| Objectives:Antimicrobial peptides play an important role in host defense. Various families of antimicrobial peptides have been identified, including the cathelicidin. Cathelicidin is characterized by a conserved N-terminal cathelin domain and a variable C-terminal part of the only human cathelicidin identified to date called human cationic antimicrobial protein (hCAP-18). LL-37is the only human cathelicidin. LL-37is a37-residue, amphipathic, helical peptide found throughout the body and has been shown to exhibit a broad spectrum of antimicrobial activity. It has been found to have defensive roles such as regulating the inflammatory response and chemoattracting cells of the immune system to wound or infection sites, binding and neutralizing LPS, and promoting re-epthelialization and wound repair. In this study, we established the LTA-induced inflammation models in vitro and analyzed the expression, function and mechanism of LL-37in macrophages.Methods:We established cellular inflammation model by treatment of RAW264.7cells and primary C57BL/6mouse peritoneal macrophage with2ug/mL LTA for24h. The expression of inflammatory cytokines were determined by ELISA and real-time quantitative PCR method, LL-37was measured by immunofluorescence and Western blot method. The mechanism of LL-37in LTA induced inflammation was deteced by Western blot.Results:1. Distribution of LL-37in macrophagesTo determine whether LL-37was expressed in macrophages and to determine its expression pattern, we did LL-37immunofluorescence staining in RAW264.7cells. The immunofluorescence staining showed that LL-37was mainly expressed in the cytoplasma but not in the nucleus. The merged figure also showed that LL-37was mainly expressed in the cytoplasma with uneven distribution.2. LTA could induce inflammatory reaction in macrophagesLTA is an exotoxin of S. aureus. In order to determine whether LTA could cause an inflammatory effect in macrophages, we used different concentrations of LTA to treat the RAW264.7cells and primary mouse peritoneal macrophages and analyzed TNF-a expression by ELISA. When the concentration of LTA is from0to2μg/mL, the con-centration of TNF-a increased from4.56±0.89to7.49±1.80ng/L in RAW264.7cells and from5.18±0.91to11.22±0.88ng/L in primary mouse peritoneal macrophages, in a concentration dependent manner while high doses of LTA from5to20μg/mL could cause acute cell death and attenuate the expression of TNF-a. This result indicates that0-2μg/mL of LTA can induce inflammation reaction in macrophages, but a high concentration of LTA could induce acute cell injury and death.3. LL-37expression in LTA treated RAW264.7cellsTo investigate the change of LL-37in LTA induced inflammation in RAW264.7cells, we checked LL-37expression in RAW264.7cells after LTA treatment. We also put chemically synthesized LL-37in the culture medium of RAW264.7cells to check its uptake efficiency by immunofluorescence staining and Western blot. There are four groups in the experiment:control group, LL-37treated positive control group, LTA treated group, and LTA and LL-37double treated group. After LTA treatment, the expression of LL-37is1.33±0.13fold compared with the control group (p<0.05). In the chemically synthesized LL-37and LTA double treated group, the expression of LL-37increases1.93±0.18fold compared with the control group (p<0.05). The LL-37expression in the double treated group is higher than that in the LTA treated group (p<0.05), but no significant difference with the LL-37treated group (p>0.05). These results indicated that LTA could effectively increase LL-37expression and chemically synthesized LL-37could be transported into RAW264.7cells.4. LL-37could attenuate LTA induced inflammation in macrophagesTo investigate whether LL-37could attenuate LTA induced inflammation reaction in macrophages, we check the TNF-a expression in different concentrations of LL-37in RAW264.7cells and primary mouse peritoneal macrophages by ELISA. We used2μg/mL of LTA treated RAW264.7cells and primary mouse peritoneal macrophages to induce inflammation. Then we used0-40μg/mL of LL-37to treat the RAW264.7 cells and primary mouse peritoneal macrophages.1-15μg/mL of LL37could effectively attenuate TNF-a expression in a concentration dependent manner, and decrease TNF-a to4.63±0.81ng/L compared with the basal level of6.46±0.36ng/L after LTA treatment in RAW264.7cells. And in primary mouse peritoneal macrophages,1-10μg/mL of LL37could effectively attenuate TNF-a expression in a concentration dependent manner, and decrease TNF-a to6.69±0.64ng/L compared with the basal level of10.52±0.92ng/L. The medium TNF-a concentration decreased to the lowest at the LL-37concentration of15μg/mL in RAW264.7cells and10μg/mL in primary mouse macrophages. On the other hand, if LL-37concentration is increased at over15μg/mL in RAW264.7cells and10μg/mL in primary mouse peritoneal macrophages, it will cause cell damage and increase TNF-a expression. Thus a moderate concentration of LL-37could attenuate LTA induced inflammation in macrophages. A high concentration of LL-37will cause cell damage. According to this, we used the optimal LL-37concentration of15μg/mL in RAW264.7cells and10μg/mL in primary mouse peritoneal macrophages for the rest of the experiments.5. LL-37attenuates LTA induced inflammatory reaction by decreasinginflammatory cytokine expression TNF-a and IL-6are two important inflammatory cytokines during inflammation. We checked TNF-a and IL-6expression by real time RT-PCR in RAW264.7cells and ELISA in primary mouse peritoneal macrophages. In the LTA treated group, the inflammatory cytokines TNF-a and IL-6mRNA increased8.75±0.47and34.12±9.68fold compared with the control group. The mRNA of TNF-a and IL-6in the LL-37and LTA double treated group decreased obviously compared with the LTA treated group.LL-37treatment alone could decrease cell TNF-a and IL-6mRNA expression. Similarly, in the LTA treated primary mouse peritoneal macrophages, the inflammatory cytokine TNF-a and IL-6expression increased to7.53±2.21ng/L and180.38±29.46ng/L compared with the basal level of4.98±1.30ng/L and16.75±9.06ng/L, respectively. The concentration of TNF-a and IL-6in the LL-37and LTA double treated group decreased obviously compared with that in the LTA treated group, and LL-37treatment alone could also decrease cell TNF-a and IL-6expression. So the exogenous chemically synthesized LL-37could attenuate LTA induced inflammatory reaction by the down regulation of TNF-a and IL-6expression in macrophages.6. LL-37attenuates LTA induced inflammation by the inhibition of p38MAPK andAkt activation in macrophages To investigate the signal transduction pathway of LL-37, we tested the activation of p38MAPK and Akt in the RAW264.7cells and primary mouse peritoneal macrophages. LTA could increase p38MAPK and Akt phosphorylation and cause the inflammatory reaction in the RAW264.7cells and primary mouse peritoneal macrophages. LL-37treatment could inhibit p38MAPK and Akt activation compared with the LTA treated group, which could attenuate inflammation reaction and modulate immune response. It suggested that LL-37could regulate inflammation by inhibition of p38MAPK and Akt signal transduction pathways in macrophages.Conclusions:1. In the cell model of Staphylococcus aureus infection induced by LTA, the expression of TNF-a, IL-6and LL-37increases, suggesting that LTA can induce macrophages to produce inflammatory reaction, LL-37is involved in the inflammatory response induced by LTA.2. LL-37reduced the expression of inflammatory factors, suggesting that LL-37can attenuate the inflammatory reaction.3. LTA could promote the phosphorylation of p38and Akt, however LL-37could inhibit the phosphorylation of p38and Akt, suggesting that LL-37takes part in the inflammatory reaction induced by LTA by inhibiting phosphorylation of p38and Akt. |
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