Inactive Parp2 causes Tp53-dependent lethal anemia by blocking replication-associated nick ligation in erythroblasts.

Poly (ADP-ribose) polymerase (PARP) 1 and 2 enzymatic inhibitors (PARPi) are promising cancer treatments. But recently, their use has been hindered by unexplained severe anemia and treatment-related leukemia. In addition to enzymatic inhibition, PARPi also trap PARP1 and 2 at DNA lesions. Here we report that, unlike Parp2-/- mice, which develop normally, mice expressing catalytically inactive Parp2 (E534A and Parp2EA/EA) succumb to Tp53- and Chk2-dependent erythropoietic failure in utero, mirroring Lig1-/- mice. While DNA damage mainly activates PARP1, we demonstrate that DNA replication activates PARP2 robustly. PARP2 is selectively recruited and activated by 5'-phosphorylated nicks (5'p-nicks), including those between Okazaki fragments, resolved by ligase 1 (Lig1) and Lig3. Inactive PARP2, but not its active form or absence, impedes Lig1- and Lig3-mediated ligation, causing dose-dependent replication fork collapse, which is detrimental to erythroblasts with ultra-fast forks. This PARylation-dependent structural function of PARP2 at 5'p-nicks explains the detrimental effects of PARP2 inactivation on erythropoiesis, shedding light on PARPi-induced anemia and the selection for TP53/CHK2 loss.

Distinct roles for PARP-1 and PARP-2 in c-Myc-driven B-cell lymphoma in mice.

Dysregulation of the c-Myc oncogene occurs in a wide variety of hematologic malignancies, and its overexpression has been linked with aggressive tumor progression. Here, we show that poly (ADP-ribose) polymerase 1 (PARP-1) and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphoma. PARP-1 and PARP-2 catalyze the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc-driven B-cell lymphoma, whereas PARP-1 deficiency accelerates lymphomagenesis in the Eµ-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in preleukemic Eµ-Myc B cells, resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc-driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1 deficiency induces a proinflammatory response and an increase in regulatory T cells, likely contributing to immune escape of B-cell lymphoma, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc-driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centered therapeutic strategies, with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc-driven tumors.

Deficiency of PARP-1 and PARP-2 in the mouse uterus results in decidualization failure and pregnancy loss.

Miscarriage is a common complication of pregnancy for which there are few clinical interventions. Deficiency in endometrial stromal cell decidualization is considered a major contributing factor to pregnancy loss; however, our understanding of the underlying mechanisms of decidual deficiency are incomplete. ADP ribosylation by PARP-1 and PARP-2 has been linked to physiological processes essential to successful pregnancy outcomes. Here, we report that the catalytic inhibition or genetic ablation of PARP-1 and PARP-2 in the uterus lead to pregnancy loss in mice. Notably, the absence of PARP-1 and PARP-2 resulted in increased p53 signaling and an increased population of senescent decidual cells. Molecular and histological analysis revealed that embryo attachment and the removal of the luminal epithelium are not altered in uterine Parp1, Parp2 knockout mice, but subsequent decidualization failure results in pregnancy loss. These findings provide evidence for a previously unknown function of PARP-1 and PARP-2 in mediating decidualization for successful pregnancy establishment.

PARP-1 protects against colorectal tumor induction, but promotes inflammation-driven colorectal tumor progression.

Colorectal cancer (CRC) is one of the most common tumor entities, which is causally linked to DNA repair defects and inflammatory bowel disease (IBD). Here, we studied the role of the DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) in CRC. Tissue microarray analysis revealed PARP-1 overexpression in human CRC, correlating with disease progression. To elucidate its function in CRC, PARP-1 deficient (PARP-1-/-) and wild-type animals (WT) were subjected to azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. Miniendoscopy showed significantly more tumors in WT than in PARP-1-/- mice. Although the lack of PARP-1 moderately increased DNA damage, both genotypes exhibited comparable levels of AOM-induced autophagy and cell death. Interestingly, miniendoscopy revealed a higher AOM/DSS-triggered intestinal inflammation in WT animals, which was associated with increased levels of innate immune cells and proinflammatory cytokines. Tumors in WT animals were more aggressive, showing higher levels of STAT3 activation and cyclin D1 up-regulation. PARP-1-/- animals were then crossed with O6-methylguanine-DNA methyltransferase (MGMT)-deficient animals hypersensitive to AOM. Intriguingly, PARP-1-/-/MGMT-/- double knockout (DKO) mice developed more, but much smaller tumors than MGMT-/- animals. In contrast to MGMT-deficient mice, DKO animals showed strongly reduced AOM-dependent colonic cell death despite similar O6-methylguanine levels. Studies with PARP-1-/- cells provided evidence for increased alkylation-induced DNA strand break formation when MGMT was inhibited, suggesting a role of PARP-1 in the response to O6-methylguanine adducts. Our findings reveal PARP-1 as a double-edged sword in colorectal carcinogenesis, which suppresses tumor initiation following DNA alkylation in a MGMT-dependent manner, but promotes inflammation-driven tumor progression.

