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Background:Macrophages are believed to be local and systemic master actors in disease, critically involved in shaping inflammation, and in disease resolution via effective efferocytosis. Impaired efferocytosis, a hallmark of failed inflammation resolution, has been reported in an increasing number of immune-mediated inflammatory diseases, like Rheumatoid Arthritis (RA). Macrophage plasticity and capability for reprogramming, make them an attractive target for novel disease-modifying therapies. While cell-based therapies currently being scrutinized for their unique potential to fulfil a lost function, including macrophage-based therapies, the use of resolution-type macrophage secretome, i.e. the molecules they release, might provide a simpler and yet potentially safer approach to restore homeostasis in RA.Objectives:The present study attempted to understand the inflammatory profile and functionality of RA patient’s monocyte-derived macrophages and evaluate their plasticity and capability for reprogramming to resolution-type macrophages as a source of RA patient-derived secretome with therapeutic potential.Methods:Patients being followed up at CHU Besançon between 2015 and 2021 were included in this prospective single center study. Inclusion criteria were 18 to 80 year-old patients with RA according to ACR/EULAR 2010 criteria, with a DAS28 ≥ 2.6, with or without csDMARDS (methotrexate, leflunomide or sulfasalazine), and naïve of biological agents or systemic corticosteroids for 6 months. Blood inflammatory cytokines and lipid mediators were quantified using CBA multiplex and MS analysis, respectively. Blood monocytes from RA patients or healthy donors (HD) were isolated by density gradient, and examined after differentiation into macrophages using M-CSF during 7 days, or not. Inflammatory responses and efferocytosis capacities were analyzed using flow cytometry.Results:A total of 28 patients and 31 HD were included. Evaluating inflammatory mediators, we found pro-inflammatory cytokines TNF-α, IL-6, IL-8, IL-1β and IL-12 increased in RA patient’s plasma compared to HD. We also detected a global increase of the pro-inflammatory lipid mediators derived from omega-3 fatty acid in patient plasma, confirming the ongoing systemic inflammatory nature of disease in RA patients.Furthermore, we observed that monocytes and dendritic cells (DC) of RA patients presented higher levels of the co-stimulatory marker CD40, as compared to HD, which was not true on plasmacytoid DC. Thus, we confirm that circulating myeloid cells exhibit an inflammatory profile in RA patients.To evaluate how inflammatory pattern affects the efferocytosis capacities of macrophages, monocytes were differentiated into macrophages and evaluated for their phagocytosis of apoptotic cells. Interestingly, we observed that the efferocytic capacities of patient monocytes-derived macrophages were not impaired ex vivo. This was confirmed by similar expression of membrane engulfment receptors at the macrophage stage. Culturing macrophages with apoptotic cells generate resolution-type macrophages secreting pro-resolutive mediators. Interestingly, when exposed to HD resolution mediators, the efferocytic capacity of RA macrophages could be further increased by 67% [5-303%, min-max].Our data show that in RA patients, monocytes remained plastic and capable to reprogram into resolution-type macrophages outside of patient inflammatory environment, and with that, could be considered not only a potential target but also a cellular source to generate autologous patient-specific resolution mediator secretome.Conclusion:Our study revealed that despite their inflammatory profile, RA patient monocytes remained plastic and conserved ex vivo their efferocytic capacities, and used to produce patient-specific autologous resolution-type secretome. Further investigations are ongoing to confirm the therapeutic properties of RA patient own macrophage-derived secretomes to be proposed as next generation disease modifying therapeutic modality/bDMARD for RA patients.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:Mélanie COUTURIER MED’INN’Pharma, F-25000 Besançon, France, MED’INN’Pharma, F-25000 Besançon, France, Emilie GAIFFE: None declared, Charline VAUCHY: None declared, Susanne BEHLKE MED’INN’Pharma, F-25000 Besançon, France, Eric Toussirot: None declared, Sylvain PERRUCHE MED’INN’Pharma, F-25000 Besançon, France, MED’INN’Pharma, F-25000 Besançon, France.

