Mechanism Research On The Role Of SPLUNC1in LPS Induced Inflammatory Reaction

It has been reported recently that the chronic inflammation is a major driver of20-25%of human malignancies. To explore the molecular machanisms of the inflammation-cancer linking has become the important focus in the fields of carcinogenesis principle investigation. Gram negative bacteria is the main pathogenic microorganisms co-existing with nasopharygneal epithelial tissue, which is common reasons for chronic inflammation in nasophargneal location. If the nasopharygneal epithelium is infected by pathogenic microorganisms for a long and last time, microorganism infection will lead to chronic inflammation in nasophargneal location. It is well known that endotoxin, such as lipopolysaccharide (LPS), is the main virulence factor of Gram-negative bacteria, which plays its biological role through binding to cell-surface receptors, mainly by CD14and TLR4.SPLUNC1as a secretory protein encoded by nasopharyngeal and respiratory tract epithelia cell is a cellular innate immune molecules. Our previous studies demonstrated that SPLUNC1contains a BPI domain, which can specifically bind to LPS. The recombinant proteins, SPLUNC1, can be secreted by SPLUNC1-transfected cells into culture serum, and also have the potential to interact with LPS and inhibit Pseudomonas aeruginosa. SPLUNC1plays an important role on Preventing infection of pathogenic microorganisms and restraining excessive inflammationIn this study, we took advantage of SPLUNC1overexpressed/downexpressed cell models and SPLUNC1gene knockout cell model and mouse model to expore the effect and molecular mechanism of SPLUNC1on prevention or blocking the reaction process of inflammation-cancer in LPS induced inflammation reaction through TLR4/NF-κB signal pathway, and provided a new molecular mechanism for the tumorgenesis of nasopharyngeal carcinoma (NPC).LPS induces the secretion of inflammatory cytokines by NPC cell and promotes its proliferationLPS is considered to be a key predisposition for chronic inflammation. So, it is very important to recognize LPS for host cells. To investigate the effects of LPS on inflammatory responses by NPC cells, we employed a proper dose of LPS (1ug/ml) to stimulate5-8F and NP-69cells, and then performed Flow Cytometry and MTT assays. The results confirmed that LPS promotes proliferation of the cells. Upon LPS stimulation, the5-8F and NP-69cells were also found to produce the inflammatory cytokines, such as IL-6, IL-8, TNF-a and IL-1β. Furthermore, we utilized LPS (1ug/ml) to stimulate5-8F/SPLUNC1or NP-69/SPLUNC1si cells, as well as respective control cells.24h poststimulation, the cell proteins were collected and detected by Western blot. The results revealed that SPLUNC1reduced increased expression levels of TLR4, MyD88, IκB, p-p65(NF-κB), induced by LPS stimulation. LPS can be recognized by TLR4, subsequently leading to activation of TLR4signials and a rapid production of inflammatory cytokines. Therefore, we explored the mechanisms of LPS in promoting inflammatory responses and NPC cell proliferation. We used Western-blot and Real-time PCR assays to determine the effects of LPS on the expression of signal molecule of TLR4signal, and found that LPS could upregulate the expression of CD14, TLR4, MyD88and p-p65. Next, we also observed that NF-κB (p65) was rapidly located into the nucleus and activated upon LPS treatment by means of luciferase reporter assays and immunofluorescence assay. Constant NF-κB activation was the main reason for chronic inflammation. From these data, we speculated that LPS may promote the secretion of inflammatory cytokines and NPC cell proliferation via activating TLR4-MyD88-NF-κB (p65).The expression and clinical significance of SPLUNC1and TLR4in NPCIn our previous study, we demonstrated that SPLUNC1proteins are absent or significantly down-regulated in NPC tissues; at the mean time, the TLR4expressed on the cell membrane could help tumor cell escape from immunological surveillance and promote the tumor development