Robust immunoglobulin class switch recombination and end joining in Parp9-deficient mice.

To mount highly specific and adapted immune responses, B lymphocytes assemble and diversify their antibody repertoire through mechanisms involving the formation of programmed DNA damage. Immunoglobulin class switch recombination (CSR) is triggered by DNA lesions induced by activation-induced cytidine deaminase, which are processed to double-stranded DNA break (DSB) intermediates. These DSBs activate the cellular DNA damage response and enroll numerous DNA repair factors, involving poly(ADP-ribose) polymerases Parp1, Parp2, and Parp3 to promote appropriate DNA repair and efficient long-range recombination. The macroParp Parp9, which is overexpressed in certain lymphomas, has been recently implicated in DSB repair, acting together with Parp1. Here, we examine the contribution of Parp9 to the resolution of physiological DSBs incurred during V(D)J recombination and CSR by generating Parp9-/- mice. We find that Parp9-deficient mice are viable, fertile, and do not show any overt phenotype. Moreover, we find that Parp9 is dispensable for B-cell development. Finally, we show that CSR and DNA end-joining are robust in the absence of Parp9, indicating that Parp9 is not essential in vivo to achieve physiological DSB repair, or that strong compensatory mechanisms exist.

Parp3 negatively regulates immunoglobulin class switch recombination.

To generate highly specific and adapted immune responses, B cells diversify their antibody repertoire through mechanisms involving the generation of programmed DNA damage. Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by the recruitment of activation-induced cytidine deaminase (AID) to immunoglobulin loci and by the subsequent generation of DNA lesions, which are differentially processed to mutations during SHM or to double-stranded DNA break intermediates during CSR. The latter activate the DNA damage response and mobilize multiple DNA repair factors, including Parp1 and Parp2, to promote DNA repair and long-range recombination. We examined the contribution of Parp3 in CSR and SHM. We find that deficiency in Parp3 results in enhanced CSR, while SHM remains unaffected. Mechanistically, this is due to increased occupancy of AID at the donor (Sµ) switch region. We also find evidence of increased levels of DNA damage at switch region junctions and a bias towards alternative end joining in the absence of Parp3. We propose that Parp3 plays a CSR-specific role by controlling AID levels at switch regions during CSR.

PARP2 deficiency affects invariant-NKT-cell maturation and protects mice from concanavalin A-induced liver injury.

Excessive or persistent inflammation and hepatocyte death are the key triggers of liver diseases. The poly(ADP-ribose) polymerase (PARP) proteins induce cell death and inflammation. Chemical inhibition of PARP activity protects against liver injury during concanavalin A (ConA)-induced hepatitis. In this mice model, ConA activates immune cells, which promote inflammation and induce hepatocyte death, mediated by the activated invariant natural killer T (iNKT) lymphocyte population. We analyzed immune cell populations in the liver and several lymphoid organs, such as the spleen, thymus, and bone marrow in Parp2-deficient mice to better define the role of PARP proteins in liver immunity and inflammation at steady state and during ConA-induced hepatitis. We show that 1) the genetic inactivation of Parp2, but not Parp1, protected mice from ConA hepatitis without deregulating cytokine expression and leucocyte recruitment; 2) cellularity was lower in the thymus, but not in spleen, liver, or bone marrow of Parp2-/- mice; 3) spleen and liver iNKT lymphocytes, as well as thymic T and NKT lymphocytes were reduced in Parp2 knockout mice. In conclusion, our results suggest that the defect of T-lymphocyte maturation in Parp2 knockout mice leads to a systemic reduction of iNKT cells, reducing hepatocyte death during ConA-mediated liver damage, thus protecting the mice from hepatitis.NEW & NOTEWORTHY The genetic inactivation of Parp2, but not Parp1, protects mice from concanavalin A hepatitis. Immune cell populations are lower in the thymus, but not in the spleen, liver, or bone marrow of Parp2-deficient mice compared with wild-type mice. Spleen and liver invariant natural killer T (NKT) lymphocytes, as well as thymic T and NKT lymphocytes, are reduced in Parp2-deficient mice.