The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome integrity and protection against cancer. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, recent studies provide evidence that PALB2 also associates with unperturbed chromatin. Here, we investigated the previously poorly described role of chromatin-associated PALB2 in undamaged cells. We found that PALB2 associates with active genes through its major binding partner, MRG15, which recognizes histone H3 trimethylated at lysine 36 (H3K36me3) by the SETD2 methyltransferase. Missense mutations that ablate PALB2 binding to MRG15 confer elevated sensitivity to the topoisomerase inhibitor camptothecin (CPT) and increased levels of aberrant metaphase chromosomes and DNA stress in gene bodies, which were suppressed by preventing DNA replication. Remarkably, the level of PALB2 at genic regions was frequently decreased, rather than increased, upon CPT treatment. We propose that the steady-state presence of PALB2 at active genes, mediated through the SETD2/H3K36me3/MRG15 axis, ensures an immediate response to DNA stress and therefore effective protection of these regions during DNA replication. This study provides a conceptual advance in demonstrating that the constitutive chromatin association of repair factors plays a key role in the maintenance of genome stability and furthers our understanding of why PALB2 defects lead to human genome instability syndromes.

DNA double-strand breaks (DSB) elicit a ubiquitylation cascade that controls DNA repair pathway choice. This cascade involves the ubiquitylation of histone H2A by the RNF168 ligase and the subsequent recruitment of RIF1, which suppresses homologous recombination (HR) in G1 cells. The RIF1-dependent suppression is relieved in S/G2 cells, allowing PALB2-driven HR to occur. With the inhibitory impact of RIF1 relieved, it remains unclear how RNF168-induced ubiquitylation influences HR. Here, we uncover that RNF168 links the HR machinery to H2A ubiquitylation in S/G2 cells. We show that PALB2 indirectly recognizes histone ubiquitylation by physically associating with ubiquitin-bound RNF168. This direct interaction is mediated by the newly identified PALB2-interacting domain (PID) in RNF168 and the WD40 domain in PALB2, and drives DNA repair by facilitating the assembly of PALB2-containing HR complexes at DSBs. Our findings demonstrate that RNF168 couples PALB2-dependent HR to H2A ubiquitylation to promote DNA repair and preserve genome integrity.

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation, limiting the success of this therapy. We previously reported that interleukin-22 (IL-22) participates to aGVHD development, but the underlying mechanisms of its contribution remain poorly understood. In this study, we analyzed the mechanism of the pathological function of IL-22 in intestinal aGVHD. Ex-vivo colon culture experiments indicated that IL-22 was able to induce Th1-like inflammation via signal transducer and activator of transcription factor-1 (STAT1) and CXCL10 induction in the presence of type I interferon (IFN). To evaluate a potential synergy between IL-22 and type I IFN in aGVHD, we transplanted recipient mice, either wild-type (WT) or type I IFN receptor deficient (IFNAR−/−), with bone marrow cells and WT or IL-22 deficient (IL-22−/−) T cells. We observed a decreased GVHD severity in IFNAR−/− recipient of IL-22−/− T cells, which was associated with a lower level of STAT1 activation and reduced CXCL10 expression in the large intestine. Finally, immunohistochemistry staining of STAT1 performed on gastrointestinal biopsies of 20 transplanted patients showed exacerbated STAT1 activation in gastrointestinal tissues of patients with aGVHD as compared with those without aGVHD. Thus, interfering with both IL-22 and type I IFN signaling may provide a novel approach to limit aGVHD.

The tumour suppressor PALB2 stimulates RAD51-mediated homologous recombination (HR) repair of DNA damage, whilst its steady-state association with active genes protects these loci from replication stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/2B, also called GCN5/PCAF), two well-known transcriptional regulators, acetylate a cluster of seven lysine residues (7K-patch) within the PALB2 chromatin association motif (ChAM) and, in this way, regulate context-dependent PALB2 binding to chromatin. In unperturbed cells, the 7K-patch is targeted for KAT2A/2B-mediated acetylation, which in turn enhances the direct association of PALB2 with nucleosomes. Importantly, DNA damage triggers a rapid deacetylation of ChAM and increases the overall mobility of PALB2. Distinct missense mutations of the 7K-patch render the mode of PALB2 chromatin binding, making it either unstably chromatin-bound (7Q) or randomly bound with a reduced capacity for mobilisation (7R). Significantly, both of these mutations confer a deficiency in RAD51 foci formation and increase DNA damage in S phase, leading to the reduction of overall cell survival. Thus, our study reveals that acetylation of the ChAM 7K-patch acts as a molecular switch to enable dynamic PALB2 shuttling for HR repair while protecting active genes during DNA replication.

