Genes and Immunity (2007) 8, 577589 & 2007 Nature Publishing Group All rights reserved 1466-4879/07 $30.

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ORIGINAL ARTICLE

Differential expression in lupus-associated IL-10 promoter single-nucleotide polymorphisms is mediated by poly(ADP-ribose) polymerase-1
EY Chung1,2,3, J Liu1,3, Y Zhang1 and X Ma1,2
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY, USA and 2Graduate Program in Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sciences, Cornell University, New York, NY, USA
1

Systemic lupus erythematosus (SLE) is a complex, multifactorial autoimmune disease characterized by the dysregulation of T and B cells that leads to hyperactivity of B cells and production of autoantibodies, and involves both environmental and genetic factors. Interleukin-10 (IL-10) is a candidate susceptibility gene in SLE. In particular, three IL-10 promoter singlenucleotide polymorphisms (SNPs; 1082A/G, 819T/C and 592A/C) are strongly associated with the pathogenesis of SLE. We found that the homozygous GCC haplotype linked to greater SLE severity confers higher IL-10 gene transcriptional activity than the ATA haplotype in macrophages that encounter apoptotic cells, because of the differential DNA binding to the 592 SNP by a nuclear protein uniquely induced by apoptotic cells. We identified this protein as poly(ADP-ribose) polymerase-1, confirmed its physiological role and characterized its molecular properties in modulating IL-10 production during phagocytosis of apoptotic cells. This study unveils a novel direct link between DNA damage repair/apoptosis pathways and IL-10-mediated immune regulation. Genes and Immunity (2007) 8, 577589; doi:10.1038/sj.gene.6364420; published online 16 August 2007
Keywords: lupus; IL-10; SNP; PARP-1; phagocytosis; apoptotic cells

Introduction
Development of systemic lupus erythematosus (SLE) involves both environmental and genetic factors. The heritability of SLE is supported by increase in concordance rate in identical twins,1 frequency of SLE in first-degree relatives,2 and risk of SLE in siblings of SLE patients.3 Disease development is the result from the accumulation of a series of events that involve the interaction between environmental and genetic factors.4,5 A number of susceptibility genes, such as HLA, mannose-binding lectin gene, C4A, FcgR, tumor-necrosis factor-a (TNF-a), programmed cell death 1 gene (PDCD1) and interleukin-10 (IL-10), have been associated with SLE among various populations.611 IL-10 is a pleiotropic cytokine produced by both T/B cells and macrophages and possesses both anti-inflammatory and immunosuppressive properties.12 Investigations in numerous inflammatory disease models including chronic enterocholitis, cutaneous inflammatory condition, endotoxic shock and Shwartzman reaction, and autoimmune encephalomyelitis in IL-10-deficient mice have
Correspondence: Professor X Ma, Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA. E-mail: [email protected] 3 These individuals are considered co-first authors. Received 26 March 2007; revised 7 May 2007; accepted 13 July 2007; published online 16 August 2007

yielded strong evidence that IL-10 plays a central role in vivo in restricting inflammatory responses.1317 However, IL-10 also processes immunostimulatory effects that have not attracted sufficient attention. IL-10 is a potent growth factor for B lymphocytes. It promotes B-cell proliferation, antibody production and class II expression.18 The B-cell-stimulating property of IL-10 is thought to be the basis of several antibody-mediated autoimmune disorders.19 Accumulating evidence suggests that IL-10 is a strong candidate gene in SLE susceptibility. Firstly, it maps to human chromosome 1q3132, which is a susceptibility region for SLE (logarithm of odds (LOD) 3.79).20 It is also homologous to a murine SLE susceptibility region.21 Secondly, IL-10 is known to be an important immunoregulatory cytokine. It enhances B-cell survival, proliferation, differentiation and autoantibodies production,22 properties that could render IL-10 a causal factor for the hyperactivity of B cells in SLE. Thirdly, high IL-10 production has been observed in B cells and macrophages from SLE patients in vitro,23 and increased serum IL-10 levels were observed in SLE patients and have been shown to be associated with disease activity.24,25 In NZB/ W F1 lupus-prone mice, T-cell cytokine imbalance toward production of interferon-g (IFN-g), and IL-10 is associated with autoantibody levels and nephritis.26 Fourthly, continuous administration of anti-IL-10 antibodies in this model delayed the onset of lupus-like autoimmunity and improved the survival rate from 10 to

