Non-homologous end joining (NHEJ) is a DNA repair pathway that senses, processes and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. During NHEJ, core Ku70 and Ku80 subunits bind DSBs as a heterodimer and promote further recruitment of accessory factors (e.g., PAXX, Mri, DNA-PKcs, Artemis) and downstream core subunits XRCC4 and DNA ligase 4 (Lig4). Inactivation of Ku70 or Ku80 genes in mice results in immunodeficiency and high levels of genomic instability; deletion of individual Dna-pkcs, Xlf, Paxx or Mri genes results in viable mice with no or modest DNA repair defects. However, combined inactivation of either Xlf and Dna-pkcs, or Xlf and Paxx, or Xlf and Mri, leads to synthetic lethality in mice, which correlates with increased levels of apoptosis in the central nervous system. Here, we demonstrated that inactivation of pro-apoptotic factor Trp53 rescues embryonic lethality of Xlf -/-Paxx -/and Xlf -/-Dna-pkcs -/double knockout mice. Moreover, combined inactivation of Paxx and Dna-pkcs results in live-born fertile Paxx -/-Dna-pkcs -/mice indistinguishable from Dna-pkcs -/knockout controls.

DNA repair consists of several cellular pathways which recognize and repair damaged DNA . The classical nonhomologous DNA end‐joining ( NHEJ ) pathway repairs double‐strand breaks in DNA . It is required for maturation of both B and T lymphocytes by supporting V(D)J recombination as well as B‐cell differentiation during class switch recombination (CSR). Inactivation of NHEJ factors Ku70, Ku80, XRCC 4, DNA ligase 4, DNA ‐ PK cs, and Artemis impairs V(D)J recombination and blocks lymphocyte development. Paralogue of XRCC 4 and XLF ( PAXX ) is an accessory NHEJ factor that has a significant impact on the repair of DNA lesions induced by ionizing radiation in human, murine, and chicken cells. However, the role of PAXX during development is poorly understood. To determine the physiological role of PAXX , we deleted part of the Paxx promoter and the first two exons in mice. Further, we compared Paxx ‐knockout mice with wild‐type (WT) and NHEJ ‐deficient controls including Ku80‐ and Dna‐pkcs ‐null and severe combined immunodeficiency mice. Surprisingly, Paxx ‐deficient mice were not distinguishable from the WT littermates; they were the same weight and size, fertility status, had normal spleen, thymus, and bone marrow. Paxx ‐deficient mice had the same number of chromosomal and chromatid breaks as WT mice. Moreover, Paxx ‐deficient primary B lymphocytes had the same level of CSR as lymphocytes isolated from WT mice. We concluded that PAXX is dispensable for normal mouse development.

To ensure genome stability, mammalian cells employ several DNA repair pathways. Nonhomologous DNA end joining ( NHEJ ) is the DNA repair process that fixes double‐strand breaks throughout the cell cycle. NHEJ is involved in the development of B and T lymphocytes through its function in V(D)J recombination and class switch recombination ( CSR ). NHEJ consists of several core and accessory factors, including Ku70, Ku80, XRCC 4, DNA ligase 4, DNA ‐ PK cs, Artemis, and XLF . Paralog of XRCC 4 and XLF ( PAXX ) is the recently described accessory NHEJ factor that structurally resembles XRCC 4 and XLF and interacts with Ku70/Ku80. To determine the physiological role of PAXX in mammalian cells, we purchased and characterized a set of custom‐generated and commercially available NHEJ ‐deficient human haploid HAP 1 cells, PAXX Δ , XRCC 4 Δ , and XLF Δ . In our studies, HAP 1 PAXX Δ cells demonstrated modest sensitivity to DNA damage, which was comparable to wild‐type controls. By contrast, XRCC 4 Δ and XLF Δ HAP 1 cells possessed significant DNA repair defects measured as sensitivity to double‐strand break inducing agents and chromosomal breaks. To investigate the role of PAXX in CSR , we generated and characterized Paxx −/− and Aid −/− murine lymphoid CH 12F3 cells. CSR to IgA was nearly at wild‐type levels in the Paxx −/− cells and completely ablated in the absence of activation‐induced cytidine deaminase ( AID ). In addition, Paxx −/− CH 12F3 cells were hypersensitive to zeocin when compared to wild‐type controls. We concluded that Paxx ‐deficient mammalian cells maintain robust NHEJ and CSR .

