Protective Effect Of Suppressing STAT3 Activity In LPS-induced Acute Respiratory Distress Syndrome

BackgroundAcute respiratory distress syndrome (ARDS), are devastating clinical conditions with mortality rates as high as 30-60%. ARDS is initiated by a variety of insults, both direct (gastric aspiration) and indirect (sepsis), and the end point of ARDS pathology is diffuse alveolar damage. Moreover, ARDS is a consequence of exposure to various pathogen-associated molecules from microbial organisms such as LPS, an outer-membrane component of Gram-negative bacteria, which is able to stimulate host innate immune responses through TLRs. Previous studies have demonstrated that excessive inflammation is the main cause of ARDS. The innate immune response plays a profound role in the pathogenesis of ARDS. Mononuclear phagocytes serve a critical role in host defense against inhaled environmental insults and toxins. Alveolar macrophages (AM), neutrophils, macrophages and dendritic cells play crucial roles in initiating inflammation and are involved in multiple immunologic processes and tissue injury of ARDS. The inflammatory cells release pro-inflammatory mediators to accelerate inflammation, activate epithelial cells, and elicit further recruitment of neutrophils and macrophages in ARDS.The JAK/STAT pathway is the predominant signaling pathway utilized by over 70 cytokines, and is critical for initiating innate and adaptive immune responses and ultimately constraining inflammatory responses. STAT3, one member of the Signal Transducers and Activators of Transcription family, plays a critical role in inflammation and tumorigenesis. STAT3 is rapidly activated in lungs (30 min) after LPS administration in mice, with maximal activation at 1-2 h. Previous studies demonstrated that STAT3 activation occurs before significant lung injury, which is related to production of the pro-inflammatory cytokines TNF-α and IL-1β, indicating that STAT3 may play a critical role in initiating pulmonary inflammation in ALL Moreover, STAT3 has been shown to be essential for the liver’s response to LPS and IL-6 via activation of downstream acute-phase genes, such as TNF-α, IL-1 and haptoglobin. Recent studies have demonstrated that STAT3 activation is critical for regulating surfactant lipid synthesis in both normal and LPS-injured lungs and the host innate immune response to ALL As a JAK-STAT3 inducible protein, Suppressor of Cytokine Signaling-3 (SOCS3) regulates STAT3 activation via a negative feed-back loop in macrophages.LLL12, a substituted anthraquinone synthesized based on the structure of curcumin using computer aided rational design, binds to the SH2 domain of STAT3 and inhibits STAT3 phosphorylation. The inhibitory efficacy of LLL12 has been characterized in cancer models that have high levels of constitutively activated STAT3. The inhibitory effect of LLL12 on STAT3 activation and subsequent downstream target genes resulted in inhibition of cancer cell viability and promotion of apoptosis. These studies suggest that LLL12 has the potential to be a therapeutic agent for the treatment of human cancers.Given the importance of STAT3 in activation of macrophages and inflammation, we hypothesized that inhibition of STAT3 activation by LLL12 treatment may have a beneficial effect in dampening inflammatory responses in the setting of lung injury. In this study, we demonstrate that STAT3 is activated in BALF macrophages and inflammatory cells in an LPS-induced ALI model. The hyperactivation of STAT3 in LysMCre-SOCS3fl/f1 mice accelerates the severity and inflammatory responses of LPS-induced ALI disease compared to SOCS3fl/fl control mice. Furthermore, the inhibitory effect is observed with both preventive and therapeutic administration of LLL12. Our findings suggest that inhibiting the STAT3 pathway by LLL12 has clinical therapeutic efficacy in lung inflammation.MethodsAnimalsC57BL/6 mice, SOCS3fl/fll mice and LysMCre-SOCS3fl/fl mice were anesthetized by inhaling isoflurane (100 mg/kg), followed by intranasal (i.n.) administration of 50 μl of 149 LPS (100 mg/kg in PBS). Control mice received i.n. instillation of 50 μl PBS. To study the effect of LLL12, LPS-induced ALI mice were treated intraperitoneally (i.p.) with LLL12 (5 mg/kg) or PBS 2 h prior to LPS administration, or 4 h after LPS administration (post-LPS treatment). The mice were sacrificed and perfused using aortic phlebotomy at 24,48,72 and 96 h after LPS administration.Specimen collection:The blood, lung tissue and BALF samples in each group of mice were collected at the same time after modeling 24h,48h,72h and 96h. Lungs were collected, and take the right lung to prepare for HE staining and immunohistochemistry. The BALF was collected to isolate PMN by flow cytometry. After centrifuged, the supernatant of BALF was collected and the expression of TNF-α, IL-1βIL-8 and