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80%, through upregulation of endogenous TNF-a.27 Conversely, continuous administration of IL-10 accelerated the onset of autoimmunity in these mice. Collectively, the above-described evidence suggests that increased IL-10 levels may play a role in SLE pathogenesis by causing hyperactivity of B cells and autoantibody production. IL-10 production is under strong genetic influence,28 and controlled at the transcriptional level.29 The 50 flanking region of the IL-10 gene, which regulates transcription, is polymorphic.30 In particular, three such single-nucleotide polymorphisms (SNPs) have been identified and shown to influence IL-10 production levels. They are: 1082 G, 819 C, 592 C (GCC); 1082 A, 819 C, 592 C (ACC); and1082 A, 819T, 592 A (ATA), in correlation with decreasing IL-10 expression levels.3133 The SNP at position 1082 is within a putative e-twenty-six (ETS)-like transcription factor-binding site.34 The SNP at 592 is located in a region that mediates negative regulatory function,34 whereas the SNP at 819 may affect an estrogen receptor element.35 In a study that involved 76 Caucasian patients with SLE and 119 controls, no significant change in the allele frequency of the three IL-10 gene promoter dimorphic polymorphisms in the SLE group compared with controls was found. However, when subgrouped according to autoantibody status and clinical features, 1082G, 819C and 592C alleles were increased in patients possessing Ro autoantibodies and those with renal involvement.34 These alleles are in preferential allelic association, namely GCC, ACC and ATA haplotypes, and the GCC haplotype was increased in these patient subgroups. A recent larger scale study examining the association of six IL-10 promoter SNPs (3575T/A, 2849G/A, 2763C/A, 1082A/G, 819T/C and 592A/C) with SLE in 554 Hong Kong Chinese SLE patients and 708 ethnically matched controls revealed that the homozygous genotype of high IL-10 production haplotypes was significantly increased in these SLE patients. Another study in a large population of Chinese patients with lupus nephritis showed a strong association of the 592A/C SNP with the disease activity and renal pathology of lupus nephritis.36 Taken together, these studies strongly suggest that SNPs within the IL-10 gene promoter that are associated with high IL-10 levels may contribute significantly to the development of certain clinical features in SLE. Multicellular organisms have evolved genetic and epigenetic mechanisms of programmed cell death (apoptosis) to eliminate cells that are no longer needed or have become damaged. Physiological apoptosis has an essential role in development, differentiation and tissue homeostasis.37 The elimination of apoptotic cells and cell bodies by phagocytes represents an evolutionarily conserved means to prevent exposure of surrounding tissue to potentially cytotoxic, immunogenic or inflammatory cellular content.38,39 Resolution of inflammation depends not only on the removal of apoptotic cells but also on active suppression of inflammatory mediator production. Aberrations in either mechanism are associated with chronic inflammatory conditions and autoimmune disorders.4042 Uptake of apoptotic cells by phagocytes is thought to suppress autoimmune responses through the release of anti-inflammatory cytokines IL-10, transforming growth factor-b, platelet-activating factor and pros-

taglandin E2, and inhibition of proinflammatory cytokines TNF-a, granulocyte-macrophage colony-stimulating factor, IL-12, IL-1b and IL-18.4345 Our group recently has shown that during phagocytosis by macrophages, apoptotic cell-derived signals trigger dephosphorylation and activation of a novel nuclear zinc finger-like protein, named GC-binding protein, which targets a specific site in the IL-12 p35 gene promoter, preventing its transcription.46 In human SLE, impaired phagocytosis of apoptotic material by macrophages has been reported.47,48 providing an explanation for increased levels of early apoptotic cells, DNA and nucleosomes observed in the circulation of SLE patients.4952 The impaired clearance of apoptotic cells resulting in an accumulation of late apoptotic and secondary necrotic cells including oligosomes might lead to an activation of autoreactive T and B cells.44 At the genetic level, how these IL-10 promoter SNPs affect IL-10 gene expression has long evaded elucidation. We therefore undertook this study to investigate the molecular mechanism whereby the 1082A/G, 819T/ C and 592A/C promoter haplotypes confer differential IL-10 gene expression in macrophages in response to apoptotic cells. The rationale for the choice of apoptotic cells as a stimulus for IL-10 production in this study was based on the observed involvement of both cellular apoptosis and high IL-10 levels in the development and pathogenesis of SLE, which compelled us to reason that there may be a connection between phagocytosis of apoptotic cells and differential IL-10 production predicated on the promoter haplotypes.

Results
Apoptotic cells induce IL-10 production in macrophages To assess the ability of macrophages to produce IL-10 during phagocytosis of apoptotic cells, we prepared murine peritoneal macrophages and treated them with apoptotic Jurkat T cells or with lipopolysaccharide (LPS) for various times. As shown in Figure 1a, both apoptotic cells and LPS potently stimulated IL-10 secretion by macrophages after 4 h. However, the kinetics were somewhat different in that LPS-stimulated IL-10 production gradually increased during the 48 h period whereas apoptotic cell-induced IL-10 reached a peak around 8 h followed by slow decreases. In contrast, TNF-a production was only stimulated by LPS, not by apoptotic cells (Figure 1b). Similar IL-10-inducing activity of apoptotic cells was observed also in primary human monocytederived macrophages that had ingested apoptotic cells (Figure 1c). Differential response of GCC- and ATA-IL-10 promoter haplotypes to apoptotic cells To understand the regulation of IL-10 gene transcription in apoptotic cell-stimulated macrophages, we used a well-established transient system in the murine macrophage-like cell line RAW264.7 and a human IL-10 promoterluciferase reporter construct containing the region between 1104 and 30 upstream of the IL-10 transcription initiation site. To determine the effect of IL-10 promoter SNPs on their response to apoptotic cells, three versions of this construct were engineered to reflect the three SNPs of interest: ACC, GCC and ATA at the

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Figure 1 Induction of IL-10 production by apoptotic cells. (a, b) A total of 1 106 thioglycolate-elicited mouse peritoneal macrophages (C57BL6) were stimulated with LPS (0.5 mg/ml) or apoptotic Jurkat cells (ac) (2:1 ratio of apoptotic cells/macrophages). Supernatants were harvested at various times as indicated after stimulation and analyzed for the production of IL-10 (a) and TNF-a (b) by ELISA. (c) The same assay was performed with primary human monocyte-derived macrophages. The data represent mean7s.d. of three individual experiments. ELISA, enzyme-linked immunosorbent assay; IL-10, interleukin-10; LPS, lipopolysaccharide; TNF-a, tumor necrosis factor-a.

