Student’s t-test of each line vs normal B cells; mean and SD are indicated; the highest P value is reported); 3-4 experiments were performed with each cell line. Results are expressed as Arbitrary Units (A) and as relative fluorescence intensity of the isotype control (B).

C) Normalized log intensity of IL-1R8 probe (218921_at; GSE43677) in DLBCL versus normal B cells (naive B cells, germinal center (GC) B cells, post GC B cells and tonsil samples). D) Normalized log intensity of IL-1R8 probe (A_23_P84344; GSE32018) in DLBCL vs FL, MCL, MALT, NMZL, CLL and lymph node samples. E) Kaplan-Meier survival curve of DLBCL patients (n=98) with low and high IL-1R8 gene expression (218921_at probe) within DLBCL specimens (HR=2.2; 95% C.I. 1.2-3.8; P=0.006).

Next, we studied IL-1R8 expression in public gene expression data of DLBCL retrieved from GEO, comparing different normal resting and activated B cell populations and lymphomas. In the first study analyzed (GSE43677), the expression of IL-1R8 resulted significantly down-regulated in DLBCL samples vs naive B cells (logFC=-0.43, Adj. P-value=1.08E-04), GC B cells (logFC=-0.21, Adj. P-value=1.70E-02), postGC B cells (logFC=-0.9, Adj P-value=1.12E-09), tonsils (logFC=-0.62, Adj. P-value=3.64E-08) (Fig. 6C). The second study (GSE32018) showed a significant down-regulation of IL-1R8 expression in DLBCL vs lymph node control samples (logFC=-1.34, Adj. P-value=1.65E-02), but also vs FL, an indolent form that may transform into DLBCL (logFC=-0.66, Adj. P-value=3.45E-02) (Fig. 6D).

In a third study (GSE4475), the expression of IL-1R8 was analyzed together with clinical data to evaluate a correlation with prognosis. DLBCL patients were divided into IL-1R8low and IL-1R8high based on the median gene expression. The resulting Kaplan-Meier curve showed that patients with IL-1R8 expression above the median value had a significant advantage in terms of overall survival (Hazard Ratio (HR)=2.2 (95% C.I. 1.2-3.8); P-value=0.006) (Fig. 6E). In addition, the GSEA analysis retrieved a total of 60 pathways significantly enriched by genes positively correlated with IL-1R8 gene expression profile (Supplementary Table 3). Among these, the apoptotic process and the DNA damage response were two of the most enriched pathways (NES=2.02, FDR q-val=0.005 for the apoptosis process; NES=1.85, FDR q-val=0.01 for the P53-dependent G1 DNA damage response) with a total of 70 and 29 genes, respectively, belonging to the core enrichment (Supplementary Table 4 and Table 5). These results show a positive co-regulation of apoptotic process and DNA damage response genes and our gene of interest, suggesting a putative activation of the apoptotic process and DNA damage response in IL-1R8 high expression profiles respect to those with low expression.

Since IL-1R8 is required for the anti-inflammatory activity of IL-37 in inflammatory conditions triggered by TLR ligands (18,46), we finally investigated whether IL-1R8 and IL-37 were co-regulated in DLBCL. In the GSE43677 and GSE32018 studies, the expression of IL-37 was significantly down-regulated in DLBCL samples compared to normal B cells (logFC=-0.18, Adj. P-value=3.73E-02 for naïve B cells, logFC=-0.25, Adj. P-value=1.65E-03 for GC B cells) or FL cells (logFC=-0.37, Adj. P-value=4.30E-03), respectively, similarly to IL-1R8. However, in contrast to what observed for IL-1R8, the overall survival was not affected by IL-37 expression in the GSE4475 study (HR=0.6 (95% C.I. 0.4-1.1); P-value=0.1), indicating that the regulatory role of IL-1R8 impacts on additional pathways.

These results indicate that IL-1R8 is poorly expressed in DLBCL compared to healthy GC B cells and other B cell lymphomas and that lower IL-1R8 expression is associated with shorter overall survival.

Discussion

IL-1R8 is known to act as a negative regulator of NF-κB and JNK activation following stimulation of IL-1R family members or TLRs. We herein show that the increased susceptibility to lymphoproliferation observed in lpr mice deficient of IL-1R8, is also associated with frequent development of DLBCL. The aggressive lymphomas developing in Il1r8-/-/lpr mice were transplantable and oligoclonal, possibly originating from multiple B cell clones. In addition, we show that IL-1R8 expression is down-regulated in human DLBCL cells in comparison with peripheral blood, GC B cells and other lymphomas, and correlates with overall survival, suggesting that IL-1R8 silencing in DLBCL might contribute to deregulated NF-κB activation, a frequent occurrence observed in DLBCL, lymphoproliferation and transformation.

