cGAS recruitment to micronuclei is dictated by pre-existing nuclear chromatin status.

Micronuclei (MN) are aberrant cytosolic compartments containing broken genomic fragments or whole lagging chromosomes. MN envelopes irreversibly rupture, allowing the viral receptor cGAS to localize to MN and initiate an inflammatory signalling cascade. Here, we demonstrate that MN envelope rupture is not sufficient for cGAS localization. Unlike MN that arise following ionizing radiation (IR), ruptured MN generated from acute transcription stressors DRB or siSRSF1 are refractory to cGAS localization. Recruitment of cGAS to MN is blocked by inhibiting the histone methyltransferase DOT1L prior to IR exposure, demonstrating that cGAS recruitment to MN is dictated by nuclear chromatin organization at the time of DNA damage. Loss of cGAS+ MN, caused either by acute transcription stressors or by preventing DOT1L-deposited histone methylation, corresponded to significantly decreased cGAS-dependent inflammatory signalling. These results implicate nuclear chromatin organization in micronuclear composition and activity, influencing the ability of damage-induced MN to retain cytosolic proteins upon rupture.

Interleukin-17 Cytokines and Receptors: Potential Amplifiers of Tendon Inflammation

Interleukin (IL)-17A, a pro-inflammatory cytokine that is linked to the pathology of several inflammatory diseases, has been shown to be upregulated in early human tendinopathy and to mediate inflammatory and tissue remodelling events. However, it remains unclear which cells in tendons can respond to IL-17A, and how IL-17A, and its family members IL-17F and IL-17AF, can affect intracellular signalling activation and mRNA expression in healthy and diseased tendon-derived fibroblasts. Using well-phenotyped human tendon samples, we show that IL-17A and its receptors IL-17RA and IL-17RC are present in healthy hamstring, and tendinopathic and torn supraspinatus tendon tissue. Next, we investigated the effects of IL-17A, IL-17F, or IL-17AF on cultured patient-derived healthy and diseased tendon-derived fibroblasts. In these experiments, IL-17A treatment significantly upregulated IL6, MMP3, and PDPN mRNA expression in diseased tendon-derived fibroblasts. IL-17AF treatment induced moderate increases in these target genes, while little change was observed with IL-17F. These trends were reflected in the activation of intracellular signalling proteins p38 and NF-κB p65, which were significantly increased by IL-17A, modestly increased by IL-17AF, and not increased by IL-17F. In combination with TNF-α, all three IL-17 cytokines induced IL6 and MMP3 mRNA expression to similar levels. Therefore, this study confirms that healthy and diseased tendon-derived fibroblasts are responsive to IL-17 cytokines and that IL-17A induces the most profound intracellular signalling activation and mRNA expression of inflammatory genes, followed by IL-17AF, and finally IL-17F. The ability of IL-17 cytokines to induce a direct response and activate diverse pro-inflammatory signalling pathways through synergy with other inflammatory mediators suggests a role for IL-17 family members as amplifiers of tendon inflammation and as potential therapeutic targets in tendinopathy.

Mini Review: Structure and Function of Nematode Phosphorylcholine-Containing Glycoconjugates

An unusual aspect of the biology of nematodes is the covalent attachment of phosphorylcholine (PC) to carbohydrate in glycoconjugates. Investigation of the structure of these molecules by ever-increasingly sophisticated analytical procedures has revealed that PC is generally in phosphodiester linkage with C6 of N-acetylglucosamine (GlcNAc) in both N-type glycans and glycosphingolipids. Up to five PC groups have been detected in the former, being located on both antenna and core GlcNAc. The PC donor for transfer to carbohydrate appears to be phosphatidylcholine but the enzyme responsible for transfer remains to be identified. Work primarily involving the PC-containing Acanthocheilonema viteae secreted product ES-62, has shown that the PC attached to nematode N-glycans possesses a range of immunomodulatory properties, subverting for example, pro-inflammatory signalling in various immune system cell-types including lymphocytes, mast cells, dendritic cells and macrophages. This has led to the generation of PC-based ES-62 small molecule analogues (SMAs), which mirror the parent molecule in preventing the initiation or progression of disease in mouse models of a number of human conditions associated with aberrant inflammatory responses. These include rheumatoid arthritis, systemic lupus erythematosus and lung and skin allergy such that the SMAs are considered to have widespread therapeutic potential.

