rhKAI1 suppresses expression of M1 macrophage phenotype-related surface markers in LPS-stimulated BMDM and RAW264.7 cells
Cytotoxic effects of rhKAI1 on primary BMDM and RAW 264.7 cells were examined with a WST-1 assay. At concentrations up to 800 ng/ml, rhKAI1 had no cytotoxic effect on BMDM or RAW 264.7 cells with or without LPS stimulation (Data not shown). Furthermore, a cell morphology comparison showed that both unstimulated-BMDM and -RAW 264.7 cells had a round shape, whereas LPS-stimulated cells had an irregular shape with polygonal spindle-shaped pseudopodia (Fig. 1A). However, such morphological change induced by LPS was diminished by rhKAI1 treatment in RAW264.7 cells, while no apparent change was observed in BMDM cells. To investigate effects of rhKAI1 on expression levels of M1 macrophage phenotype-related surface markers in LPS-stimulated condition, populations of F4/80+CD86+ cells and F4/80+CD80+ cells were examined by flow cytometry. Results of flow cytometry indicated that 100 ng/ml LPS significantly increased F4/80+CD86+ cells to 4.60-fold of those in the control BMDM. However, such increase in the population of LPS-induced F4/80+CD86+ cells was markedly decreased by rhKAI1 pre-treatment in a concentration-dependent manner (Fig. 1B-D). In addition, the population of F4/80+CD86+ and F4/80+CD80+ cells was substantially enhanced by LPS treatment to 90.47-fold of that in control RAW 264.7 cells, while such increase was significantly suppressed by pretreatment with rhKAI1 (Fig. 1E, F). Quantitative mean fluorescence intensity results were similar to results of population analysis using M1 surface markers (Fig. 1G). Results of morphological observation and flow cytometry for F4/80+CD86+ cells and F4/80+CD80+ cells in BMDM and RAW264.7 cells revealed that RAW 264.7 cells were more sensitive to rhKAI1 than BMDM. Therefore, RAW264.7 cells were selected and used in subsequent experiments to investigate the protective effect of rhKAI1 on LPS-induced M1 polarization. To re-verify the inhibitory effect of rhKAI1 on the increase of F4/80+CD86+ cells induced by LPS treatment, we carried out an immunofluorescence analysis for CD86 in RAW 264.7 cells. As shown in Fig. 1H, the expression of CD86, a surface marker of M1 macrophage polarization, was markedly increased by LPS stimulation. However, such increase of CD86 induced by LPS was noticeably suppressed in the presence of rhKAI1, consistent with results obtained by flow cytometry.
rhKAI1 inhibits LPS-induced inflammatory response in RAW 264.7 cells
LPS is considered an initiator of M1 macrophages polarization. It can induce the production of key inflammatory mediators such as inflammatory cytokines and chemokines (3, 4). Extensive studies have shown that LPS can enhance pro-inflammatory cytokines (such as IL-1β, IL-6, IL-12 and TNF-α) and representative inflammatory mediators (e.g., NO and PGE2) in macrophages, ultimately promoting an inflammatory response (18). Inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) are enzymes responsible for the production of NO and PGE2-that are also positively correlated with pro-inflammatory cytokines (19). Therefore, levels of pro-inflammatory cytokines and inflammatory mediators have been applied as indicators of anti-inflammatory efficacy (18, 19). In the present study, we assessed effects of rhKAI1 on expression levels of inflammatory cytokines in LPS-stimulated RAW 264.7 cells. Real-time qPCR results (Fig. 2A) revealed that LPS remarkably increased mRNA expression levels of pro-inflammatory cytokines and mediators including IL-1β, IL-6, TNF-α, and COX-2. However, such increases were substantially down-regulated by rhKAI1 pre-treatment. Especially, expression levels of IL-1β, IL-6, and COX-2 following rhKAI1 treatment were similar to those in untreated control cells. Next, to determine whether rhKAI1 could also regulate the release of inflammatory cytokines and mediators, secretion levels of NO, IL-1β, IL-6, and PGE2 in cell supernatants were analyzed. As shown in Fig. 2B, secretion levels of NO were markedly increased in LPS-stimulated cells (10.90 ± 1.13 μM, P < 0.001) compared to those in untreated cells (1.69 ± 0.47 μM). However, the secretion of NO was significantly reduced in 800 ng/ml rhKAI1 pre-treated cells (8.39 ± 0.76 μM, P < 0.05) compared with that in LPS-stimulated cells. Release levels of other inflammatory mediators such as PGE2, IL-1β, and IL-6 were also greatly increased by LPS, whereas such increases were suppressed by rhKAI1 pre-treatment before LPS treatment (Fig. 2C). These results suggest that rhKAI1 can suppress LPS-induced inflammatory response by modulating the production of pro-inflammatory cytokines and mediators. Findings of the present study are consistent with multiple previous studies suggesting that some members of the tetraspanin family, CD9, CD53, CD63, and CD81 can suppress inflammatory responses (20).