New readers and interpretations of poly(ADP-ribosyl)ation.

Poly(ADP-ribosyl)ation (PARylation), a protein post-translational modification that was originally connected to the DNA damage response, is now known to engage in a continuously increasing number of biological processes. Despite extensive research and ceaseless, important findings about its role and mode of action, poly(ADP-ribose) remains an enigma regarding its structural complexity and diversity. The recent identification and structural characterization of four different poly(ADP-ribose) binding motifs represents a quantum leap in the comprehension of how this molecule can be decoded. Moreover, the recent discovery of a direct connection between PARylation and poly-ubiquitylation in targeting proteins for degradation by the proteasome has paved the way for a new interpretation of this protein modification. These two novel aspects, poly(ADP-ribose) recognition and readout by the ubiquitylation/proteasome system are developed here.

PARG deficiency is neither synthetic lethal with BRCA1 nor PTEN deficiency.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) inhibitors have entered the clinics for their promising anticancer effect as adjuvant in chemo- and radiotherapy and as single agent on BRCA-mutated tumours. Poly(ADP-ribose) glycohydrolase (PARG) deficiency was also shown to potentiate the cytotoxicity of genotoxic agents and irradiation. The aim of this study is to investigate the effect of PARG deficiency on BRCA1- and/or PTEN-deficient tumour cells. METHODS: Since no PARG inhibitors are available for in vivo studies, PARG was depleted by siRNA in several cancer cell lines, proficient or deficient for BRCA1 and/or PTEN. The impact on cell survival was evaluated by colony formation assay and short-term viability assays. The effect of simultaneous PARG and BRCA1 depletion on homologous recombination (HR) efficacy was evaluated by immunodetection of RAD51 foci and using an in vivo HR assay. RESULTS: The BRCA1-deficient cell lines MDA-MB-436, HCC1937 and UWB1.289 showed mild sensitivity to PARG depletion, whereas no sensitivity was observed for the BRCA1-proficient MDA-MB-231, MDA-MB-468, MCF10A and U2OS cell lines. However, the BRCA1-reconstituted UWB1.289 cell lines was similarly sensitive to PARG depletion than the BRCA1-deficient UWB1.289, and the simultaneous depletion of PARG and BRCA1 and/or PTEN in MDA-MB-231 or U2OS cells was not more cytotoxic than depletion of BRCA1 or PTEN only. CONCLUSIONS: Some tumour cells displayed slight sensitivity to PARG deficiency, but this sensitivity could not be correlated to BRCA1- or PTEN-deficiency. Therefore, PARG depletion cannot be considered as a strategy to kill tumours cells mutated in BRCA1 or PTEN.

PARG is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(ADP-ribose) upon prolonged replicative stress.

Poly(ADP-ribosyl)ation is involved in numerous bio-logical processes including DNA repair, transcription and cell death. Cellular levels of poly(ADP-ribose) (PAR) are regulated by PAR polymerases (PARPs) and the degrading enzyme PAR glycohydrolase (PARG), controlling the cell fate decision between life and death in response to DNA damage. Replication stress is a source of DNA damage, leading to transient stalling of replication forks or to their collapse followed by the generation of double-strand breaks (DSB). The involvement of PARP-1 in replicative stress response has been described, whereas the consequences of a deregulated PAR catabolism are not yet well established. Here, we show that PARG-deprived cells showed an enhanced sensitivity to the replication inhibitor hydroxyurea. PARG is dispensable to recover from transient replicative stress but is necessary to avoid massive PAR production upon prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR accumulation impairs replication protein A association with collapsed forks resulting in compromised DSB repair via homologous recombination. Our results highlight the critical role of PARG in tightly controlling PAR levels produced upon genotoxic stress to prevent the detrimental effects of PAR over-accumulation.

PARP3 affects the relative contribution of homologous recombination and nonhomologous end-joining pathways.