Monocyte Regulation By Soluble Uric Acid

<p>Hyperuricaemia is a chronic condition associated with diseases of the metabolic syndrome. However, the cause and effect relationship between increased serum uric acid (UA) levels and the pathophysiology of metabolic dysfunction is far from clear. From an immunological angle hyperuricaemia has been shown to modulate inflammatory signalling in both immune and nonimmune cell types. Blood monocytes are constantly exposed to soluble UA in the circulation but the direct effect of this exposure has not been examined. This research focuses on the how soluble UA alters blood monocyte responses to inflammatory stimuli using in vitro, in vivo and clinical manipulation of UA levels. The Harper group previously found that blood monocytes from hyperuricaemic individuals produced lower levels of inflammatory cytokines compared to monocytes from healthy controls when stimulated ex vivo with LPS. My research began by studying the direct effect of soluble UA on human blood monocytes in vitro. I found that soluble UA reduced monocyte production of pro-inflammatory cytokines and increased IL-10 in response to stimulation with LPS. I identified two inflammatory signalling pathways modulated by soluble UA that could be contributing to this suppressive monocyte phenotype: MAP kinase phosphorylation was reduced alongside increased expression of the regulatory protein DUSP10 and reduced ASC; there was a switch towards anti-inflammatory NFκB signalling illustrated by decreased p65 and increased p50 nuclear translocation. To study the modulation of soluble UA levels in a physiological context I raised serum UA levels in vivo with a model of acute hyperuricaemia and lowered serum UA using two clinically relevant medications: allopurinol and rasburicase. Consistent with in vitro UA treatment, raising serum UA levels in vivo suppressed pro-inflammatory cytokine responses to LPS, increased IL-10 and down-regulated monocyte MAP kinase and NFκB signalling pathways. Acute urate-lowering therapy (ULT) with allopurinol or rasburicase reversed this suppressive inflammatory cytokine and signalling pattern. The PLT2 mouse strain has had the purine metabolic pathway disrupted by random mutagenesis of the gene encoding 5-hiydroxyisourate hydrolase, the enzyme responsible for degradation of the molecule directly downstream of UA, 5-hydroxyisourate. I found that this mutation resulted in chronic hyperuricaemia with an average 2-fold increase in serum UA over C57 mice. LPS challenge resulted in increased IL-10 production in PLT2 mice compared to C57, however no differences in monocyte inflammatory signalling were observed between the two strains. Acute ULT with rasburicase reduced serum UA in the PLT2 strain and subsequent LPS challenge increased monocyte inflammatory signalling. Finally, I studied the effects of ULT on the inflammatory phenotype of human blood monocytes from patients with hyperuricaemia. ULT significantly reduced serum UA levels, which coincided with reduced blood monocyte percentages and adhesion molecule expression (CD11b and ICAM1). ULT increased the inflammatory potential of human blood monocytes: Monocytes stimulated with LPS produced less IL-10; MAP kinase phosphorylation increased alongside increased ASC expression; nuclear translocation of NFκB p65 was increased. ULT also increased expression of the NLRP3 inflammasome components procaspase1, pro-IL-1β and NLRP3. Taken together these results demonstrate a previously unidentified role for soluble UA in moderating monocyte immune responses to inflammatory stimuli. In vitro, in vivo and clinical experimentation all confirmed the immunosuppressive function of soluble UA. This potentially places UA in the centre of innate immune control through the dichotomy of its suppressive soluble effects, demonstrated herein, and the widely reported inflammatory crystalline effects. Importantly, this research illustrates that serum UA levels can be manipulated in a clinical setting to control the inflammatory phenotype of circulating immune cells.</p>