1082, 819 and 592 positions. These constructs are identical otherwise. After transient transfection of these reporter constructs, RAW264.7 cells were stimulated with apoptotic cells. As shown in Figure 2a, the ACC and GCC haplotype IL-10 promoter constructs exhibited very similar responses to apoptotic cells, whereas the ATA construct was significantly less responsive to apoptotic cells, by B30% compared to the GCC haplotype. This result suggests that 1082A/G is not crucial for the differential transcriptional response to apoptotic cells. The non-essential nature of the 1082A/G SNPs in conferring haplotype-specific differential IL-10 gene transcription induced by apoptotic cells was confirmed by deleting this region from the IL-10 promoter. Without the 1082 site, the differential transcriptional response of the IL-10 promoter bearing the two downstream SNPs remained (Figure 2b). The degree of difference we observed in IL-10 transcriptional activity between the two haplotypes is highly similar to the B33% difference in concanavalin A-stimulated IL-10 production of human peripheral blood lymphocytes in vitro in the general population reported in the original study.33 We also compared LPS-stimulated IL-10 transcription in these cells with the same three constructs. As shown in Figure 2c, like in the apoptotic cell response, the GCC construct was also more responsive to LPS than the ATA haplotype. Interestingly, unlike the apoptotic cell response, the ACC construct was less responsive to LPS than the GCC type. Since the only difference between the two constructs is the A/G at 1082, this result strongly suggests that the 1082 SNP confers differential LPS response while having no effect on the apoptotic cell

response of the IL-10 promoter. In contrast, the 592 SNP confers differential apoptotic cell response while being indifferent to LPS response. Particularly, 1082G confers higher transcriptional activity on the IL-10 promoter than 1082A. The critical importance of 1082G in enhancing LPS response was confirmed by its deletion, which diminished the ability of the remaining IL-10 promoter sequence containing 819C and 592C to respond to LPS compared with the same construct bearing 819T and 592A (Figure 2d). Differential DNA binding to the 592 SNPs by a novel nuclear factor induced by apoptotic cells Underlying the differential responses of the ATA and GCC promoter haplotypes to apoptotic cells, there might be differential binding by transcription factors to these sites that drive IL-10 gene transcription to different degrees. We performed electrophoretic mobility-shift assays (EMSA) using nuclear extracts from macrophages exposed to apoptotic cells and oligonucleotide probes spanning the three polymorphic regions with one nucleotide difference at each of the SNPs. As shown in Figure 3a, strong and equivalent binding activities were observed at both the 1082G and 1082A haplotype sites in unstimulated cells (lanes 2 and 5, indicated by arrows), and these activities were strongly reduced by apoptotic cells (lanes 3 and 6). In contrast, very little constitutive binding activities were observed at both 819C/T and 592C/A sites (lanes 8, 11, 14 and 17), whereas specific binding activities were strongly induced by apoptotic cells at these sites (lanes 9, 12, 15 and 18, indicated by *). Little difference in binding was
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Figure 2 Differential response of IL-10 promoter haplotypes to apoptotic cells. (a) The ACC, GCC and ATA haplotype IL-10 promoter constructs in the backbone of 1104/ 30 sequence were linked to the firefly luciferase reporter gene and transiently transfected into RAW264.7 cells. Transfected cells were incubated with no (medium), apoptotic (ac), live (lc) or necrotic (nc) Jurkat cells (2:1 ratio) for 7 h. Luciferase activities were measured from cell lysate. Transfection efficiency was normalized to the expression of a co-transfected LacZ plasmid. Results represent mean7s.d. of three individual experiments. P-value was obtained by Students t-test. (b) The same experimental approach was applied to the analysis of the 819C/592C and 892T/592A haplotype IL-10 promoter constructs in the backbone of 1044/ 30 sequence lacking the upstream 1082 SNP. Ac, apoptotic cells. (c) Response of the ACC, GCC and ATA haplotype IL-10 promoter constructs to LPS. These three constructs were transiently transfected into RAW264.7 cells and stimulated with LPS (1 mg/ml) for 24 h before harvesting for luciferase activity measurement. Results represent three separate experiments each with quadruplets (s.d. is shown). (d) The 819C/592C and 892T/592A haplotype IL-10 promoter constructs used in (b) were analyzed for their LPS response as described in (c). Results represent two separate experiments each with quadruplets (s.d. is shown). IL-10, interleukin-10; LPS, lipopolysaccharide.

observed at the 819 SNPs, either to the C- or Tcontaining sequence under the apoptotic cell-stimulated condition (compare lanes 9 and 12), whereas a consistently greater binding activity was observed at the 592A SNP than at the 592C sequence (compare lanes 15 and 18). Moreover, this binding activity appears rather unique to apoptotic cells because neither necrotic cells nor LPS could induce it (Figure 3b). Since the binding activities with the 819C/T and 592C/A probes were very similar in terms of mobility, we performed Supershift and competitive EMSA to further characterize them (Figure 3c). These experiments are summarized as follows: (i) this binding activity was competed off most efficiently by itself (592A), much less by 592C, or other oligos (819C, 819T and NFY); (ii) supershift EMSA revealed that the binding activity induced by apoptotic cells is unlikely related to NFY, Sp1 or Ets1 and Ets2 despite some resemblance of the sequence to the consensus binding sites of these transcription factors (data not shown). Human primary monocyte-derived macrophages also exhibited similarly preferential binding activities to
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the 592A site than at the 592C site (Figure 3d, right panel). These preferential binding activities were not observed with the 819C or T probes (Figure 3d, left panel). Given that the ATA haplotype is less active transcriptionally than the GCC haplotype, we hypothesize that this novel binding activity at 592A may represent a negative regulatory factor for IL-10 gene expression. Thus, this apoptotic cell-induced DNA-binding activity was designated as XR. DNA ligand-mediated pull-down assay for XR and its biochemical identification We carried out a series of biochemical experiments to identify XR by DNA affinity binding followed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis analysis. As shown in Figure 4a, a band of B24 kDa was uniquely present in nuclear extracts derived from apoptotic cell-exposed macrophages (pointed to by a black arrow that was bound to 592A and 592C, but not in cells unexposed to apoptotic cells or exposed to necrotic cells. This band was excised and analyzed by