rhKAI1 attenuates JNK/NF-κB p65 signaling pathway in LPS-stimulated RAW 264.7 cells
Inhibiting nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and mitogen-activated protein kinases (MAPK) pathways has been suggested as the main mechanism underlying the attenuation of LPS-induced inflammatory cytokine production (19, 21). Among various intracellular signaling pathways, NF-κB has been identified as the key transcription factor in regulating the expression of inflammatory factors under LPS stimulation (19). Inactive NF-κB heterodimer p65/50 can interact with IκB-α in the cytosol to form a complex. Upon stimulation with LPS, IκB-α is phosphorylated and degraded subsequently. Released NF-κB dimers then translocate to the nucleus to initiate inflammatory gene transcription (21). Therefore, we further assessed whether the anti-inflammatory effect of rhKAI1 involved the NF-κB signaling pathway through immunofluorescence analysis. As shown in Fig. 2D, NF-κB p65 expression was significantly increased in the nuclei of LPS-stimulated RAW 264.7 cells than in control cells. However, such increase of NF-κB p65 expression induced by LPS was greatly reversed in the presence of rhKAI1. Next, to investigate the molecular mechanism by which rhKAI1 regulated LPS-induced inflammation, MAPKs signaling pathways including extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinases (JNK), and p38 MAPK were analyzed. MAPKs can phosphorylate different intracellular proteins and transcription factors, subsequently regulating gene expression. ERK proteins are activated by endogenous or exogenous mitogens, cytokines, and growth factors to regulate cell proliferation (22). JNK and p38 MAPK are predominantly activated by inflammatory cytokines and bacterial LPS (23). In the present study, we found that phosphorylation levels of ERK, JNK, and p38 MAPK were substantially increased by LPS treatment (Fig. 2E, F). However, phosphorylation level of JNK was significantly reduced by rhKAI1 pre-treatment in a dose-dependent manner, while phosphorylation levels of ERK and p38 were not affected by rhKAI1. This result indicates that rhKAI1 can attenuate LPS-induced inflammatory response by blocking the JNK/NF-κB p65 signaling pathway.
rhKAI1 prevents binding of LPS to TLR4s on the cell surface
TLRs expressed on the plasma and endosomal membranes of immune cells including macrophages are of interest to immunologists because of their front-line role in the initiation of innate immunity against invading pathogens (24, 25). Among various TLRs, TLR4 plays a crucial role in inflammatory and immune responses. It can efficiently sense Gram-negative bacterial infections through recognition of LPS, a bacterial membrane component (26). LPS binding to TLR4 leads to the activation and translocation of NF-κB into the nucleus, which then triggers the production of pro-inflammatory cytokines and type-I interferons (26, 27). To determine whether rhKAI1 could interact with TLR4 or prevent binding of LPS with TLR4, an immunofluorescence analysis was carried out to calculate molecular docking of each element. As shown in Fig. 3A, increment of TLR4 expression by LPS was markedly suppressed in the presence of rhKAI1. Subsequently, to predict the interaction between TLR4 and rhKAI1, 3-D structures of TLR4 and rhKAI1 were obtained from the PDB (PDB ID code of TLR4: 3VQ2) and Alphafold (AF ID code of hKAI1: P277701). Binding affinity was then analyzed and visualized with PyMOL molecular graphics system. As a result, we found that LPS could bind with four TLR4 residues: Glu 214, Lys341, Lys360, Lys367, and Arg434 (28). Among them, Glu 214 of rhKAI1 residue could strongly interact with Lys 360 of TLR4 residue. The distance between them was 2.9 Å (Fig. 3B-E). To validate this finding, we assessed the effect of rhKAI1 mutant (Glu 214) protein on LPS-stimulated macrophage polarization in RAW 264.7 cells. As shown in Fig. 1G, pretreatment with rhKAI1 mutant (Glu 214) protein markedly reversed down-regulation of mRNA expression of pro-inflammatory cytokines by wild type rhKAI1. The population and MFI of F4/80+CD86+ M1 surface marker were also reversed in rhKAI1 mutant (Glu 214) protein-treated cells (Fig. 3H, I). These results demonstrate that mutation of Glu 214 in rhKAI1 can decrease the anti-inflammatory effect of rhKAI1. Furthermore, we carried out a surface plasmon resonance binding analysis using BLItz, an optical technique for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time (29). Results of BLItz analysis showed that rhKAI1 strongly bound to recombinant human TLR4 in a highly specific fashion (Fig. 3F). These results indicate that rhKAI1 can directly interact with TLR4, which is involved in the suppression of LPS/TLR4-mediated M1 macrophage polarization. In a previous study, Khan et al. have demonstrated that KAI1 could interact with TLR9, an endosomal innate immune receptor, and modulate TLR9-dependent NF-κB nuclear translocation as a response to cytosine-phosphate-guanine stimulation in immune cells (30). Khan et al.’s results and our findings suggest that KAI1 could directly interact with TLR9 and TLR4 to block TLR-dependent downstream signaling pathway. We further determine whether down-regulation of TLR4 was mediated by the anti-inflammatory effect of rhKAI1 in LPS-stimulated cells. We found that the protective ability of rhKAI1 on increased cytokine secretion and TLR4 expression was enhanced in the presence of TAK-242, an inhibitor of TLR4 (Supplementary Fig. 1). These results demonstrate that KAI1 is associated with the TLR4 signaling pathway in the inhibition of inflammatory responses.
In conclusion, the current study showed that rhKAI1 could attenuate LPS-induced inflammatory responses in RAW 264.7 macrophages-like cells and primary mouse BMDMs. The anti-inflammatory effect of rhKAI1 was supported by reduced M1 macrophage phenotype-related surface markers and inhibited production of pro-inflammatory cytokines and mediators. However, many questions remain unsolved regarding macrophage polarization. More efforts are needed to establish specific markers and steps of the differentiation process leading to different subpopulation of macrophages (31). We noted that rhKAI1 also suppressed nuclear translocation of NF-κB p65 and phosphorylation of JNK in LPS-stimulated RAW 264.7 cells. In addition, rhKAI1 inhibited the expression of TLR4 on the cellular surface and bound strongly to TLR4. Taken together, these findings suggest that rhKAI1 has an anti-inflammatory effect in LPS-polarized macrophages through its interaction with TLR4 and down-regulation of the JNK/NF-κB signaling pathway (Fig. 4). Although further studies are required to determine the effect of rhKAI1 in vivo, our findings indicate a pharmacological potential of rhKAI1 in the prevention of various inflammatory diseases.