Abstract
Proinflammatory cytokines such as IL-6 induce endothelial cell (EC) barrier disruption and trigger an inflammatory response in part by activating the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. The protein suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of JAK-STAT, but its role in modulation of lung EC barrier dysfunction caused by bacterial pathogens has not been investigated. Using human lung ECs and EC-specific SOCS3 knockout mice, we tested the hypothesis that SOCS3 confers microtubule (MT)-mediated protection against endothelial dysfunction. SOCS3 knockdown in cultured ECs or EC-specific SOCS3 knockout in mice resulted in exacerbated lung injury characterized by increased permeability and inflammation in response to IL-6 or heat-killed Staphylococcus aureus (HKSA). Ectopic expression of SOCS3 attenuated HKSA-induced EC dysfunction, and this effect required assembled MTs. SOCS3 was enriched in the MT fractions, and treatment with HKSA disrupted SOCS3–MT association. We discovered that—in addition to its known partners gp130 and JAK2—SOCS3 interacts with MT plus-end binding proteins CLIP-170 and CLASP2 via its N-terminal domain. The resulting SOCS3–CLIP-170/CLASP2 complex was essential for maximal SOCS3 anti-inflammatory effects. Both IL-6 and HKSA promoted MT disassembly and disrupted SOCS3 interaction with CLIP-170 and CLASP2. Moreover, knockdown of CLIP-170 or CLASP2 impaired SOCS3–JAK2 interaction and abolished the anti-inflammatory effects of SOCS3. Together, these findings demonstrate for the first time an interaction between SOCS3 and CLIP-170/CLASP2 and reveal that this interaction is essential to the protective effects of SOCS3 in lung endothelium.
Highlights
Cytokine-derived signaling plays an essential role in modulating all kinds of host immune responses
heat-killed Staphylococcus aureus (HKSA)-induced endothelial permeability evidenced by increased FITC fluorescence intensity was much greater in HKSA-treated endothelial cell (EC) transfected with suppressor of cytokine signaling-3 (SOCS3) siRNA (Fig. 1B)
We observed a significant increase of all three EC inflammatory marker proteins following HKSA challenge of SOCS3 siRNA-transfected cells, as compared with NS siRNA-treated groups (Fig. 1C)
Summary
SOCS3 is a known negative regulator of inflammation [10], but its role in modulation of inflammatory lung endothelial barrier dysfunction is not known. We observed a significant increase of all three EC inflammatory marker proteins following HKSA challenge of SOCS3 siRNA-transfected cells, as compared with NS siRNA-treated groups (Fig. 1C). Actin staining with Texas Red Phalloidin revealed increased formation of paracellular gaps in HKSA-challenged EC with SOCS3 knockdown, as compared with NS siRNA-transfected controls (Fig. 1, D and E). These data suggest a critical role of SOCS3 in negative regulation of HKSA- and IL-6-induced EC permeability and inflammation. A higher number of cells and increased protein content were observed in BAL from SOCS3 KO mice following IL-6 injection as compared with matching WT controls (Fig. 2D). The results demonstrated that SOCS3 overexpression diminished HKSA-induced upregulation of these proteins
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