FAS-deficient lpr mice are a model of ALPS and SLE. FAS is a pro-apoptotic TNF receptor superfamily member, highly expressed on GC B cells. Mutations in the genes encoding FAS or its ligand (FASL) cause massive accumulation of autoreactive B and T cells, resulting in ALPS in humans (12). In addition, FAS mutation has been found associated with perforin deficiency in one case of ALPS and lymphoma (47), whereas in mice, increased lymphoma development was observed in SPARC-deficient lpr mice (48). In a previous study, we demonstrated that IL-1R8-deficiency was associated with a more severe lymphadenopathy at 6 month of age in FAS-deficient lpr mice (16). This phenotype was mainly due to over-activation of DC, B cells and CD4+ T cells upon stimulation with lupus autoantigens, possibly through TLR7 engagement (16). Indeed, chromatin antigens in immune complexes can potently engage both the BCR and TLRs in B cells, leading to over-stimulation and defective apoptosis of B cells, as well as to secondary inflammation (5). FAS-mutations have also been observed in human lymphomas, indicating that longer lymphocyte survival may allow accumulation of additional oncogenic events (4).

In addition to these autoimmunity-dependent mechanisms, genetic alterations affecting components of the NF-κB signaling pathways have been shown to occur frequently in DLBCL. Constitutive NF-κB pathway activity is observed in almost all activated B-cell-like (ABC) type of DLBCL and in a large fraction of germinal center B-cell (GCB)-DLBCL, and is liked to proliferation, differentiation, and survival of malignant lymphoid cells (49,50). Among mutations of the NF-κB signaling pathway in B cell lymphomas, MYD88 mutations have recently emerged as one of the most frequently recurring (40). MyD88 is an adaptor protein that mediates TLR and IL-1R signaling. Gain of function mutations of MYD88 confer a cell survival advantage during evolution of DLBCL by promoting NF-κB and JAK/STAT3 signaling (40). IL-1R8 tunes TLR and IL-1R-dependent signaling by interfering with the recruitment of TIR-containing adaptor molecules (51) and IL-1R8-deficiency in mice is associated with uncontrolled inflammatory responses both in infectious and sterile conditions (42). Furthermore, genetic inactivation of IL-1R8 was observed to cause earlier, more disseminated and aggressive leukemia in the Eμ-TCL1 mouse model of CLL (26). In this model, the neoplastic transformation of B cell has an incidence of 100% and is mediated by the overexpression of the TCL1 oncogene; the absence of IL-1R8 exacerbated CLL progression, but its impact on the B cell transformation was not investigated (26). Finally, IL-1R8 in association with IL-18Rα serves a receptor chain for IL-37, an anti-inflammatory cytokine, which provides an anti-inflammatory environment in the aging bone marrow, preventing oncogenic transformation of B cell progenitors (52). Our results show that Il1r8+/+/lpr mice spontaneously developed DLBCL at a very low frequency and at late age (12-18 months), and IL-1R8-deficiency increased the frequency and the severity of the disease and anticipated this occurrence at 8-12 months of age, indicating that IL-1R8 has also a role in neoplastic transformation of B cells, and not only in the progression of established B cell leukemia or lymphoma.

The pathogenesis of lymphoma seen in patients with autoimmune diseases is complex and involves different factors contributing to lymphomagenesis, including both disease activity and immunosuppression, as well as disease-specific mechanisms and mechanisms unique to lymphoma subtype (8). With our study, we showed that the Il1r8-/-/lpr mouse model recapitulates autoimmunity-associated lymphomagenesis, suggesting that the absence of a negative regulator of the ILR- or TLRMyD88 axis in an autoimmune-prone background is sufficient for the neoplastic transformation of B cells. These results are in line with data in humans, showing that aggressive B-cell lymphomas (particularly DLBCL) are more frequently associated with autoimmune conditions than more indolent lymphomas (particularly FL) (4). It is important to note that IL-1R8-deficiency in this model is not restricted to B cells and might also impact on anti-tumor immunity, as shown in other models (27,28). Therefore, our results may underestimate the effect of selective IL-1R8-deficiency in tumor B cells.

Western blot analysis of p52 and phospho-p65 demonstrated that both canonical and non-canonical NF-κB pathways are constitutively activated in Il1r8+/+/lpr mice. Moreover, the TLR-induced NF-κB-regulated Nfkbiz gene is constitutively activated in Il1r8-/- mice. A combination of these pathways may contribute to the activation of distinct NF-κB target genes and B cell activation observed in lymphoma prone Il1r8-/-/lpr mice. We previously observed that Il1r8-/-/lpr B cells had increased proliferation rate after stimulations with autoantigens acting through TLR7 and TLR9 and other TLR ligands, compared to lpr B cells (16). In the present manuscript, we described a high mitotic rate and diffusely elevated Ki67 immunoreactivity in Il1r8-/-/lpr spleen, indicating increased proliferation rate, associated with immunoreactivity for Bcl-2, suggesting activation of an antiapoptotic machinery. Thus, the results presented here are in line with the view that the lack of a tuner of TLR and IL-1R signaling-dependent NF-κB activation could impinge upon B cell transformation in a context of lymphoproliferative syndrome. Indeed, other oncogenic events circumventing negative feedback mechanisms that attenuate NF-κB signaling, such as inactivation of the deubiquitinase A20, are associated with autoimmunity and lymphoma development (40,53).

