Lysosome-associated Small GTPase Rab7b Negatively Regulates Toll-like Receptor Signaling In Macrophages

Innate immune cells recognize pathogen-associated molecular patterns (PAMPs) conserved in microbes by pattern recognition receptors (PRRs) to trigger host immune responses against invading pathogens. As an important kind of PRRs, Toll-like receptors (TLRs) play critical roles in the initiation of immune responses. However, prolonged or excessive immune responses may cause acute and chronic diseases, such as septic shock and rheumatoid arthritis. Thus, how to bring the TLR signaling under tight control is urgent to be answered. In this study, we investigated the role of mouse small GTPases Rab7b in TLRs signal pathway in macrophages.Part â…  Identification and characterization of the mouse homologue of small Rab GTPase Rab7bWe previously reported a novel human small GTPase Rab7b, which was isolated from a human dendritic cell cDNA library by large-scale random sequencing. Herein, we cloned the mouse homologue of Rab7b under the GenBank accession Number of NM-145509. The clone shares 92% overall similarity with Rab7b, and also potentially encodes a 199-amino acid proteins, designated as mouse Rab7b (mRab7b).mRab7b was cloned from mouse macrophage cell line RAW264.7. The full length of mRab7b cDNA is 2909bp, including a 600bp open reading frame (ORF). Via searching the database of HTGS, we found mRab7b located in the 1E4 region of mouse chromosome 1. The predicted protein is about 22.5KD, potentially being anacid protein (pl=5.83). There is no distinct transmembrane domain in the structure of mRab7b, suggesting that it may be a cytosol protein. Through motif analysis, a conserved Rab domain was found in mRab7b. In particular, there are three protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, one tyrosine kinase phosphorylation site, one ATP/GTP binding motif, and one N-myristoylation site in mRab7b. The two cysteines in the C terminal of mRab7b may be associated with its location.The effects of stimulation with TLR ligands including LPS, CpG ODN, Polyl: C on mRab7b expression in RAW cells were examined. Expression of mRab7b mRNA decreased at 4-6h after stimulation with LPS (lOOng/ml). Although the expression of mRab7b began to restore at 12h, it was still lower than the basal level. When RAW264.7 cells were stimulated with TLR9 ligand, CpG ODN, 50% decline of mRab7b mRNA occurred at 6h, and the decline maintained till 24h. Similarly, a time-dependent downregulation of mRab7b was visualized by treatment with polyl: C, a ligand for TLR3. However, TNFa failed to affect the expression of mRab7b. These results suggested that TLR signaling may downregulate mRab7b expression in macrophages.Part II mRab7b negatively regulates TLR signaling in macrophagesTo address mRab7b's role in TLR signaling, we generated His-tagged wild-type and mutant mRab7b vectors, namely mRab7b, mRab7bT22N and mRab7bACC, respectively. All the constructs were transfected into RAW264.7 cells, then the stable cell lines were selected by G418 . As verified by RT-PCR and Western blotting, we successfully established the cell lines stably overexpressing of mRab7b and its mutants. We further observed the subcellular localization of mRab7b. GFP-mRab7b exhibited a cytosolic and perinuclear region distribution and partially colocalized with the lysosome-specific fluorescent dye LysoTracker. However, the two mutants of mRab7b failed to colocalize to lysosomes, indicating that both the GTP bindingability and the integrity of C terminal are required for lysosomal location of mRab7b.We examined the biological effects of mRab7b on TLR signaling in macrophages using different stable transfectants. After stimulation with lOOng/ml LPS for 8 hours, forced expression of mRab7b inhibited IL-6 production by maximally about 50%. However, mRab7bT22N macrophage transfectant sustained the level of IL-6, and only slightly attenuated IL-6 secretion was observed in mRab7bACC macrophage transfectant. Taken together, these results suggested that the inhibitory effect of mRab7b depends on its active station and partially associates with its localization. Similarly, wild-type mRab7b inhibited the TLR-induced accumulation of IFN-B in macrophages. Since mRab7b prevented both TLR-induced IL-6 and IFN-B production, we proposed that mRab7b may affect both the MyD88-dependent and TRIF-dependent pathways in macrophages.Engaging TLRs activates both NF-kB and mitogen-activated protein kinase (MAPK) signaling pathways. So, the effects of mRab7 on MAPKs activation in macrophages was detected. mRab7b transfection inhibited LPS-induced ERK1/2 and JNK activation, as compared with controls. On the other hand, dominant negative mRab7bT22N partially blocked the above effects of mRab7b, with increase in ERK1/2 and JNK phosphorylation. ERK1/2, JNK and p-38 activations triggered by CpG ODN were also prevented by mRab7b. Collectively, we concluded that mRab7b negatively regulates MAPK pathways activated by TLR4 and TLR9 ligands.We investigated the role of mRab7b in TLR-triggered NF-kB activation in macrophages by a NF-kB reporter gene assay. As expected, mRab7b overexpression inhibited NF-kB luciferase reporter activity as well as the phosphorylation of IkBo induced by LPS and CpG ODN. Since the TRIF pathway was also regulated by mRab7b, we detected phosphorylation and translocation of IRF-3 upon LPS stimulation. Marked IRF-3 phosphorylation appeared at 120 minutes in the control cells. However, IRF-3 phosphorylation failed to be detected inmRab7b-overexpressing cells. To further examine the effect of mRab7b on IRF-3 activation, we extracted the nuclear proteins to observe the translocation of IRF-3. In fact, overexpression of mRab7b resulted in far less IRF3 nucleus translocation .The molecular mechanism for mRab7b-mediated suppression of TLR signaling was investigated. Because mRab7b could not interact with IRAKI, TRAF6 and MyD88 (data not shown), we investigated the effect of mRab7 on TLR4 expression by RT-PCR , FACS and western blotting. Neither the transcription nor the membrane TLR4 expression was affected by mRab7b. However, the total TLR4 expression was inhibited by mRab7b.These results indicated that mRab7b may affect the traffic and degradation of TLR4.In summary, we demonstrate that mRab7b inhibits TLR4-induced IL-6 and IFN-B production in macrophages. mRab7b inhibits TLR4 and TLR9-induced ERK, JNK and NF-kB activation. In addition, mRab7b also suppresses the activation of IRF3 in macrophages activated by TLR signaling. Therefore, we identify mRab7b as a novel negative regulator of TLR signaling.