Monocyte Regulation By Soluble Uric Acid

<p>Hyperuricaemia is a chronic condition associated with diseases of the metabolic syndrome. However, the cause and effect relationship between increased serum uric acid (UA) levels and the pathophysiology of metabolic dysfunction is far from clear. From an immunological angle hyperuricaemia has been shown to modulate inflammatory signalling in both immune and nonimmune cell types. Blood monocytes are constantly exposed to soluble UA in the circulation but the direct effect of this exposure has not been examined. This research focuses on the how soluble UA alters blood monocyte responses to inflammatory stimuli using in vitro, in vivo and clinical manipulation of UA levels. The Harper group previously found that blood monocytes from hyperuricaemic individuals produced lower levels of inflammatory cytokines compared to monocytes from healthy controls when stimulated ex vivo with LPS. My research began by studying the direct effect of soluble UA on human blood monocytes in vitro. I found that soluble UA reduced monocyte production of pro-inflammatory cytokines and increased IL-10 in response to stimulation with LPS. I identified two inflammatory signalling pathways modulated by soluble UA that could be contributing to this suppressive monocyte phenotype: MAP kinase phosphorylation was reduced alongside increased expression of the regulatory protein DUSP10 and reduced ASC; there was a switch towards anti-inflammatory NFκB signalling illustrated by decreased p65 and increased p50 nuclear translocation. To study the modulation of soluble UA levels in a physiological context I raised serum UA levels in vivo with a model of acute hyperuricaemia and lowered serum UA using two clinically relevant medications: allopurinol and rasburicase. Consistent with in vitro UA treatment, raising serum UA levels in vivo suppressed pro-inflammatory cytokine responses to LPS, increased IL-10 and down-regulated monocyte MAP kinase and NFκB signalling pathways. Acute urate-lowering therapy (ULT) with allopurinol or rasburicase reversed this suppressive inflammatory cytokine and signalling pattern. The PLT2 mouse strain has had the purine metabolic pathway disrupted by random mutagenesis of the gene encoding 5-hiydroxyisourate hydrolase, the enzyme responsible for degradation of the molecule directly downstream of UA, 5-hydroxyisourate. I found that this mutation resulted in chronic hyperuricaemia with an average 2-fold increase in serum UA over C57 mice. LPS challenge resulted in increased IL-10 production in PLT2 mice compared to C57, however no differences in monocyte inflammatory signalling were observed between the two strains. Acute ULT with rasburicase reduced serum UA in the PLT2 strain and subsequent LPS challenge increased monocyte inflammatory signalling. Finally, I studied the effects of ULT on the inflammatory phenotype of human blood monocytes from patients with hyperuricaemia. ULT significantly reduced serum UA levels, which coincided with reduced blood monocyte percentages and adhesion molecule expression (CD11b and ICAM1). ULT increased the inflammatory potential of human blood monocytes: Monocytes stimulated with LPS produced less IL-10; MAP kinase phosphorylation increased alongside increased ASC expression; nuclear translocation of NFκB p65 was increased. ULT also increased expression of the NLRP3 inflammasome components procaspase1, pro-IL-1β and NLRP3. Taken together these results demonstrate a previously unidentified role for soluble UA in moderating monocyte immune responses to inflammatory stimuli. In vitro, in vivo and clinical experimentation all confirmed the immunosuppressive function of soluble UA. This potentially places UA in the centre of innate immune control through the dichotomy of its suppressive soluble effects, demonstrated herein, and the widely reported inflammatory crystalline effects. Importantly, this research illustrates that serum UA levels can be manipulated in a clinical setting to control the inflammatory phenotype of circulating immune cells.</p>

Loss of LKB1-NUAK1 Signalling Enhances NF-κB Activity in a Spheroid Model of High-Grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy often diagnosed at an advanced stage. Although most HGSOC patients respond initially to debulking surgery combined with cytotoxic chemotherapy, many ultimately relapse with platinum-resistant disease. Thus, improving outcomes requires new ways of limiting metastasis and eradicating residual disease. We identified previously that Liver kinase B1 (LKB1) and its substrate NUAK1 are implicated in EOC spheroid cell viability and are required for efficient metastasis in orthotopic mouse models. Here, we sought to identify additional signalling pathways altered in EOC cells due to LKB1 or NUAK1 loss-of-function. Transcriptome analysis revealed that inflammatory signalling mediated by NF-κB transcription factors is hyperactive due to LKB1-NUAK1 loss in HGSOC cells and spheroids. Upregulated NF-κB signalling due to NUAK1 loss suppresses reactive oxygen species (ROS) production and sustains cell survival in spheroids. NF-κB signalling is also activated in HGSOC precursor fallopian tube secretory epithelial cell spheroids, and is further enhanced by NUAK1 loss. Finally, immunohistochemical analysis of OVCAR8 xenograft tumors lacking NUAK1 displayed increased RelB expression and nuclear staining. Our results support the idea that NUAK1 and NF-κB signalling pathways together regulate ROS and inflammatory signalling, supporting cell survival during each step of HGSOC pathogenesis. We propose that their combined inhibition may be efficacious as a novel therapeutic strategy for advanced HGSOC.