The repair of toxic double-strand breaks (DSB) is critical for the maintenance of genome integrity. The major mechanisms that cope with DSB are: homologous recombination (HR) and classical or alternative nonhomologous end joining (C-NHEJ versus A-EJ). Because these pathways compete for the repair of DSB, the choice of the appropriate repair pathway is pivotal. Among the mechanisms that influence this choice, deoxyribonucleic acid (DNA) end resection plays a critical role by driving cells to HR, while accurate C-NHEJ is suppressed. Furthermore, end resection promotes error-prone A-EJ. Increasing evidence define Poly(ADP-ribose) polymerase 3 (PARP3, also known as ARTD3) as an important player in cellular response to DSB. In this work, we reveal a specific feature of PARP3 that together with Ku80 limits DNA end resection and thereby helps in making the choice between HR and NHEJ pathways. PARP3 interacts with and PARylates Ku70/Ku80. The depletion of PARP3 impairs the recruitment of YFP-Ku80 to laser-induced DNA damage sites and induces an imbalance between BRCA1 and 53BP1. Both events result in compromised accurate C-NHEJ and a concomitant increase in DNA end resection. Nevertheless, HR is significantly reduced upon PARP3 silencing while the enhanced end resection causes mutagenic deletions during A-EJ. As a result, the absence of PARP3 confers hypersensitivity to anti-tumoral drugs generating DSB.

Poly(ADP-ribose) polymerase 1 (PARP1) associates with E3 ubiquitin-protein ligase UHRF1 and modulates UHRF1 biological functions.

Poly(ADP-ribose) polymerase 1 (PARP1, also known as ARTD1) is an abundant nuclear enzyme that plays important roles in DNA repair, gene transcription, and differentiation through the modulation of chromatin structure and function. In this work we identify a physical and functional poly(ADP-ribose)-mediated interaction of PARP1 with the E3 ubiquitin ligase UHRF1 (also known as NP95, ICBP90) that influences two UHRF1-regulated cellular processes. On the one hand, we uncovered a cooperative interplay between PARP1 and UHRF1 in the accumulation of the heterochromatin repressive mark H4K20me3. The absence of PARP1 led to reduced accumulation of H4K20me3 onto pericentric heterochromatin that coincided with abnormally enhanced transcription. The loss of H4K20me3 was rescued by the additional depletion of UHRF1. In contrast, although PARP1 also seemed to facilitate the association of UHRF1 with DNMT1, its absence did not impair the loading of DNMT1 onto heterochromatin or the methylation of pericentric regions, possibly owing to a compensating interaction of DNMT1 with PCNA. On the other hand, we showed that PARP1 controls the UHRF1-mediated ubiquitination of DNMT1 to timely regulate its abundance during S and G2 phase. Together, this report identifies PARP1 as a novel modulator of two UHRF1-regulated heterochromatin-associated events: the accumulation of H4K20me3 and the clearance of DNMT1.

Parp-2 is required to maintain hematopoiesis following sublethal γ-irradiation in mice.

Hematopoietic stem cells self-renew for life to guarantee the continuous supply of all blood cell lineages. Here we show that Poly(ADP-ribose) polymerase-2 (Parp-2) plays an essential role in hematopoietic stem/progenitor cells (HSPC) survival under steady-state conditions and in response to stress. Increased levels of cell death were observed in HSPC from untreated Parp-2-/- mice, but this deficit was compensated by increased rates of self-renewal, associated with impaired reconstitution of hematopoiesis upon serial bone marrow transplantation. Cell death after γ-irradiation correlated with an impaired capacity to repair DNA damage in the absence of Parp-2. Upon exposure to sublethal doses of γ-irradiation, Parp-2-/- mice exhibited bone marrow failure that correlated with reduced long-term repopulation potential of irradiated Parp-2-/- HSPC under competitive conditions. In line with a protective role of Parp-2 against irradiation-induced apoptosis, loss of p53 or the pro-apoptotic BH3-only protein Puma restored survival of irradiated Parp-2-/- mice, whereas loss of Noxa had no such effect. Our results show that Parp-2 plays essential roles in the surveillance of genome integrity of HSPC by orchestrating DNA repair and restraining p53-induced and Puma-mediated apoptosis. The data may affect the design of drugs targeting Parp proteins and the improvement of radiotherapy-based therapeutic strategies.

Poly(ADP-ribose) polymerases in double-strand break repair: focus on PARP1, PARP2 and PARP3.