DLBCL developing in Il1r8-/-/lpr mice was characterized by the presence of a monomorphic population of large B cells in lymphoid tissues. Neoplastic B cells displayed high proliferation rate and showed widespread involvement of distant organs including gut, liver, lung, and kidney. Histopathologic analysis and immunostainings for CD3, B220, Bcl-2, and Ki67 of spleen and lymph node specimens of Il1r8-/-/lpr mice documented sharply separated masses constituted by DLBCL arising within a background represented by atypical lymphoproliferative disorder. Excessive lymphoproliferation associated with activation of anti-apoptotic mechanisms were potentially responsible of multiple independent transformation events resulting in polyclonal (or oligoclonal) development of different primary foci of DLBCL, as suggested by the detection of different bands of IgH rearrangement in the same mouse or in the same organ. Our results suggest that FAS-deficiency was responsible of polyclonal B cell expansion and very rarely lymphoma transformation, and the addition of the deficiency of IL-1R8 causing hyperactivation of the MyD88-NF-κB axis, reasonably in response to autoantigens, resulted in tumors that resemble human ABC DLBCL. In Il1r8-/-/lpr mice, these tumors emerge sporadically and thus are likely to have acquired the additional oncogenic hits necessary to give rise to DLBCL.

In the present study, we showed that the expression of IL-1R8 in human DLBCL is downmodulated compared to peripheral blood or GC B cells from healthy donors, and correlates with overall survival. The molecular mechanisms underlying IL-1R8 downmodulation in human DLBCL are still undefined, and could include promoter methylation, as observed in human gastric carcinomas (54), alternative splicing leading to aberrant protein expression, as described in colorectal cancer (55), or promoter hypo-acetylation as suggested by the analysis of hematological cancer cell-lines from public available datasets (Ensembl, UCSC Genome Browser). In addition, it was recently reported that genomic methylation affected IL-1R8 expression since Azacytidine treatment of CLL cell lines restored IL-1R8 mRNA levels (56). Very rarely, non-sense and somatic non-synonymous mutations have been observed in IL-1R8 coding sequence as emerged by Whole Exome Sequencing data from The Cancer Genome Atlas (TCGA) and in sequenced samples within DLBCL patients (Dalla-Favera R., personal communication), but the functional consequences of these mutations or polymorphisms need to be investigated. The apoptotic process and DNA damage response resulted among the pathways significantly enriched by genes positively correlated with IL-1R8 gene expression, suggesting that higher IL-1R8 expression might be associated with increased apoptotic activity and better control of DNA damage, and as a consequence, with a less aggressive phenotype of lymphoma cells, thus leading to better prognosis.

Patients affected by DLBCL show different clinical courses, making difficult to predict a prognosis and program successful therapy, leading to only 50% of curable patients (57). Our results demonstrate that IL-1R8 activity limits B-cell activation and malignant transformation induced by autoimmune stimulation and contribute to the identification of genes and molecular pathways that could represent targets for novel therapeutic approaches in DLBCL treatment.

Supplementary Material

Supplemental material includes Supplementary Table 1, 2, 3, 4, 5.

Supplementary Tables

Finacial support

The study was supported by the European Commission (ERC project PHII-669415; FP7 project TIMER HEALTH-F4-2011-281608), Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) (project PRIN 2015YYKPNN; project FIRB RBAP11H2R9), Associazione Italiana Ricerca sul Cancro (AIRC IG 19014 to AM and AIRC 5x1000 9962 to AM and CG; AIRC IG 16777 and 13042 to MMu; AIRC 5x1000 9965 to MMu and FCC), CARIPLO (project 2010-0795 to FCC, MMu and CG), the Italian Ministry of Health (Ricerca Finalizzata, RF-2013-02355470 to CG) and the Deutsche Forschungsgemeinschaft (AN 372/24-1 and 27-1 to HJA).

Footnotes

Authorship

Contribution: F.R, F.C-C., A.M, M.Mu., and C.G. conceived the study; F.R., M.P., D.S., S.B., M.Ma., A.I., H.J.A. and M.Mu. designed and/or conducted experiments, performed data analysis and interpretation, and informed study direction; F.P., N.P., A.A., T.V-R., and G.S. helped with experimental work; R.C. performed bioinformatics analyses; F.R., M.Mu. and C.G. drafted the manuscript; and all authors discussed the results and commented on the manuscript.

Conflict-of-interest disclosure: All the authors declare no competing financial interests.

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