For decades, radiotherapy and chemotherapy were the two only approaches exploiting DNA repair processes to fight against cancer. Nowadays, cancer therapeutics can be a major challenge when it comes to seeking personalized targeted medicine that is both effective and selective to the malignancy. Over the last decade, the discovery of new targeted therapies against DNA damage signalling and repair has offered the possibility of therapeutic improvements in oncology. In this review, we summarize the current knowledge of DNA damage signalling and repair inhibitors, their molecular and cellular effects, and future therapeutic use.

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation (allo-HCT), limiting the success of this therapy. Many proinflammatory cytokines secreted following the conditioning regimen have been linked to aGVHD initiation. Interleukin-22 (IL-22) is a cytokine related to IL-10 for its structure and is secreted by T helper type 17 (TH17) cells and innate immune cells. Given the paradoxical role of IL-22 in inflammation with both protective or proinflammatory functions, we investigated whether IL-22 could have a role in aGVHD pathophysiology in a mouse allo-HCT model. In this study, we show that IL-22 deficiency in donor T cells can decrease the severity of aGVHD, while limiting systemic and local inflammation in aGVHD target organs. In addition, we found that Foxp3+ regulatory T cells (Treg cells) were increased in recipient mice that received IL-22-deficient T cells, suggesting that Treg were involved in the reduced severity of GVHD. Finally, we found that the graft-versus-leukemia (GVL) effect mediated by donor T cells was preserved in the absence of IL-22. Overall, these data suggest that targeting of IL-22 may represent a valid approach towards decreasing aGVHD severity after allo-HCT while preserving the GVL effect.

BackgroundPro-resolving mediators produced by macrophages eliminating apoptotic effector cells during the resolution of inflammation have been shown to stop inflammation, favor tissue repair and return to homeostasis. In immune-mediated inflammatory diseases such as rheumatoid arthritis (RA), one may believe that reintroducing pro- resolving mediators would allow the control of the disease. We have developed a cell culture system of macrophages and apoptotic cells that mimics this phase of resolution. The supernatant of this cell culture system called SuperMApo (SUPERnatant issued from Macrophage APOptotic cell culture) is enriched in pro-resolving factors including anti- inflammatory cytokines like TGF-b.Objectivesto evaluate the effects of SuperMApo in the treatment of collagen-induced arthritis (CIA).MethodsSuperMApo is obtained after 48h of culturing macrophages from peritoneal cavity with apoptotic cells obtained from thymus after X-ray irradiation. CIA was used as a model of RA. SuperMApo (200 μL of 5x concentrated SuperMApo, IV, for 2 days) was injected in score 8 CIA mice. The effect of SuperMApo was first evaluated on APC and T cells from naive mice in terms of phenotype, profile and properties such as maturation of APC before or after TLR-ligand stimulation and T cell polarization assays. Then, SuperMApo was evaluated in vivo in CIA mice.ResultsMacrophages, cDC and pDC submitted to SuperMApo demonstrated robust insensitivity to TLR ligand-induced activation, particularly in terms of co-stimulatory molecules expression. Spleen cells cultured with SuperMApo demonstrated a strong Treg increase. This was confirmed using naive CD4+CD25– T cells cultured in the presence of SuperMApo showing Foxp3 expression. CIA mice received 2 injections of SuperMApo (over 48h) and demonstrated a significant long term decrease of CIA (joint score before and after SuperMApo: 8–10 and 1–4, respectively; sustained response to 60 days). This clinical improvement was associated with a higher suppressive function of Treg. Treg transfer from mice treated by SuperMApo to arthritic mice induced joint improvement. Finally, we tested SuperMApo effect on PBMC issued from 4 patients with RA, with and without TLR ligand activation. Our results were that monocytes from 3 patients showed a decreased inflammatory phenotype after overnight exposure to SuperMApo (CD80, CD86, CD40 and HLA-DR expression).Conclusionsthese data demonstrated that macrophages eliminating apoptotic cells produced pro-resolving mediators affecting APC properties therefore allowing the control of arthritis in the CIA model.Disclosure of InterestNone declared