CCL2 were measured. Bone marrow cells were flushed from the femurs and tibiae of 7-8 wk old C57BL/6, SOCS3fl/fl, or LysMCre-SOCS3fl/fl mice, and the cells were cultured in RPMI 1640 medium. Western blot and RT-PCR were performed to analyze the activation of STAT3 the production of inflammatory cytokines.Results1. LLL12 Treatment Diminishes Lung Inflammatory Responses in LPS-induced ARDS. Unchallenged (UN) mice exhibited a normal pulmonary architecture. There, is a striking infiltration of inflammatory cells, pulmonary edema, and alveolar collapse in lung tissues from LPS challenged mice at the indicated time points up to 96 h compared to unchallenged mice. Notably, all of the lung injury features, including infiltration of inflammatory cells, pulmonary edema, and alveolar collapse, were substantially attenuated in LLL12 treated mice compared to PBS treated mice. Unchallenged (UN) SOCS3fl/fl and LysMCre-SOCS3fl/fl mice exhibited normal pulmonary architecture without evidence of inflammatory features. Inflammation, lung hemorrhage, edema, and macrophage and neutrophil infiltration were enhanced in LPS-challenged LysMCre-SOCS3fl/fl mice compared to SOCS3fl/fl mice. Pre-LPS treatment with LLL12 diminished the remarkable alveolar wall thickness, hemorrhage, alveolar collapse, and inflammatory cell infiltration at the 48 h time point.2. The changes of total cells and CDllb+CD45+and CDllb+/CDllc+ ratio in BALF of ARDS mice. We examined the accumulation of inflammatory cells in lung tissues from LPS-induced ALI mice. A significant increase in total cell numbers and the percentage of lung CD11b+CD45+ macrophages in BALF was observed after LPS administration, compared with unchallenged mice. And a significant increase in total cells and the percentage of CD11b+CD45+ macrophages in BALF was observed in LysMCre-SOCS3fl/fl mice compared to SOCS3fl/fl mice after LPS administration. Pre-LPS treatment with LLL12 significantly reduced these responses. Our results demonstrate that LPS induces an expansion in the CD11b+/CDl1c+ ratio, and pre-LPS treatment with LLL12 decreases this, indicating that inhibition of STAT3 activity suppresses the infiltration of macrophages from the periphery in ALI. The percentage of CD11b+CD45+Ly6C+ cells was significantly enhanced (over 80%) in the LPS-induced ALI model at all time points. Concordant with injury attenuation, the percentage of CD11b+CD45+Ly6C+ macrophages in BALF was markedly decreased in pre-LPS treatment with LLL12 mice.3. The expression of TNF-a, IL-1β and LLL12 in serum and BALF of ARDS mice. LPS administration significantly induced TNF-α, IL-1β and CCL2 expression in serum compared with PBS control mice, and pre-LPS treatment with LLL12 markedly inhibited expression in serum. Similar results were observed in BALF. These data suggest that inhibition of STAT3 activity plays a protective role in LPS-induced ALI. LPS administration significantly induced TNF-α, IL-1β and CCL2 expression in serum and BALF from LysMCre-SOCS3fl/fl mice compared to SOCS3fl/fl mice. Hence, these data suggest that SOCS3 plays a protective role in LPS-induced lung inflammatory responses.4. LLL12 Selectively Blocks STAT3 Activation and Pro-inflammatory Mediators Production in Macrophages. LLL12 at a concentration of 0.5 μM efficiently inhibited LPS-induced STAT3 phosphorylation, while activation of STAT1 or ERK1/2 was not affected. Furthermore. pre-LPS treatment with LLL12 inhibited LPS-induced TNF-α, IL-1β and iNOS mRNA levels in macrophages at 4,8 and 12 h, and at the protein level at 12,24 and 48 h. Deletion of SOCS3 in macrophages resulted in enhanced LPS-induced STAT3 activation compared to cells from SOCS3fl/fl mice, and STAT3 phosphorylation was abolished by LLL12 treatment. Macrophages with deletion of SOCS3 had increased expression of TNF-a and iNOS compared to macrophages from SOCS3 mice. Furthermore, pre-LPS treatment with LLL12 attenuated LPS-stimulated inflammatory gene expression in macrophages from SOCS3fl/fl and LysMCre-SOCS3fl/fl mice.Conclusions1. The activation of STAT3 induced by LPS could regulate the expression and release of cytokines such as TNF-α, IL-1β and CCL2, and hyper-activation of STAT3 in mice accelerates the severity of inflammation in the ALI model.2. Both pre and post-LPS therapeutic treatment with LLL12 decreases LPS-induced inflammatory responses in mice with ALI. Importantly, LLL12 treatment attenuates STAT3 phosphorylation in human peripheral blood mononuclear cells induced by plasma from ARDS patients, which suggests the feasibility of targeting the STAT3 pathway therapeutically for ALI and ARDS patients.3. SOCS3 negatively regulates LPS-induced STAT3 activation and production of inflammatory mediators in BALF, and LLL12 effectively suppresses enhanced lung inflammation in LPS-induced lung injury in LysMCre-SOCS3fl/fl mice with hyper-activation of STAT3.