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Figure 3 Differential DNA binding to the 592 SNPs by a novel nuclear factor induced by apoptotic cells. (a) Nuclear extracts were isolated from non-stimulated (M) or apoptotic cell (A)-stimulated RAW264.7 cells. EMSA was performed with the 1082G/A, 819C/T and 592C/ A haplotype probes. The apoptotic cell-induced binding activity is indicated by *. (b) EMSA was performed as described for (a) with nuclear extracts also isolated from necrotic cell-stimulated (N)- or LPS-stimulated (L) RAW264.7 cells. (c) Competitive EMSA was performed with nuclear extracts from non-stimulated (M) or apoptotic cell (A)-stimulated RAW264.7 cells and the 592A probe. Unlabeled (cold) competitors were used at 50 times molar excess over the labeled probe. (d) EMSA was performed with nuclear extracts from apoptotic cellstimulated RAW264.7 (RAW) cells and human peripheral blood monocyte-derived macrophages (Mf). EMSA probes used were the 819C/T and 592C/A haplotype sequences. EMSA; electrophoretic mobility-shift assay; FP, free probe; LPS, lipopolysaccharide.

mass spectrometry (MS) (Figure 4b). The result revealed, by a prominently high search score, that it was a cleavage product of Poly(ADP-ribose) polymerase-1 (PARP-1). The identity of PARP-1 was further confirmed by Western blot analysis using a commercial polyclonal antibody directed toward the N-terminal PARP-1 (Figure 4c). It appeared that the 592A probe bound to uncleaved PARP-1 to varying degrees under all three conditions (medium, apoptotic and necrotic cells), whereas only the apoptotic cell-stimulated macrophages displayed binding of the cleaved PARP-1 (pointed to by an arrow). Consistent with this identification, EMSA analysis of primary human macrophages using several specific antiPARP-1 antibodies resulted in significantly reduced XR binding to the 592A probe (lanes 57, Figure 4d). The fact that these antibodies only reduced XR binding rather than changing the mobility of XR suggests that PARP-1 may be the major component, if not the only component, of XR. Thus, we have identified PARP-1 as the key component of XR which binds differentially to the 592C and 592A haplotypes.

PARP-1 transcriptionally represses IL-10 gene expression PARP-1 is a nicotinamide adenine dinucleotide (NAD )dependent nuclear enzyme that detects and repairs damage to DNA in response to genotoxic stress. It is a 113-kDa protein composed of an N-terminal DNAbinding domain, containing two zinc-finger motifs, a C-terminal NAD -binding domain, catalyzing the synthesis of ADP-ribose polymers from its substrate, NAD , and an automodification site, which links the N- and C-terminal domains.53 During apoptosis, PARP-1 is cleaved by caspase 3, resulting in the N-terminal 24-kDa DNA-binding fragment and the C-terminal 89 kDa catalytic fragment. This cleavage is important for the regulation of inflammatory responses by PARP-1.54 It has been shown that the 24-kDa fragment can act to compete against the full-length PARP-1 and inhibit DNA repair, ADP-ribose polymer formation and damagedependent upregulation of transcription.55,56 We investigated the direct role of PARP-1 in the regulation of IL-10 gene transcription by using a chemical inhibitor of PARP-1 activity, 3-aminobenzamide (3-AB)57 and by overexpressing PARP-1. As shown in
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Figure 4 DNA pull-down assay, PAGE and MS analyses. (a) DNA pull-down assay was performed with complementary biotinylated oligonucleotides encompassing the 592A/C-binding sites, as described in Experimental Procedures. Eluted proteins were separated by 10 or 12% SDS-polyacrylamide gel. The gel was visualized by silver staining. This experiment was repeated two more times independently with identical results. Med, medium; nc, necrotic cell; ac, apoptotic cell. (b) Mass spectrometric analysis. The two gel slices were analyzed by MS. The highest scored protein (75.49) is shown here. Altogether, five peptides were sequenced between the two gel slices that hit the same protein, PARP-1. (c) Western blot analysis. The same samples that had been through the procedure described in (a) were subject to Western blot analysis using PARP-1 (A-20), a goat polyclonal antibody raised against a peptide mapping at the N terminus of PARP-1 of mouse origin (Santa Cruz Biotechnologies). The intact (113 kDa) and cleaved (24 kDa) PARP-1 products are indicated by an * and a black arrow, respectively. This analysis was performed twice. (d) EMSA was performed using nuclear extract isolated from primary human monocytederived macrophages following exposure to apoptotic Jurkat cells, and the 592A probe. Various antibodies (1 mg each) were also added to the binding reaction: control goat and rabbit IgGs, and three PARP-1-specific antibodies from Santa Cruz Biotechnologies. Lane 1 contains free probe (FP), and lanes 27 contain nuclear extracts (2.5 mg each). PARP-1-binding is pointed by an arrow. EMSA, electrophoretic mobilityshift assay; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis; PARP-1, poly(ADP-ribose) polymerase-1; SDS, sodium dodecyl sulfate.

