Chronic low-grade inflammation contributes to the pathology and complications of type 2 diabetes (T2D). Interleukin-10 (IL10), an anti-inflammatory cytokine, is suggested to play a protective role in T2D. However, the impact of T2D on IL10 function has not been previously assessed. We examined the ability of IL10 to inhibit inflammation in human T2D immune cells and explored underlying mechanisms using macrophage models. IL10 was less effective at inhibiting tumour necrosis factor (TNF)-α secretion in T2D whole blood cultures, which was not explained by altered IL10 receptor surface expression. These findings were observed in macrophages exposed to high glucose, which demonstrated similar IL10 resistance or hyporesponsiveness. These findings were also not explained by changes in IL10 receptor protein or other downstream signaling proteins. High glucose was also shown to impair the ability of IL10 to activate STAT3, a downstream signaling protein of IL10. Treatment with the SHIP1 agonist, AQX-MN100, reversed IL10 hyporesponsiveness in macrophages cultured in high glucose and showed equal effectiveness at different glucose conditions. This data supports the idea that IL10 hyporesponsiveness may contribute to chronic inflammation in T2D. These novel findings suggest that strategies aimed to overcome IL10 hyporesponsiveness may hold therapeutic potential for reducing inflammation in T2D.

The anti-inflammatory cytokine interleukin-10 (IL-10) is essential for attenuating the inflammatory response, which includes reducing the expression of pro-inflammatory microRNA-155 (miR-155) in lipopolysaccharide (LPS) activated macrophages. miR-155 enhances the expression of pro-inflammatory cytokines such as TNFα and suppresses expression of anti-inflammatory molecules such as SOCS1. Therefore, we examined the mechanism by which IL-10 inhibits miR-155. We found that IL-10 treatment did not affect the transcription of the miR-155 host gene nor the nuclear export of pre-miR-155, but rather destabilized both pri-miR-155 and pre-miR-155 transcripts, as well as interfered with the final maturation of miR-155. This inhibitory effect of IL-10 on miR-155 expression involved the contribution of both the STAT3 transcription factor and the phosphoinositol phosphatase SHIP1. This is the first report showing evidence that IL-10 regulates miRNA expression post-transcriptionally.

Summary The anti-inflammatory actions of interleukin-10 (IL10) are thought to be mediated primarily by the STAT3 transcription factor, but pro-inflammatory cytokines such as interleukin-6 (IL6) also act through STAT3. We now report that IL10, but not IL6 signaling, induces formation of a complex between STAT3 and the inositol polyphosphate-5-phosphatase SHIP1 in macrophages. Both SHIP1 and STAT3 translocate to the nucleus in macrophages. Remarkably, sesquiterpenes of the Pelorol family, which we previously described as allosteric activators of SHIP1 phosphatase activity, could induce SHIP1/STAT3 complex formation in cells and mimic the anti-inflammatory action of IL10 in a mouse model of colitis. Using crystallography and docking studies we identified a drug-binding pocket in SHIP1. Our studies reveal new mechanisms of action for both STAT3 and SHIP1 and provide a rationale for use of allosteric SHIP1-activating compounds, which mimic the beneficial anti-inflammatory actions of IL10. Video Abstract

The anti-inflammatory cytokine interleukin-10 (IL10) is essential for attenuating inflammatory responses, which includes reducing the expression of pro-inflammatory microRNA-155 (miR155) in lipopolysaccharide (LPS) activated macrophages. miR155 enhances the expression of pro-inflammatory cytokines such as TNFα and suppresses expression of anti-inflammatory molecules such as SHIP1 and SOCS1. We previously found that IL10 interfered with the maturation of pre-miR155 to miR155. To understand the mechanism by which IL10 interferes with pre-miR155 maturation we isolated proteins that associate with pre-miR155 in response to IL10 in macrophages. We identified CELF2, a member of the CUGBP, ELAV-Like Family (CELF) family of RNA binding proteins, as protein whose association with pre-miR155 increased in IL10 treated cells. CRISPR-Cas9 mediated knockdown of CELF2 impaired IL10's ability to inhibit both miR155 expression and TNFα expression.

