Sex differences in IL-10's anti-inflammatory function: greater STAT3 activation and stronger inhibition of TNF-α production in male blood leukocytes ex vivo.

Interleukin-10 (IL-10) inhibits proinflammatory cytokine production in blood leukocytes-an effect mediated by signal transducer and activator of transcription 3 (STAT3) activation. To examine potential sex-based differences in IL-10's anti-inflammatory function, we treated whole blood from healthy males and females (n = 16 participants of each sex; age: 28 ± 6 yr; body mass index: 23.5 ± 2.3 kg/m2) with increasing concentrations of IL-10 (1-100 ng/mL) and quantified changes in phosphorylated STAT3 (pSTAT3) in CD14+ monocytes and CD4+ lymphocytes via flow cytometry. In parallel, liposaccharide (LPS)-stimulated whole blood cultures were used to assess sex-based differences in IL-10's ability to inhibit tumor necrosis factor (TNF)-α production. IL-10 concentration dependently increased pSTAT3 median fluorescent intensity (MFI) in CD14+ and CD4+ cells (main effects of concentration, P < 0.01) with males exhibiting larger changes in pSTAT3 MFI in both cell types (main effects of sex, P < 0.01). Accordingly, IL-10-mediated inhibition of TNF-α production was more pronounced in males (main effect of sex, P < 0.01) with changes in other monocyte-derived cytokines (IL-1ß, IL-1RA, and IL-15) also supporting a sexual dimorphism in IL-10 action (P < 0.05). These sex-based differences were not explained by differences in circulating plasma IL-10 concentrations, basal IL-10 receptor expression in unstimulated CD14+ and CD4+ cells, nor the basal expression of IL-10 signaling proteins (STAT3, SHIP1, and p38 MAPK) in unstimulated peripheral blood mononuclear cells. We conclude that IL-10's anti-inflammatory function differs between male and female blood leukocytes ex vivo. This sexual dimorphism should be considered in future work investigating IL-10's anti-inflammatory action in humans as it may represent a mechanism contributing to sex differences in overall immune function.

Interleukin-10 Induces Expression of Neuroendocrine Markers and PDL1 in Prostate Cancer Cells.

Interleukin-10 (IL10) is best studied for its inhibitory action on immune cells and ability to suppress an antitumour immune response. But IL10 also exerts direct effects on nonimmune cells such as prostate cancer epithelial cells. Elevated serum levels of IL10 observed in prostate and other cancer patients are associated with poor prognosis. After first-line androgen-deprivation therapy, prostate cancer patients are treated with androgen receptor antagonists such as enzalutamide to inhibit androgen-dependent prostate cancer cell growth. However, development of resistance inevitably occurs and this is associated with tumour differentiation to more aggressive forms such as a neuroendocrine phenotype characterized by expression of neuron specific enolase and synaptophysin. We found that treatment of prostate cancer cell lines in vitro with IL10 or enzalutamide induced markers of neuroendocrine differentiation and inhibited androgen receptor reporter activity. Both also upregulated the levels of PDL1, which could promote tumour survival in vivo through its interaction with the immune cell inhibitory receptor PD1 to suppress antitumour immunity. These findings suggest that IL10's direct action on prostate cancer cells could contribute to prostate cancer progression independent of IL10's suppression of host immune cells.

Interleukin-10 contributes to PGE2 signalling through upregulation of EP4 via SHIP1 and STAT3.

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 E2 (PGE2). In macrophages, EP4 is a Gαs-protein 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.

Interleukin-10 control of pre-miR155 maturation involves CELF2.

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.

Interleukin-10 and Small Molecule SHIP1 Allosteric Regulators Trigger Anti-inflammatory Effects through SHIP1/STAT3 Complexes.

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.

Topical tranexamic acid inhibits fibrinolysis more effectively when formulated with self-propelling particles.

BACKGROUND: Endogenous fibrinolytic activation contributes to coagulopathy and mortality after trauma. Administering tranexamic acid (TXA), an antifibrinolytic agent, is one strategy to reduce bleeding; however, it must be given soon after injury to be effective and minimize adverse effects. Administering TXA topically to a wound site would decrease the time to treatment and could enable both local and systemic delivery if a suitable formulation existed to deliver the drug deep into wounds adequately. OBJECTIVES: To determine whether self-propelling particles could increase the efficacy of TXA. METHODS: Using previously developed self-propelling particles, which consist of calcium carbonate and generate CO2 gas, TXA was formulated to disperse in blood and wounds. The antifibrinolytic properties were assessed in vitro and in a murine tail bleeding assay. Self-propelled TXA was also tested in a swine model of junctional hemorrhage consisting of femoral arteriotomy without compression. RESULTS: Self-propelled TXA was more effective than non-propelled formulations in stabilizing clots from lysis in vitro and reducing blood loss in mice. It was well tolerated when administered subcutaneously in mice up to 300 to 1000 mg/kg. When it was incorporated in gauze, four of six pigs treated after a femoral arteriotomy and without compression survived, and systemic concentrations of TXA reached approximately 6 mg/L within the first hour. CONCLUSIONS: A formulation of TXA that disperses the drug in blood and wounds was effective in several models. It may have several advantages, including supporting local clot stabilization, reducing blood loss from wounds, and providing systemic delivery of TXA. This approach could both improve and simplify prehospital trauma care for penetrating injury.