The Role Of Growth Factor Pgrn In Endotoxin Induced Inflammatory Response

Inflammation is a complex physiological or pathological reactions stimulated by internal or external environment, characterized by degeneration, exudation and proliferation.The clinical symptoms are known as redness and swelling with heat and pain. Inflammation is in general a protective defensive response. Meanwhile inflammation could be a common pathway to various diseases, and plays an important role in the occurrence and progression of such many diseases:infections, cancer, cardiovascular diseases, Alzheimer’s and neurodegenerative diseases, allergic diseases, mental illness and other major diseases. Sepsis is a severe systemic inflammatory response syndrome(SIRS) caused by infection characterized by a natural immune response disorders, showed strongly inflammatory response accompanied with enhanced inflammatory cytokines production ("cytokine storm") in the early stage, and result in apoptosis of T lymphocytes, which lead to immune suppression and oxidative damage and following organ failure and body death. Because of the complex pathogenesis, sepsis possesses high morbidity and mortality. Progranulin (PGRN) is a growth factor, with a wide range of physiological functions. It is not only able to regulate the growth, homeostasis and repair of healthy tissue, but also involved in many pathological processes such as tumor. PGRN has anti-inflammatory action, it is a potential inhibitor of tumour necrosis factor a (TNF-a), can inhibit neutrophil respiratory burst induced by TNF-a. PGRN also plays a key role in the autoimmune diseases such as rheumatoid arthritis. Recent studies reported that macrophages from PGRN-deficient mice released less interleukin-10and more inflammatory cytokines than wild type (WT) when exposed to lipopolysaccharide. PGRN-deficient mice failed to clear Listeria monocyiogenes infection as quickly as WT. In this study, we used peretoneal macrophage (PM), cecal ligation and puncture (CLP) and endotoxic shock models to investigate the effects of PGRN in the inflammatory responses in vitro and vivo.1. Inflammatory responses of PGRN-deficient peritoneal macrophage to bacterial lipopolysaccharide in vitro.Objective To investigate the effects of PGRN in the inflammatory responses of peritoneal macrophages (PMs) to bacterial lipopolysaccharide (LPS) in vitro.Methods Peritoneal exudate cells (PECs) were induced and extracted from wild-type (WT) mice and PGRN gene knock-out mice (KO), then the number, morphology and classes of PECs were subsequently evaluated. Surface markers CD11b and F4/80of PMs were tested by flow cytometry. PMs derived from WT or KO mice were treated with LPS and WT PMs were treated with PBS, LPS, recombinant PGRN or LPS plus recombinant PGRN respectively. Supernatants of cultivation were collected after24-hours incubation and concentration of TNF-α, IL-1β\IL-12and production of NO were detected by ELISA or Griess assay respectively. Results There were no significant differences in cell number, classes and expression of surface makers CD11b and F4/80between WT and KO mice-derived PECs. Higher concentration of TNF-α, IL-1β, IL-12and more NO production were detected in the supernatants of KO PMs stimulated by LPS compared to those of WT PMs. Additionally, recombinant PGRN dramatically inhibited the production of pro-inflammatory cytokines TNF-α, IL-1β, IL-12and inflammatory intermediate NO of WT PMs stimulated by LPS. Conclusion PGRN KO PECs are no significant differences with WT PMs in phenotypes but PGRN KO PMs display a stronger inflammatory response than WT PMs when treated with LPS. In addition, recombinant PGRN powerfully inhibits LPS stimulating production of TNF-α, IL-1β, IL-12and NO of PMs.2. Inflammatory responses of PGRN-deficient mice in CLP and endotoxic shock models.Objective To investigate the effects of PGRN in the inflammatory responses in vivo.Methods PGRN KO and WT mice were operated with CLP for sepsis and built endotoxin shock models by intraperitoneal injection with different doses of LPS, then monitored the survival of mice for7d. In CLP models, the infections were monitored by counting the bacterial colony-forming units in the blood and peritoneal lavage at24h after surgery. TNF-a, IL-10in serum of two models were detected by ELISA.Results The mortality of PGRN KO mice caused by CLP was higher than that of WT mice. Bacterial colonizations of PGRN KO group both in blood and peritoneal lavage were more than that of WT group. Serum from KO mice24h after CLP surgery displayed higher concentration of TNF-a, and lower concentration of IL-10compared with that from WT mice. In endotoxic shock model, KO and WT groups were both completely died when mice were injected with lethal dose of LPS, but mice of KO group died faster. At the same time higher concentration of TNF-a were detected in KO groups. While LPS in median lethal dose (LD50) of WT group were injected to KO group, mortality of KO mice was100%and obviously occurred earlier. Concentration of TNF-a were evidently higher in KO group. Conclusion PGRN KO mice have increased susceptibility to sepsis and endotoxin shock with higher inflammatory cytokines production and stronger inflammatory response.In summary, we observed (1) There are no significant differences in cell number, classes and expression of surface makers CD11b and F4/80between WT and KO mice-derived PECs;(2) PGRN KO PMs display a stronger inflammatory response than WT PMs when treated with LPS;(3) The mortality of PGRN KO mice caused by CLP or endotoxic shock is higher than that of WT mice. Hence, to compare the immune responses with WT mice and PGRN KO mice in vivo and in vitro, this paper proves PGRN gene knockout results in more sensitive and stronger immune response in vitro and in vivo by endotoxin stimulation, which provides a theoretical and experimental basis to further explore the role of PGRN in the anti-inflammatory mechanism and inflammatory signaling pathways.