Figure 5a, inhibiting PARP-1 activity with 3-AB dose dependently increased IL-10 promoter activity induced by apoptotic cells. Both GCC and ATA promoter types responded to the drug, although the latter type responded more at lower concentration of the inhibitor. Conversely, when PARP-1 was overexpressed by cotransfection with a PARP-1-expression vector,58 both the GCC and ATA promoters activities were inhibited dose dependently (Figure 5b). This inhibition was not seen with an enzymatically inactive mutant of PARP-1 (with a point mutation, E988K, in the catalytic domain)59 on the GCC promoter whereas the mutant PARP-1 enhanced the ATA promoter activity. It suggests that inhibition of IL-10 transcription requires the enzymatic activity of PARP-1 and that this mutant may have dominant-negative effect on the endogenous wild-type PARP-1. It should also be pointed out 5 mM 3-AB is generally regarded as the upper limit of the chemical for target specificity beyond which nonspecific effects are observed.60 Importantly, the enhancement of IL-10 transcriptional activity by the use of 3-AB in RAW264.7 cells was also observed to a very similar degree in primary human monocytes stimulated with apoptotic cells (Figure 5c). Consistent with the enhanced promoter activity, use of the inhibitor 3-AB reduced PARP-1-binding activities
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at 592C and 592A to the degree similar to their transcriptional enhancement (Figure 5d), supporting a major role of PARP-1 in the inhibition of IL-10 transcription through direct physical interaction. The fact that the use of 3-AB only reduced PARP-1 binding, as opposed to altering its mobility, again suggests that PARP-1 may be the major component of XR whereas other potentially present components do not contribute significantly to the mobility of the complex. PARP-1-deficient macrophages display enhanced IL-10 production induced by apoptotic cells To investigate further the physiological role of PARP-1 in the regulation of IL-10 production induced by apoptotic cells, we carried out experiments with primary macrophages derived from PARP-1-deficient mice. Compared to control wild-type mice, PARP-1-knockout (KO) macrophages were not impaired in phagocytosis of apoptotic cells (Figure 6a), and were equally responsive to blockade of phagocytosis by cytochalasin D, an inhibitor of actin polymerization (Figure 6b). Their response to LPS was normal with respect to IL-10 production (Figure 6c), whereas their response to apoptotic cells was enhanced by B33%, which again was very similar to the enhancing effect of 3-AB in primary human monocytes (Figure 5c). These data demonstrate that PARP-1 is

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Figure 5 Role of PARP-1 in IL-10 transcription. (a) The GCC or ATA IL-10 promoterreporter construct described in Figure 2 was transfected into RAW264.7 cells by electroporation. Cells were treated with varying amounts of 3-AB as indicated with or without apoptotic cells for 7 h. Luciferase activity was measured from cell lysate. (b) The two haplotype IL-10 promoter constructs (reporter) were co-transfected with the wild-type (WT) PARP-1 (effector) or an enzymatically inactive mutant E988K at 1:1 and 2:1 molar ratios (effector to reporter). (c) Freshly isolated primary human monocytes were treated with apoptotic Jurkat T cells (1:2 ratio) for 24 h in the presence or absence of 5 mM 3-AB. Culture supernatant as harvested and analyzed for human IL-10 secretion by ELISA. Data represents mean of three individual donors with s.d. and P-value. The genetic background of the donors was not determined. (d) PARP-1 inhibitor decreases XR binding to 592C/A. EMSA was performed as described above using nuclear extract isolated from RAW264.7 cells after exposure to apoptotic Jurkat cells in the presence of 5 mM of 3-AB, and the 592C and 592A probes. FP, free probe; M, medium; A, apoptotic cells. (e) Quantification of PARP-1-binding activities in (d) by densitometric analysis. Results shown represent three independent experiments with s.d. 3-AB, aminobenzamide; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility-shift assay; IL-10, interleukin-10; PARP-1, poly(ADP-ribose) polymerase-1.

indeed a physiological regulator of IL-10 uniquely in response to apoptotic cells.

Discussion
In this study, we for the first time present strong molecular evidence that the differential apoptotic cellinduced IL-10 gene expression in individuals with the GCC or ATA promoter haplotypes is determined at the level of transcription mediated by PARP-1. Although a 30% difference in IL-10 production between the two promoter types seems small, it recapitulates the difference in human populations based on their genotypes.31 Moreover, chronically, these different levels of IL-10 could well impact on the sensitivity of the immune system to external and internal danger signals, especially given the presence of other significant SLE-associated factors. PARP-1 has been linked to multiple events for transcriptional regulation in development.6164 A large

body of evidence shows that PARP-1 is activated during the inflammatory response, contributing to tissue damage. Accordingly, pharmacological inhibition of PARP1 has shown therapeutic efficacy in animal models of inflammation such as ischemiareperfusion,60 chronic colitis,65 asthma,66 autoimmune encephalomyelitis,67 diabetes mellitus,68 and PARP/ mice are protected from endotoxic shock.69,70 Interestingly, in an ovalbumin sensitization model of asthma in mice, inhibition of PARP-1 with the potent water soluble inhibitor PJ-34 reduced the number of inflammatory cells (especially of neutrophils) in the bronchoalveolar lavage fluid. Accompanying the reduction in cellular infiltration was the reduction in the production of inflammatory cytokines TNF-a, IL-12, and the chemokine MIP-1. The level of IL-10 in the lung was slightly but significantly increased in PJ-34-treated asthmatic animals, but not those of IL-5 and IL-13.66 The degree of increase of IL-10 (B30%) was very similar to that seen in vitro in primary human monocytes in response to apoptotic cells in the presence of 3-AB (Figure 5c). This study provides strong physiological
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Figure 6 Apoptotic cell-induced IL-10 production in PARP-1 KO mice. Macrophages were elicited by thioglycolate from the peritoneal cavity of PARP-1 KO mice (Parp1tm1Zqw) and the control wild-type mice 129/SvImJ. (a) Phagocytosis assay was performed as described in Experimental Procedures. (b) Cytochalasin D was added at the indicated concentrations to macrophage cultures at the time of addition of apoptotic cells for 12 h. (c) IL-10 analysis by ELISA from peritoneal macrophages elicited in wild-type and PARP-1 KO mice after stimulation with LPS (0.5 mg/ml) or with apoptotic Jurkat cells (AC in 2:1 ratio) for 12 h. Data represent mean s.d. of three mice per group. ELISA, enzyme-linked immunosorbent assay; IL-10, interleukin-10; KO, knockout; LPS, lipopolysaccharide; PARP-1, poly(ADP-ribose) polymerase-1.

