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Acute lung injury (ALI) is an acute respiratory failure that is associated with excessive neutrophil recruitment into the bronchoalveolar space and results in severe mortality. To evaluate 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) as a therapeutic agent for ALI, PLAG was administered orally to mice intranasally challenged with lipopolysaccharide (LPS). The excessive neutrophil infiltration in the bronchoalveolar lavage fluid (BALF) was detected in LPS-treated mice. Intranasally introduced LPS especially stimulates tissue-resident macrophages in the pulmonary tissue and induces neutrophil-attracting chemokine production such as MIP-2. Co-administrated PLAG dramatically ameliorated neutrophil infiltration into the bronchoalveolar region via modification of TLR4 signaling. We found that PLAG effectively accelerated endocytosis of LPS-TLR4 complex and promoted the NADPH oxidase activity through the formation of Rac, p47phox assembly into membrane for ROS production which results in elimination of exotoxin in Raw264.7 cells. PLAG also triggered a prompt TLR4 signals occurred in endosome mediated by TRIF and terminated signaling including MIP-2 expression when endocytosed exotoxin is cleared. Moreover, immunofluorescence microscopes of RAW264.7 cells showed that PLAG accelerated the endocytosis of the LPS-TLR4 complex and the clearance of the internalized LPS. Our results suggest that PLAG promotes the clearance of invaded exotoxin and eventually triggers an earlier termination of MIP-2 secretion in the exotoxin-cleared macrophages, and might be used as a potential therapeutic agent to prevent ALI through speedy resolution of exotoxin.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen and can cause an acute and chronic infection disease in immunocompromised condition. P. aeruginosa has a potent pathogen; causes invasion diseases in hospitalized patients and complicates 90% of cystic fibrosis deaths. NF-κB and STAT3 are important signal molecules in inflammatory responses. Recently, research on the interaction between NF-κB and STAT3 in inflammation has been actively conducted. Previously, we reported that PLAG (1-palmityoyl-2-linoleoyl-3-acetyl-rac- glycerol) has an effect on regulation of STAT3 activation in diverse disease model. Therefore, we investigated whether PLAG has an effect on bacteria-infected mice with chemotherapy-induced neutropenia. To establish neutropenic mouse model, we used Cyclophosphamide and doxorubicin (AC regimen). And P. aeruginosa was administrated by intranasal injection in AC regimen induced neutropenic mice and followed by oral administration of PLAG during the experiment. We observed that survival rate of P. aeruginosa infected neutropenic mice is increased and bacteria in BALF is decreased by PLAG at early time. To reveal the function of PLAG on increase of infected bacteria elimination, we checked out neutrophil recruitment and NETosis. We observed that neutrophil recruitment in BALF was not increased significantly, but NETosis occured by P. aeruginosa at early time. And PLAG enhanced NETosis through NF-κB activation via down-regulation of STAT3. Based on these data, we suggest that role of PLAG on bacteria clearance, regulates interaction between NF-κB and STAT3 activation through P. aeruginosa induced pneumonia model with CIN and PLAG could be help immunocompromised patients.

Chemotherapy-induced neutropenia (CIN) is a common side effect that necessitate dose reductions during treatment of cancer patients. Preventing CIN is critical in chemotherapy because a rapid decline of neutrophil counts increase susceptibility to infection of cancer patients. In spite of its importance, the mechanism of CIN still remains unclear. This study is aimed to investigate the mechanism of CIN in an aspect of neutrophil activation by chemotaxis in gemcitabine-treated breast tumor model. We hypothesized that gemcitabine-generated ROS promotes CXCL8 or MIP-2 production by upregulating ERK pathway in PKCδ-activated intratumoral macrophages and thus neutrophil recruitment to the chemokine gradient. In 4T1 breast tumor mouse model, we found that the basal level of PKCδ activity was higher than in normal mice. The number of blood and peritoneal leukocytes was analyzed at 24h after intraperitoneal administration of gemcitabine. The neutrophil counts decreased in the blood but increased in the peritoneal cavity. In addition, intratumoral neutrophils were elevated in the gemcitabine-treated group. The mRNA levels of neutrophil attracting chemokines/receptors were increased in the peritoneal and tumoral cells. ROS inhibitors N-Acetyl-L-cysteine and Apocynin blocked gemcitabine-induced neutrophil migration, mRNA expression of the chemokines and receptors and PKCδ/ERK phosphorylation. CXCR1/2 inhibitor reparixin decreased neutrophil recruitment to the peritoneal cavity and the tumor tissue. Together, our results suggest that gemcitabine treatment promotes neutrophil chemotactic activity by increasing ROS/PKCδ/ERK/CXCL8 pathway. These findings will provide new insights for developing a therapeutic for CIN.

Increased number of eosinophils in the circulation and sputum is associated with the severity of asthma. The respiratory epithelium produces chemokine (C-C motif) ligands (CCL) which recruits and activates eosinophils. A chemically synthesized monoacetyl-diglyceride, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) is a major constituent in the antlers of Sika deer (Cervus nippon Temminck) which has been used in oriental medicine. This study was aimed to investigate the molecular mechanism of PLAG effect on the alleviation of asthma phenotypes. A549, a human alveolar basal epithelial cell, and HaCaT, a human keratinocyte, were activated by the treatment of interleukin-4 (IL-4), and the expression of chemokines, known to be effective on the induction of eosinophil migration was analyzed by RT-PCR. The expression of IL-4 induced genes was modulated by the co-treatment of PLAG. Especially, CCL26 expression from the stimulated epithelial cells was significantly blocked by PLAG, which was confirmed by ELISA. The transcriptional activity of signal transducer and activator of transcription 6 (STAT6), activated by IL-4 mediated phosphorylation and nuclear translocation, was down-regulated by PLAG in a concentration-dependent manner. In ovalbumin-induced mouse model, the infiltration of immune cells into the respiratory tract was decreased by PLAG administration. Cytological analysis of the isolated bronchoalveolar lavage fluid (BALF) cells proved the infiltration of eosinophils was significantly reduced by PLAG. In addition, PLAG inhibited the migration of murine bone marrow-derived eosinophils, and human eosinophil cell line, EoL-1, which was induced by the addition of A549 culture medium.

Inappropriate regulation of leukocyte trafficking can lead to impaired neutrophil clearance and increased tissue damage from the accumulation of neutrophil-secreted proteases and reactive oxygen species at the site of inflammation. In this study, we have discovered that 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) regulates the activity of signal transducer and activator of transcription 3 (STAT3), which is a master regulator of IL-6 expression. PLAG has been isolated from the antlers of Sika deer (Cervus nippon Temminck), which were known to have immunosuppressive and anti-arthritic activities. PLAG induced a significant decrease of IL-6 production in a macrophage cell line, RAW264.7, and rheumatoid arthritis-fibroblast-like synoviocyte (RA-FLS) via the regulation of STAT3 signaling. IL-6, a multifunctional pro-inflammatory cytokine, plays a critical role in the pathogenesis of joint and systemic inflammation in RA. In collagen-induced arthritis (CIA) mouse model, arthritic symptoms were recapitulated with an increase of IL-6 in the synovium, which was recuperated by the treatment of PLAG to the level comparable with commercial therapeutics (such as Remicade or Methotrexate). When the joint tissues were stained with neutrophil-specific antibodies, PLAG significantly reduced the infiltration of neutrophils into the joint synovium of CIA mouse. Therefore, PLAG inhibits the infiltration of destructive neutrophils into inflammatory sites, and can be utilized as a therapeutic agent for the treatment of sustained inflammation and joint destruction.