BackgroundPro-resolving mediators produced by macrophages eliminating apoptotic effector cells during the resolution of inflammation have been shown to stop inflammation, favor tissue repair and return to homeostasis. In immune-mediated inflammatory diseases such as rheumatoid arthritis (RA), one may believe that reintroducing pro-resolving mediators would allow the control of the disease. We have developed a cell culture system of macrophages and apoptotic cells that mimics this phase of resolution. The supernatant of this cell culture system called SuperMApo (SUPERnatant issued from Macrophage APOptotic cell culture) is enriched in pro-resolving factors including anti-inflammatory cytokines like TGF-b.ObjectivesTo evaluate the effects of SuperMApo in the treatment of collagen-induced arthritis (CIA).MethodsSuperMApo is obtained after 48h of culturing macrophages from peritoneal cavity with apoptotic cells obtained from thymus after X-ray irradiation. CIA was used as a model of RA. SuperMApo (200 μL of 5x concentrated SuperMApo, IV, for 2 days) was injected in score 8 CIA mice. The effect of SuperMApo was first evaluated on APC and T cells from naïve mice in terms of phenotype, profile and properties such as maturation of APC before or after TLR-ligand stimulation and T cell polarization assays. Then, SuperMApo was evaluated in vivo in CIA mice.ResultsMacrophages, cDC and pDC submitted to SuperMApo demonstrated robust insensitivity to TLR ligand-induced activation, particularly in terms of co-stimulatory molecules expression. Spleen cells cultured with SuperMApo demonstrated a strong Treg increase. This was confirmed using naïve CD4+CD25- T cells cultured in the presence of SuperMApo showing Foxp3 expression. CIA mice received 2 injections of SuperMApo (over 48h) and demonstrated a significant long term decrease of CIA (joint score before and after SuperMApo: 8-10 and 1-4, respectively; sustained response to 60 days). This clinical improvement was associated with a higher suppressive function of Treg. Treg transfer from mice treated by SuperMApo to arthritic mice induced joint improvement. Finally, we tested SuperMApo effect on PBMC issued from 4 patients with RA, with and without TLR ligand activation. Our results were that monocytes from 3 patients showed a decreased inflammatory phenotype after overnight exposure to SuperMApo (CD80, CD86, CD40 and HLA-DR expression).ConclusionsThese data demonstrated that macrophages eliminating apoptotic cells produced pro-resolving mediators affecting APC properties therefore allowing the control of arthritis in the CIA model.Disclosure of InterestNone declared

Background Filamentous fungi are potent biomass degraders due to their ability to thrive in ligno(hemi)cellulose-rich environments. During the last decade, fungal genome sequencing initiatives have yielded abundant information on the genes that are putatively involved in lignocellulose degradation. At present, additional experimental studies are essential to provide insights into the fungal secreted enzymatic pools involved in lignocellulose degradation. Results In this study, we performed a wide analysis of 20 filamentous fungi for which genomic data are available to investigate their biomass-hydrolysis potential. A comparison of fungal genomes and secretomes using enzyme activity profiling revealed discrepancies in carbohydrate active enzymes (CAZymes) sets dedicated to plant cell wall. Investigation of the contribution made by each secretome to the saccharification of wheat straw demonstrated that most of them individually supplemented the industrial Trichoderma reesei CL847 enzymatic cocktail. Unexpectedly, the most striking effect was obtained with the phytopathogen Ustilago maydis that improved the release of total sugars by 57% and of glucose by 22%. Proteomic analyses of the best-performing secretomes indicated a specific enzymatic mechanism of U. maydis that is likely to involve oxido-reductases and hemicellulases. Conclusion This study provides insight into the lignocellulose-degradation mechanisms by filamentous fungi and allows for the identification of a number of enzymes that are potentially useful to further improve the industrial lignocellulose bioconversion process.