Serotonin is a highly conserved and ubiquitous signalling molecule involved in a vast variety of biological processes. Approximately 90% of serotonin is produced in the gastrointestinal tract, where it is suggested to act as a prominent regulatory molecule in the inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC). Extracellular and circulating serotonin levels are thought to be elevated during intestinal inflammation, but the underlying mechanisms has been poorly understood. The data on human material is limited, contradictory and in need of further investigation and substantiating. In this study we show a potent and significant downregulation of the dominant serotonin reuptake transporter (SERT) mRNA (SLC6A4) in active CD ileitis, CD colitis and UC colitis, compared to healthy controls. The mRNA of the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase 1 was unregulated. Immunohistochemistry showed expression of SERT protein in both the epithelium and the lamina propria and localised the downregulation to the epithelial monolayer. Laser capture microdissection followed by RNA sequencing confirmed downregulation of SLC6A4 in the epithelial monolayer during intestinal inflammation. Patient-derived colon epithelial cell lines (colonoids) incubated with the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) reduced SERT expression. In summary, these results show that intestinal inflammation potently reduces the expression of SERT in both CD and UC, and that TNFα alone is sufficient to induce a similar reduction in colonoids. The reduced serotonin reuptake capacity may contribute to the increased interstitial serotonin level associated with intestinal inflammation.

In recent years it has become apparent that the epithelium is highly involved in inflammatory bowel disease (IBD) pathophysiology. The majority of gene expression studies of IBD are generated from heterogeneous biopsies, providing no distinction between immune cells, the epithelium and other mucosal cells. By using laser capture microdissection (LCM) coupled with RNA sequencing, we aimed to characterize the expressional changes of the isolated colonic epithelial monolayer from ulcerative colitis (UC) and Crohn’s disease (CD) patients compared to healthy controls (HC). The analysis identified 3706 genes as differentially expressed between active IBD epithelium and HC. Weighted gene co-expression network analysis was used to stratify genes into modules, which were subsequently characterized using enrichment analysis. Our data show a distinct upregulation of the antigen presentation machinery during inflammation, including major histocompatibility complex class II molecules (e.g. HLA-DPA1, HLA-DPB1, HLA-DRA) and key transcription factors/activators (STAT1, IRF1, CIITA). We also see an epithelial downregulation of retinoic acid-responsive nuclear receptors (RARA, RARB, RXRA), but upregulation of retinoid-metabolizing enzymes (RDH11, ALDH1A2, ALDH1A3), which together suggest a perturbation of epithelial vitamin A signaling during active IBD. Lastly, we identified a cluster of stress-related genes, including activator protein 1 components JUNB and ATF3, as significantly upregulated in active UC but not in CD, revealing an interesting aspect of IBD heterogeneity. The results represent a unique resource for enhanced understanding of epithelial involvement in IBD inflammation and is a valuable tool for further studies on these processes.

Background Gene expression analyses on IBD tissue are dominated by analyses of biopsy homogenates that are highly heterogeneous due to a difference in cell populations when contrasting inflamed vs. healthy mucosa. We wished to characterise the inflammatory activity of the isolated epithelial monolayer (EM), avoiding the infiltrating inflammatory cells, thereby form a comprehensive understanding of the epithelium’s role in IBD pathobiology. Methods Total RNA was isolated from laser capture microdissected (LCM) colon EM, and sequencing libraries were prepared using TruSeq RNA access kit (Illumina, CA, USA). Gene cluster analysis (WGCNA) was used to stratify the expression data in genetic modules. Following this, we characterised the modules correlated to IBD status, using Metacore pathway analysis, in order to suggest what underlying biological processes contribute to the observed correlation of expression data. Finally, we used immunohistochemistry to verify the protein expression pattern of key players in the suggested pathways, thereby confirming EM involvement in these processes during inflammation. Results Biopsies from active (a) IBD (7 UCa, 5 CDa) and from un-inflamed (u) IBD and healthy control (7 UCu, 5 CDu and 6 HC) were included in the analysis. 6386 genes were differentially expressed (adjusted p-value (pval.) <0.05) in EM from active inflamed colon vs. un-inflamed (Figure 1). Six gene modules showed a significant correlation between inflammatory status and gene expression (Figure 2). Of note was a broad, significant up-regulation of MHC class II presentation machinery, e.g. STAT1 (log2 fold change (lfc) 0.93, p < 0.001), CIITA (lfc 2.57, p < 0.001) and HLA-DRB1 (lfc 3.69, p < 0.001), and a down-regulation unique to epithelial cells of the retinoic acid receptors RARA (lfc −0.79, p < 0.001) and RXRA (log fc −0.56, p < 0.001). The most significantly up-regulated pathway was ‘Immune Response’, induction of antigen presentation machinery by IFN-γ (FDR 2.5 × 10–18), while ‘RXR-dependent regulation of lipid metabolism’ (FDR 2.6 × 10–8) was the most significantly down-regulated pathway. Conclusion Our analyses suggest that the involvement of the EM in IBD inflammation goes far beyond acting as a barrier. The largest genetic modules are dominated by genes important for inflammatory regulation, suggesting that the EM is much more involved in these processes than what is commonly understood. This study lends support to further analyses of interplay between epithelial cells and immune cells, particularly in a pro-inflammatory environment.