evidence that inhibiting PARP-1 activity in vivo can directly and specifically increase IL-10 production. The association of SLE with PARP-1 has long been suspected. The genetic loci located on chromosome 1 are associated with SLE in humans.71 Located in these loci are genes encoding TNFR2, complement component C1q, Fcg receptors, TCR z chain, HRES-1 (an endogenous retrovirus), and interestingly, IL-10 and PARP-1. In patients with SLE, the highly ordered signal transduction cascade of apoptosis is disturbed. SLE patients show reduced PARP activity. PARP cleavage products are mainly found in association with either antinuclear and/ or anti-dsDNA antibodies. Serum samples from SLE patients and other autoimmune diseases display antiPAR and anti-PARP autoantibodies.72 In particular, autoantibodies to the catalytic fragment of PARP-1 were found in the sera of nearly 50% patients with SLE whereas they were not present in the sera of patients with rheumatoid arthritis, systemic sclerosis or healthy donors.73,74 It is noted that in the standard supershift experiment in Figure 4d, all three antibodies caused a neutralizing effect rather than a supershifting one. This suggests that the antibodies likely interfered directly with the binding of PARP-1 to DNA by, for example, recognizing epitopes located within the region of the protein that interact with DNA. The A-20 and N-20 antibodies target epitopes in the N termini of PARP-1, where the DNA-binding
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domain is located. H-250, however, targets amino acids 7641014 at the C terminus of PARP-1 where the catalytic domain is located. Thus, how this antibody interferes with PARP-1 binding to DNA is not immediately apparent. During apoptosis, PARP-1 is cleaved by caspases to generate 89 and 24-kDa fragments. This cleavage is thought to be a regulatory event for cellular death. The biological significance of PARP-1 cleavage is shown in a PARP-1 knockin (PARP-1(KI/KI)) mouse model, in which the caspase cleavage site of PARP-1, DEVD(214), was mutated to render the protein resistant to caspases during apoptosis.54 Although PARP-1(KI/KI) mice developed normally, they were highly resistant to endotoxic shock and to intestinal and renal ischemia reperfusions, which were associated with reduced inflammatory responses in the target tissues and cells due to the compromised production of specific inflammatory mediators. The DNA pull-down assay (Figure 4c) showed that the 592A/C probe binds not only to cleaved PARP1 but also to the full-length protein. It appears that the fulllength protein is predominant in medium-stimulated macrophages and is downregulated in apoptotic cellstimulated macrophages. This is very relevant because it could explain the decreased levels of PARP1, previously described in SLE patients with high levels of circulating apoptotic cells. However, the binding activity of the

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uncleaved protein is not apparent in EMSA assays (Figure 3). It is our belief that both the intact and cleaved PARP-1 bind the DNA in EMSA. This assertion is based on the observation that the H-250 antibody recognizes amino acids 7641014 at the C terminus of the human PARP-1, which is not present in the cleaved 24-kDa PARP-1, suggesting that the intact PARP-1 is present in the complex (Figure 4d) (the C-terminal portion of the cleaved PARP-1 does not have a DNA-binding domain, thus unable to bind DNA on its own). We think the major DNA-binding complex is composed of the uncleaved PARP-1, and the addition of the small, cleaved PARP-1 would not alter the mobility of this complex in a native gel. In unstimulated cells, PARP-1 is intact with no cleavage. This intact PARP-1 still binds to the target DNA but with many other proteins such that the large complex(es) does not even enter the native gel in EMSA, being trapped in the well (see lanes 14 and 16 in Figure 3a). This is probably also why there are many proteins being pulled down by the target DNA (Figure 4a, lanes 1 and 4), and abundant intact PARP-1 could be seen in lanes 1 and 4 in Figure 4c. Upon stimulation by apoptotic cells, PARP-1 is cleaved and disassociated from the other factors. Then, their DNA binding becomes a resolvable complex visible in EMSAs, and their detection by SDSpolyacrylamide gel electrophoresis following DNA pull down becomes much less complex (lanes 1 and 4 in Figures 4a and c). It has been shown that the 24-kDa fragment can act to compete against the full-length PARP-1 and inhibit DNA repair, ADP-ribose polymer formation and damagedependent upregulation of transcription.55,56 Thus, it would be of interest to determine the relative role of the full-length PARP-1 vs its derivative with respect to IL-10 transcriptional regulation. There have been reports of decreased PARP-1 activity in SLE patients.72 Since our study shows that PARP-1 is a critical regulator of IL-10 gene expression in the context of SLE, it will be of significance to investigate the expression and activity of PARP-1 in SLE patients macrophages following phagocytosis of apoptotic cells, as a way to understand the genetic basis and pathophysiology of SLE. In a separate study, we have determined that IL-10 production stimulated by apoptotic cells is regulated primarily at the level of transcription in a manner dependent on the p38 mitogen-activated protein kinase, partially on the scavenger receptor CD36, and requires cellcell contact but not actual phagocytosis. Furthermore, we have also determined that the major promoter sequence element that mediates apoptotic cell-induced IL-10 transcription is localized at 106/98, which interacts with the homeodomain protein and Hox cofactor pre-B-cell leukemia transcription factor-1b (Pbx-1b) (manuscript submitted). However, this element does not dictate the haplotype-specific differential IL-10 gene transcription in individuals. It is possible that the cleaved PARP-1 could potentially originate from either macrophages, or apoptotic cells that have been ingested by the macrophages. Cleaved PARP1 from the uningested apoptotic cells is unlikely to defuse into macrophages. The experiment described in Figure 6, however, seems to argue that the active function of PARP-1 originates from the macrophages, instead of the apoptotic cells, since it is reversed in