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins catalysed by Poly(ADP-ribose) polymerases (PARP). A wealth of recent advances in the biochemical and functional characterization of the DNA-dependent PARP family members have highlighted their key contribution in the DNA damage response network, the best characterized being the role of PARP1 and PARP2 in the resolution of single-strand breaks as part of the BER/SSBR process. How PARylation contributes to the repair of double-strand breaks is less well defined but has become recently the subject of significant research in the field. The aim of this review is to provide an overview of the current knowledge concerning the role of the DNA-activated PARP1, PARP2 and PARP3 in cellular response to double-strand breaks (DSB). In addition, we outline the biological significance of these properties in response to programmed DNA lesions formed during physiological processes such as antibody repertoire assembly and diversification.

Poly(ADP-ribose) polymerase 3 (PARP3), a newcomer in cellular response to DNA damage and mitotic progression.

The ADP ribosyl transferase [poly(ADP-ribose) polymerase] ARTD3(PARP3) is a newly characterized member of the ARTD(PARP) family that catalyzes the reaction of ADP ribosylation, a key posttranslational modification of proteins involved in different signaling pathways from DNA damage to energy metabolism and organismal memory. This enzyme shares high structural similarities with the DNA repair enzymes PARP1 and PARP2 and accordingly has been found to catalyse poly(ADP ribose) synthesis. However, relatively little is known about its in vivo cellular properties. By combining biochemical studies with the generation and characterization of loss-of-function human and mouse models, we describe PARP3 as a newcomer in genome integrity and mitotic progression. We report a particular role of PARP3 in cellular response to double-strand breaks, most likely in concert with PARP1. We identify PARP3 as a critical player in the stabilization of the mitotic spindle and in telomere integrity notably by associating and regulating the mitotic components NuMA and tankyrase 1. Both functions open stimulating prospects for specifically targeting PARP3 in cancer therapy.

Enhancing cisplatin drug sensitivity through PARP3 inhibition: The influence on PDGF and G-coupled signal pathways in cancer.

Drug resistance poses a significant challenge in cancer treatment despite the clinical efficacy of cisplatin. Identifying and targeting biomarkers open new ways to improve therapeutic outcomes. In this study, comprehensive bioinformatic analyses were employed, including a comparative analysis of multiple datasets, to evaluate overall survival and mutation hotspots in 27 base excision repair (BER) genes of more than 7,500 tumors across 23 cancer types. By using various parameters influencing patient survival, revealing that the overexpression of 15 distinct BER genes, particularly PARP3, NEIL3, and TDG, consistently correlated with poorer survival across multiple factors such as race, gender, and metastasis. Single nucleotide polymorphism (SNP) analyses within protein-coding regions highlighted the potential deleterious effects of mutations on protein structure and function. The investigation of mutation hotspots in BER proteins identified PARP3 due to its high mutation frequency. Moving from bioinformatics to wet lab experiments, cytotoxic experiments demonstrated that the absence of PARP3 by CRISPR/Cas9-mediated knockdown in MDA-MB-231 breast cancer cells increased drug activity towards cisplatin, carboplatin, and doxorubicin. Pathway analyses indicated the impact of PARP3 absence on the platelet-derived growth factor (PDGF) and G-coupled signal pathways on cisplatin exposure. PDGF, a critical regulator of various cellular functions, was downregulated in the absence of PARP3, suggesting a role in cancer progression. Moreover, the influence of PARP3 knockdown on G protein-coupled receptors (GPCRs) affects their function in the presence of cisplatin. In conclusion, the study demonstrated a synthetic lethal interaction between GPCRs, PDGF signaling pathways, and PARP3 gene silencing. PARP3 emerged as a promising target.

Inactive Parp2 causes Tp53-dependent lethal anemia by blocking replication-associated nick ligation in erythroblasts.