and progression. Here, we used NPC TMA and immunohistochemical staining to detect the protein expression of SPLUNC1and TLR4in NPC. And we also performed the correlation analysis between them. Nasopharyngeal epithelial tissues expressed high levels of SPLUNC1, while the proteins were strongly down-regulated or even absent in NPC tissues. However, TLR4showed an inverse expression model, that was, TLR4was upregulated in NPC and inflammatory tissue samples compared with nasopharyngeal epithelial tissues. Moreover, increased TLR4expression correlates reversely with decreased SPLUNC1expression in NPC by correlation analysis. Importantly, Kaplan-Meier’s analysis indicated that the lower level of SPLUNC1expression and the higher level of TLR4expression, the worse the patients’prognosis was.SPLUNC1reduces the secretion of LPS-induced inflammatory cytokines and NPC cell proliferation via its suppression of TLR4/NF-KB activation.We previously demonstrated that SPLUNC1contains a BPI domain, which can bind with LPS. Therefore, we assumed that SPLUNC1exerts the inhibitory effects on LPS-induced inflammatory responses. To explore the roles of SPLUNC1,5-8F/SPLUNC1(5-8F stably expressing SPLUNC1) and NP-69/SPLUNC1si (NP-69stably silencing SPLUNC1) cells were constructed. Similarly, we employed LPS to stimulate5-8F/SPLUNC1or NP-69/SPLUNC1si cells, as well as respective control cells, and then detected cell prolifection by using MTT assay.5-8F/SPLUNC1cells displayed a decreased cell proliferation rate compared with5-8F/control cells, while on the stimulation with LPS, NP60/SPLUNC1si cells showed an increased proliferation rate with respect to NP/Control si cells. Furthermore, we revealed that SPLUNC1inhibited LPS-induced NPC cell proliferation, and induced cell cycle G0/G1arrest by Flow Cytometry. By means of qRT-PCR and ELISA assay, we observed that SPLUNC1suppressed the secretion of LPS-induced inflammatory cytokines, such as IL-6, IL-8, TNF-α and IL-1β. These data indicated that SPLUNC1reduces the secretion of LPS-induced inflammatory cytokines and inhibits NPC cell proliferation.Our above results demonstrated that LPS LPS may promote the secretion of inflammatory cytokines and NPC cell proliferation via activating TLR4-MyD88-NF-KB (p65). Therefore, we next investigated whether SPLUNC1reduces the synthesis of inflammatory cytokines induced by LPS via inhibiting TLR4/NF-κB. Luciferase reporter assays and immunofluorescence assay indicated that SPLUNC1could inhibit LPS-induced NF-κB location into nucleus and its activity. ELISA assays indicated that SPLUNC1reduced the production of IL-6, IL-8, TNF-a and IL-1β induced by LPS.To confirm that SPLUNC1has inhibitory effect on LPS-induced inflammatory response, we constructed a Splunc1-knockout mouse model for further study in vivo. We gained the lung tissue and MEF cell of Splunc1-knockout-type mice and wt-type mice to detect the expression of the key molecular of Tlr4/Nf-Kb pathway. and then Splunc1-knockout-type mice and wt-type mice were subjected to the acute inflammation caused by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS,5mg/kg). After24h, lung sections were assessed by HE staining, and serum were isolated for ELISA assays. These results showed that after Spluncl was konckout, the expresion of Tlr4、Myd88、 p-p65(Nf-Kb) in knockout mice was higher than WT mice, and the expresion of Ikba was downregulated in knockout mice, IHC showed that lung section from Splunc1-/-mice expressed higher levels of Tlr4、 Myd88、p-p65(Nf-Kb) than WT mice. In addition, ELISA assays indicated that Spluncl-/-mice has an increased expression of11-6and11-8.In conclusion, SPLUNC1can reduce LPS-induced inflammatory response and NPC cell proliferation, and it exerts its inhibitory effect mainly by down-regulating expression of TLR4and its downstream molecules, and inhibiting NF-κB acitivity. Therefore, we provide evidence that SPLUNC1, as a key molecule of innate immunity, can prevent inflammatory responses induced by bacteria infection, and maintain microenvironment stability.