Macrophage cells form part of our first line defense against pathogens. Macrophages become activated by microbial products such as lipopolysaccharide (LPS) to produce inflammatory mediators, such as TNFα and other cytokines, which orchestrate the host defense against the pathogen. Once the pathogen has been eradicated, the activated macrophage must be appropriately deactivated or inflammatory diseases result. Interleukin-10 (IL10) is a key anti-inflammatory cytokine which deactivates the activated macrophage. The IL10 receptor (IL10R) signals through the Jak1/Tyk2 tyrosine kinases, STAT3 transcription factor and the SHIP1 inositol phosphatase. However, IL10 has also been described to induce the activation of the cyclic adenosine monophosphate (cAMP) regulated protein kinase A (PKA). We now report that IL10R signalling leads to STAT3/SHIP1 dependent expression of the EP4 receptor for prostaglandin E 2 (PGE 2). In macrophages, EP4 is a G αsprotein coupled receptor that stimulates adenylate cyclase (AC) production of cAMP, leading to downstream activation of protein kinase A (PKA) and phosphorylation of the CREB transcription factor. IL10 induction of phospho-CREB and inhibition of LPS-induced phosphorylation of p85 PI3K and p70 S6 kinase required the presence of EP4. These data suggest that IL10R activation of STAT3/SHIP1 enhances EP4 expression, and that it is EP4 which activates cAMP-dependent signalling. The coordination between IL10R and EP4 signalling also provides an explanation for why cAMP elevating agents synergize with IL10 to elicit anti-inflammatory responses.

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency 1 , potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli. Video LinkThe video component of this article can be found at

28The anti-inflammatory actions of interleukin-10 (IL10) are thought to be mediated primarily by the STAT3 29 transcription factor, but pro-inflammatory cytokines such as interleukin-6 (IL6) also act through STAT3. 30We now report that IL10, but not IL6 signaling, induces formation of a complex between STAT3 and the 31 inositol polyphosphate-5-phosphatase SHIP1 in macrophages. Both SHIP1 and STAT3 translocate to the 32 nucleus in macrophages. Remarkably, sesquiterpenes of the Pelorol family we previously described as 33 allosteric activators of SHIP1 phosphatase activity, could induce SHIP1/STAT3 complex formation in cells, 34 and mimic the anti-inflammatory action of IL10 in a mouse model of colitis. Using crystallography and 35 docking studies we identified a drug-binding pocket in SHIP1. Our studies reveal new mechanisms of 36 action for both STAT3 and SHIP1, and provide a rationale for use of allosteric SHIP1-activating compounds 37 which mimic the beneficial anti-inflammatory actions of IL10. 38 39 1999) much like an IL10 -/mouse (Kuhn et al., 1993, Zigmond et al., 2014. However, STAT3 becomes 63 tyrosine phosphorylated and activated by many stimuli including the pro-inflammatory cytokine 64 interleukin-6 (IL6) (Garbers et al., 2015), so STAT3 activation must differ downstream of IL10 and IL6 65 signaling in order to mediate their opposing actions. 66The SHIP1 phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase is a cytoplasmic protein expressed 67 predominantly in hematopoietic cells (Hibbs et al., 2018, Fernandes, 2013 #1400, Huber et al., 1999 68 Krystal, 2000, Pauls and Marshall, 2017). In response to extracellular signals, SHIP1 can be recruited to 69 the cell membrane and one of its actions can be to turn off phosphoinositide 3-kinase (PI3K) signaling 70 (Brown et al., 2010) by dephosphorylating the PI3K product PIP3 into PI(3,4)P2 (Fernandes et al., 2013, 71 Huber et al., 1999, Krystal, 2000, Pauls and Marshall, 2017. We have shown that SHIP1 phosphatase 72 activity is allosterically activated by its product PI(3,4)P2 and that small molecules of the pelorol family 73 (ZPR-MN100 and ZPR-151) also allosterically enhance SHIP1 phosphatase activity (Meimetis et al., 2012, 74 Ong et al., 2007. These data suggest that stimulating SHIP1 phosphatase activity with small molecule 75 SHIP1 activators could be used to treat inflammatory diseases caused by inappropriately sustained PI3K 76 production of PI(3,4)P2. 77However, in addition to its enzymatic function in hydrolyzing PIP3, SHIP1 can also act as a docking protein 78 for assembly of signaling complexes (Pauls and Marshall, 2017). We previously showed that IL10R 79 signaling requires SHIP1 to inhibit TNFα translation (Chan et al., 2012) but whether SHIP1 and STAT3 80 worked independently or together was not determined. We now report that a SHIP1 protein containing 81 point mutations, which inactivates its phosphatase activity, could still mediate the anti-inflammatory 82 action of IL10, and that SHIP1 and STAT3 associate with each other in respo...

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency 1 , potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.