PARP1&2 enzymatic inhibitors (PARPi) are promising cancer treatments. But recently, their use has been hindered by unexplained severe anemia and treatment-related leukemia. In addition to enzymatic inhibition, PARPi also trap PARP1&2 at DNA lesions. Here, we report that unlike Parp2 -/- mice, which develop normally, mice expressing catalytically-inactive Parp2 (E534A, Parp2 EA/EA ) succumb to Tp53- and Chk2 -dependent erythropoietic failure in utero , mirroring Lig1 -/- mice. While DNA damage mainly activates PARP1, we demonstrate that DNA replication activates PARP2 robustly. PARP2 is selectively recruited and activated by 5'-phosphorylated nicks (5'p-nicks) between Okazaki fragments, typically resolved by Lig1. Inactive PARP2, but not its active form or absence, impedes Lig1- and Lig3-mediated ligation, causing dose-dependent replication fork collapse, particularly harmful to erythroblasts with ultra-fast forks. This PARylation-dependent structural function of PARP2 at 5'p-nicks explains the detrimental effects of PARP2 inhibition on erythropoiesis, revealing the mechanism behind the PARPi-induced anemia and leukemia, especially those with TP53/CHK2 loss. Significance: This work shows that the hematological toxicities associated with PARP inhibitors stem not from impaired PARP1 or PARP2 enzymatic activity but rather from the presence of inactive PARP2 protein. Mechanistically, these toxicities reflect a unique role of PARP2 at 5'-phosphorylated DNA nicks during DNA replication in erythroblasts.

Purification of Recombinant Human PARG and Activity Assays.

The purification of poly(ADP-ribose) glycohydrolase (PARG) from overexpressing bacteria Escherichia coli is described here as a fast and reproducible one chromatographic step protocol. After cell lysis, GST-PARG-fusion proteins from the crude extract are affinity purified by a glutathione 4B sepharose chromatographic step. The PARG proteins are then freed from their GST-fusion by overnight enzymatic cleavage using the preScission protease. As described in the protocol, more than 500 µg of highly active human PARG can be obtained from 1.5 L of E. coli culture.

Purification of Recombinant Human PARP-3.

The purification of poly(ADP-ribose) polymerase-3 (PARP-3) from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible two-chromatographic-step protocol. After cell lysis, PARP-3 protein from the crude extract is affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute PARP-3 from the previous affinity column are removed on the high-performance strong cation exchanger MonoQ™ matrix. This step allows also the concentration of the protein. The columns connected to an A° KTA™ purifier system allow the purification of the protein in three days with a high-yield recovery. As described in the protocol, more than 3 mg of pure and active human PARP-3 can be obtained from 1.5 L of E. coli culture.

PARP2 downregulation in T cells ameliorates lipopolysaccharide-induced inflammation of the large intestine.

Introduction: T cell-dependent inflammatory response with the upregulation of helper 17 T cells (Th17) and the downregulation of regulatory T cells (Treg) accompanied by the increased production of tumor necrosis alpha (TNFa) is characteristic of inflammatory bowel diseases (IBD). Modulation of T cell response may alleviate the inflammation thus reduce intestinal damage. Poly(ADP-ribose) polymerase-2 (PARP2) plays role in the development, differentiation and reactivity of T cell subpopulations. Our aim was to investigate the potential beneficial effect of T cell-specific PARP2 downregulation in the lipopolysaccharide (LPS) induced inflammatory response of the cecum and the colon. Methods: Low-dose LPS was injected intraperitoneally to induce local inflammatory response, characterized by increased TNFa production, in control (CD4Cre; PARP2+/+) and T cell-specific conditional PARP2 knockout (CD4Cre; PARP2f/f) mice. TNFa, IL-1b, IL-17 levels were measured by ELISA, oxidative-nitrative stress was estimated by immunohistochemistry, while PARP1 activity, p38 MAPK and ERK phosphorylation, and NF-kB expression in large intestine tissue samples were examined by Western-blot. Systemic & local T cell subpopulation; Th17 and Treg alterations were also investigated using flowcytometry and immunohistochemistry. Results: In control animals, LPS induced intestinal inflammation with increased TNFa production, while no significant elevation of TNFa production was observed in T cell-specific PARP2 knockout animals. The absence of LPS-induced elevation in TNFa levels was accompanied by the absence of IL-1b elevation and the suppression of IL-17 production, showing markedly reduced inflammatory response. The increase in oxidative-nitrative stress and PARP1-activation was also absent in these tissues together with altered ERK and NF-kB activation. An increase in the number of the anti-inflammatory Treg cells in the intestinal mucosa was observed in these animals, together with the reduction of Treg count in the peripheral circulation. Discussion: Our results confirmed that T cell-specific PARP2 downregulation ameliorated LPS-induced colitis. The dampened TNFa production, decreased IL-17 production and the increased intestinal regulatory T cell number after LPS treatment may be also beneficial during inflammatory processes seen in IBD. By reducing oxidative-nitrative stress and PARP1 activation, T cell-specific PARP2 downregulation may also alleviate intestinal tissue damage.