PARP-1-deficient mice. The definitive experiment to address this question is to generate apoptotic cells from the PARP-1 KO mice and feed them to both wild-type and PARP-1 KO macrophages. If it is the cleaved PARP-1 from the apoptotic cells that regulates IL-10 production, then the PARP-1 status (sufficient or deficient) of the phagocytes would not matter. Conversely, if the cleaved PARP-1 comes from the phagocytes, then the PARP-1 status of the apoptotic cells should not make any difference. The second scenario implies that upon phagocytosis of apoptotic cells, macrophages may also undergo caspase activation that results in the cleavage of PARP-1. On the basis of these and other data, we propose a working model. We hypothesize that the major determinant of IL-10 production induced by apoptotic cells in phagocytes is located at 106/98 where the homeodomain protein Pbx-1 transactivates the IL-10 promoter in a p38 mitogen-activated protein kinase-dependent manner. The overproduction of IL-10 in certain SLE patients with the GCC haplotype IL-10 promoter SNPs, on the other hand, is due to their weaker interaction with the nuclear protein PARP-1 at 592. PARP-1, upon encountering apoptotic cells and perhaps other yet to be defined extracellular stress signals, acts as a transcriptional repressor of IL-10 gene expression by directly binding the 592A/C sequence, repressing IL-10 transcription. The binding of PARP-1 is differential at the 592 SNP such that it exerts weaker repression on the 592C sequence (weaker binding) than on the 592A sequence (stronger binding), thus allowing higher IL-10 production in the 592C haplotype. This differential IL-10 production in a chronic manner contributes to heightened autoreactive B-cell responses characteristic of SLE carrying the GCC polymorphism. Our findings here also have implications in tumor pathogenesis. For example, Burkitts lymphoma (BL) cells have an inherent tendency to undergo apoptosis at a high rate and significant macrophage infiltration. It has been shown that macrophages, regulated by IL-10, have the potential to promote BL pathogenesis, firstly, through suppression of antitumor immunity following enhanced engulfment of apoptotic tumor cells and, secondly, through increased production of tumor cell growth/ survival factors.75 In summary, this study reveals a novel physiological role of PARP-1 in the regulation of IL-10 induced uniquely by apoptotic cell-derived signals, and provides a clear molecular mechanism whereby individual IL-10 promoter haplotypes confer differential IL-10 production. Further investigation and elucidation of the molecular nature and characteristics of PARP-1-mediated immunoregulation will likely benefit the design of effective therapeutic strategies against a wide range of inflammatory and pathological conditions such as SLE, arthritis, asthma, sepsis, cardiomyopathy, stroke, inflammatory bowel disease, diabetes and cancer.

585

Materials and methods

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586

Harbor, ME, USA). RAW264.7 was obtained from American Type Culture Collection (Manassas, VA, USA). Jurkat, a leukemic human T-cell line, was kindly provided by Dr Gary Koretzky of the University of Pennsylvania (Philadelphia, PA, USA). Both cell lines were maintained in RPMI 1640 (Gibco, Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.2 mM L-glutamine (Cellgro, Mediatech; Hendon, VA, USA). Reagents and antibodies Staurosporine (Cayman Chemical, Ann Arbor, MI, USA) was used to induce apoptosis on Jurkat T cells, autologous human T cells and murine splenic CD4 T cells. 3-AB was purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies against PARP-1 and control antibodies were purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA, USA). Mouse peritoneal macrophages Three percent sterile thioglycolate broth (1 ml; Sigma) was injected into the peritoneal cavity of each mouse (n 3). Macrophages were harvested 4 days after injection. They were plated directly onto tissue culture plates containing Dulbeccos modified Eagles medium (Gibco), supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.2 mM L-glutamine (Cellgro). Human peripheral blood monocytes and macrophages Leukocytes from healthy human donors were purchased from the New York Blood Center. Preparation of monocytes and monocyte-derived macrophages was carried out as described previously.46 Induction of apoptosis and necrosis Jurkat T cells were the source of apoptotic cells. Staurosporine (0.5 mg/ml; Cayman Chemical) was added at (0.5 ng/ml) to T cells resuspended at (4 106 cells/ml) with complete RPMI 1640 (Gibco, Invitrogen). After incubation for 6 h at 371C in the presence of 5% CO2, the cells were harvested and washed three times with incomplete RPMI 1640. At this time, 65% of the population was Annexin V-positive and PI-negative as determined by fluorescence-activated cell sorter staining. Cell viability by trypan blue staining was 490%. Necrosis was generated by five times repeated freeze-thawing with o10% cell viability by trypan blue staining. Cell stimulation and measurement of cytokines by enzyme-linked immunosorbent assay Macrophages were plated at 0.5 105 cells/well in 96well culture plates (BD-Falcon, Franklin Lakes, NJ, USA). Cells were stimulated with LPS at (0.5 mg/ml) or with 1.0 105 apoptotic cells/well. Supernatants were centrifuged at 1000 r.p.m. for 10 min at 41C to remove particulate debris and were stored in aliquots at 801C. Human and mouse IL-10 and TNF-a OptEIAt ELISA sets were purchased from BD PharMingen (San Diego, CA, USA). Construction of luciferase reporter gene vectors The human IL-10 promoterluciferase construct pIL-10 (1044/ 30)-luc was generously provided by

