Abstract
Interleukin 6 (IL-6), acting via the IL-6 receptor (IL6R) and signal transducer and activator of transcription-3 (STAT3), limits neutrophil recruitment once bacterial infections are resolved. Bovine endometritis is an exemplar mucosal disease, characterized by sustained neutrophil infiltration and elevated IL-6 and IL-8, a neutrophil chemoattractant, following postpartum Gram-negative bacterial infection. The present study examined the impact of the IL6R/STAT3 signaling pathway on IL-8 production by primary endometrial cells in response to short- or long-term exposure to lipopolysaccharide (LPS) from Gram-negative bacteria. Tyrosine phosphorylation of STAT3 is required for DNA binding and expression of specific targets genes. Immunoblotting indicated constitutive tyrosine phosphorylation of STAT3 in endometrial cells was impeded by acute exposure to LPS. After 24 h exposure to LPS, STAT3 returned to a tyrosine phosphorylated state, indicating cross-talk between the Toll-like receptor 4 (TLR4) and the IL6R/STAT3 signaling pathways. This was confirmed by short interfering RNA targeting the IL6R, which abrogated the accumulation of IL-6 and IL-8, induced by LPS. Furthermore, there was a differential endometrial cell response, as the accumulation of IL-6 and IL-8 was dependent on STAT3, suppressor of cytokine signaling 3, and Src kinase signaling in stromal cells, but not epithelial cells. In conclusion, positive feedback through the IL6R amplifies LPS-induced IL-6 and IL-8 production in the endometrium. These findings provide a mechanistic insight into how elevated IL-6 concentrations in the postpartum endometrium during bacterial infection leads to marked and sustained neutrophil infiltration.
Highlights
suppressor of cytokine signaling 3 (SOCS3) is tyrosine phosphorylated in response to many growth factors and upon phosphorylation interacts with the Ras inhibitor p120 (RasGAP), resulting in sustained Ras-dependent activation of ERK1/2.16 Considering ERK1/2 may be important for SOCS3 stability, we explored whether SOCS3 interacted with RasGAP using co-immunoprecipitation and immunoblot analysis
Using targeted short interfering RNA (siRNA), IL-6 receptor (IL6R) proved to be critical in maintaining Interleukin 6 (IL-6) and IL-8 production in epithelial and stromal cells
Reducing signal transducer and activator of transcription 3 (STAT3) or SOCS3 in epithelial cells, did not affect IL-6 or IL-8 production, whereas STAT3 and SOCS3 were essential for IL-6 production in stromal cells
Summary
The mechanisms of response to pathogens and their pathogenassociated molecular patterns, such as Escherichia coli and lipopolysaccharide (LPS), is orchestrated by pattern-recognition receptors, including the Toll-like receptors (TLRs), and the subsequent downstream activation of nuclear factor-kB (NF-kB) and mitogen-activated protein kinase (MAPK) signaling pathways, resulting in the production of inflammatory cytokines.[1,2] The signal transducer and activator of transcription 3 (STAT3) pathway orchestrates the inflammatory response through cross-talk with pattern-recognition receptor pathways, such as the TLR family, inducing the production of proinflammatory signaling cytokines, including interleukin (IL)-6.3,4 The multifunctional cytokine IL-6 is produced by many cells, including endometrial cells, in response to infection and damage, and is critical for the pattern of leukocyte recruitment and tissue homeostasis.[1,2,5,6,7] During this process, IL-6 signals and activates STAT3 via the cognate IL-6 receptor (IL6R) complex, which consists of a heterodimer of IL6Ra and gp[130].
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