Dr L Zaiegler-Heitbrock of the University of Leicester (Leicester, UK).76 The more extended promoter construct harboring 1104/ 30 was generated by ligating the extra upstream sequence with the 1044/ 30-luc reporter construct. Transient transfection of RAW264.7 cells and measurement of luciferase activity Transfection of RAW264.7 cells with plasmids containing the full-length or various hIL-10 promoter fragments was performed using electroporation followed by luciferase assay as described previously.77 All statistical analyses were performed with two-tailed Students t-tests. Data were considered significant if P was o0.05. EMSA EMSA was performed as described previously.46 The probe sequences are as follows: 1082, TTCTTTGGGAG/ AGGGGAAGTA; 819, GGTGATGTAAC/TATCTCTG TG; 592, CCCCGCCTGTC/ACTGTAGGAA (the critical SNPs are in bold and underlined). DNA pull-down assay and polyacrylamide gel electrophoresis analysis Complementary biotinylated oligonucleotides encompassing the 592A-binding site, CCCCGCCTGTA/ CCTGTAGGAA (the critical SNP A or C is in bold and underlined) were synthesized and annealed to form double-stranded DNA. Biotinylated double-stranded DNA (2 mg) were conjugated to 100 ml streptavidin-bound magnetic beads (Dynabeads, M280; Dynal) in binding/ washing buffer (10 mM TrisHCl, pH 8.0, 1 mM EDTA and 0.1 M NaCl) for 30 min at room temperature. Conjugated DNA was collected with a magnetic particle concentrator. DNA-conjugated beads were then blocked with 0.5% bovine serum albumin in TGEDN buffer (120 mM TrisHCl, pH 8.0, 1 mM EDTA, 0.1 M NaCl, 1 mM dithiothreitol, 0.1% Triton X-100 and 10% glycerol) at room temperature for 1 h. Beads were washed once in TGEDN buffer and resuspended in 50 ml TGEDN. Ten-microliter beads conjugated to 2 mg DNA were equilibrated with TGEDN buffer and incubated with 500 mg RAW264.7 cell nuclear extracts and 20 mg herring sperm DNA (Sigma-Aldrich) at 41C for 2 h. Beads were washed in TGEDN buffer and bound materials were eluted in 20 ml of the same buffer supplemented with 0.5% SDS and 1 M NaCl. Eluted proteins were separated by 10 or 12% SDS-polyacrylamide gel. The gel was visualized by silver staining. Liquid chromatography-tandem MS Nanoflow liquid chromatography-tandem mass MS analysis (nLC-MS/MS) was performed using an 1100 series LC/MSD Ultra Plus ion trap mass spectrometer (Agilent Technologies, Foster City, CA, USA). The system was equipped with an Agilent Chip Cube interface and a silicon wafer chip-column that integrates a C18 enrichment column, C18 resolving column and nanospray emitter. Samples were loaded on the enrichment column at a flow rate of 5 ml/min and then resolved at a flow rate of 0.3 ml/min on 40 mm 75 mM of ZORBAX 300 C18 resin (5 mM particle size). The LC gradient was 1040% solvent B for 30 min, followed by 4090% solvent B for 20 min. Solvent A contained 0.1% formic acid in 3% acetonitrile (ACN) and solvent B contained 0.1% formic

Genes and Immunity

PARP-1 regulates IL-10 SNP-mediated transcription EY Chung et al

acid in 90% ACN. Electrospray ionisation (ESI) conditions included a needle voltage of 2 kV, nitrogen gas flow rate of 4 l/min and a capillary temperature of 3001C. MS spectra were acquired at a scan speed of 20 000 m/z/s and the four most intense precursor ions at intervals of 0.5 s were selected for MS/MS fragmentation. The fragmentation amplitude was 1.15 V and the skimmer voltage was 30 V. Database search of MS/MS data for peptide sequence identification Analysis of MS/MS spectra for peptide identification was performed by protein database searching using Spectrum Mill software (Agilent Technologies). Raw MS/ MS spectra were first processed to extract MS/MS spectra that could be assigned to at least two y- or bseries ions, and only those spectra were searched against the mouse SwissProt protein database. Key search parameters were a minimum-matched peak intensity of 50%, a precursor mass tolerance of 2.0 Da and a product mass tolerance of 0.6 Da. The maximum number of missed cleavages allowed was one. The threshold used for peptide identification was a Spectrum Mill score greater than 13.0 and SPI% (the percentage of assigned spectrum intensity of total spectrum intensity) greater than 70%. All MS/MS spectra were validated by manual inspection. Western blot analysis RAW264.7 cells or HMDM were cultured at 2 106 cells/ well in six-well culture plates (Falcon). After 6 h of stimulation, macrophages were washed three times with 1 PBS followed by lysis with 200 ml of 1 reducing buffer. Total whole-cell lysates (50 ml) were boiled for 10 min and subjected to electrophoresis on 10% polyacrylamide SDS gels. Proteins were transferred to PolyScreen PVDF transfer membrane (Perkin Elmer Life Sciences, Boston, MA, USA) for 1 h at 100 V. The membranes were blocked for 1 h with 8% non-fat milk in 1 TBS (Tween-20, 1.0%) in the cold room overnight. The membranes were probed overnight with antibodies against PARP-1. Immunoblots were developed by ECL (Perkin Elmer Life Sciences) according to the manufacturers instructions. Phagocytosis assay Peritoneal macrophages were plated in six-well culture plates (Falcon) at 1 106 cells/ml with 2 ml of culture medium per well. Apoptotic cells were stained with 5-(and -6) carboxytetramethylrhodamine, succinimidyl ester (TAMRA; Molecular Probes, Eugene, OR, USA) according to the manufacturers protocol. Cytochalasin D (Sigma-Aldrich) was added 30 min before or at the same time as the addition of TAMRA-stained apoptotic cells. Macrophages were stimulated with apoptotic cells for 1 h. Unbound apoptotic cells were washed off three times with 1 PBS (Gibco) followed by harvest with trypsin/EDTA. The number of macrophages that had undergone phagocytosis was counted under the hemocytometer. Statistical analysis All transfection and cytokine studies were performed in duplicates. The results were expressed as mean7s.d. from at least three independent experiments. All

statistical analyses were performed with two-tailed Students t-test. Data were considered significant if the Po0.05.

587

Acknowledgements
This work was supported by a grant from the NIH (AI45899) to XM and a grant from the Mary Kirkland Foundation for Lupus Research to XM.

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