A unique hybrid characteristic having both pro- and anti-inflammatory phenotype transformed by repetitive low-dose lipopolysaccharide in C8-B4 microglia

Although lipopolysaccharide (LPS) is regarded as an inducer of inflammation, previous studies have suggested that repetitive low-dose LPS has neuroprotective effects via immunomodulation of microglia, resident macrophages of brain. However, microglia transformed by the stimulus of repetitive low-dose LPS (REPELL-microglia) are not well characterized, whereas microglia transformed by repetitive high-dose LPS are well studied as an endotoxin tolerance model in which the induction of pro-inflammatory molecules is suppressed. In this study, to characterize REPELL-microglia, the gene expression and phagocytic activity of REPELL-microglia were analyzed with the murine C8-B4 microglia cell line. The REPELL-microglia were characterized by a high expression of pro-inflammatory molecules (Nos2, Ccl1, IL-12B, and CD86), anti-inflammatory molecules (IL-10, Arg1, Il13ra2, and Mrc1), and neuroprotective molecules (Ntf5, Ccl7, and Gipr). In addition, the phagocytic activity of REPELL-microglia was promoted as high as that of microglia transformed by single low-dose LPS. These results suggest the potential of REPELL-microglia for inflammatory regulation, neuroprotection, and phagocytic clearance. Moreover, this study revealed that gene expression of REPELL-microglia was distinct from that of microglia transformed by repetitive high-dose LPS treatment, suggesting the diversity of microglia transformation by different doses of LPS.

Maintaining promotion of phagocytic activity in REPELL-microglia. Phagocytosis is one of the pivotal functions of microglia to remove xenobiotics and waste products, maintaining brain homeostasis. To assess phagocytic activity, REPELL-microglia were incubated with fluorescent latex beads, and the phagocytosis rate and mean fluorescence intensity (MFI) of phagocytosed beads in the cells were measured (Fig. 4). Compared with untreated controls (53.0% ± 1.9%), the phagocytosis rate of REPELL-microglia (67.4% ± 0.9%) was promoted to that of SINGLL-microglia (67.2% ± 0.6%). The MFI of phagocytosed beads in the cells also showed same tendency. The MFI of REPELL-microglia was 1.4 times higher than the untreated controls, and was comparable to the MFI of SINGLL-microglia. These results therefore demonstrated that REPELL-microglia have high phagocytic activity against foreign substances.

The gene expression pattern of REPELL-microglia is unique to the low-dose LPS condition.
To test whether the heretofore mentioned characteristics of REPELL-microglia were unique to low-dose LPS treatment, the mRNA expression of REPELL-microglia (1 ng/mL LPS) was compared with that of microglia transformed by repetitive high-dose LPS (100 ng/mL LPS) (Fig. 5). Gene expression patterns of Ccl1, Il1rn, Fpr2, and Trem2 were different between the two groups. The level of Ccl1 was upregulated in REPELL-microglia but suppressed during repetitive high-dose LPS treatment (Fig. 5a). The level of Il1rn was suppressed in REPELL-microglia compared with that of SINGLL-microglia, whereas it was promoted by repetitive high-dose LPS to as high as the single high-dose LPS group (Fig. 5b). The level of Fpr2 was suppressed in REPELL-microglia compared with SINGLL-microglia and in contrast to its upregulation by repetitive high-dose LPS. Trem2 level was not changed in REPELL-microglia, but it was downregulated by repetitive high-dose LPS (Fig. 5c). Expression of the other genes showed the same tendency between low-dose and high-dose LPS treatments. Thus, gene expression induced by repetitive LPS differed depending on LPS concentration, indicating that the gene expression pattern of REPELL-microglia is unique to its low-dose LPS treatment.    www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Previous studies suggest that administration of repetitive low-dose LPS contributes to neuroprotection by inducing microglia with neuroprotective phenotype [7][8][9][10]16 . However, few studies have focused on the characterization of REPELL-microglia. In the present study, we characterized REPELL-microglia by high expression of pro-inflammatory molecules (Nos2, Ccl1, IL-12B, and CD86: Fig. 1), anti-inflammatory molecules (IL-10, Arg1, Il13ra2, and Mrc1: Fig. 2), and neuroprotective molecules (Ntf5, Ccl7, and Gipr: Fig. 3). For these representative molecules such as IL-6, TNF-α, IL-12B, and IL-10, protein expression levels (IL-6, TNF-α, IL-12B: Fig. 1e, and IL-10: Fig. 2d) were confirmed to be almost correlative with mRNA expression (Il6, Tnfa, Il12b: Fig. 1b, and Il10: Fig. 2a). It is generally known that a single LPS treatment induces pro-inflammatory molecules, while repetitive LPS treatment suppresses the induction of pro-inflammatory molecules, a phenomenon known as "LPS tolerance". Indeed, it has been reported that pro-inflammatory molecules such as IL-1β, IL-6, and TNF-α are suppressed in microglia by repetitive high-dose LPS treatment [17][18][19][20][21][22] . Although IL-1β, IL-6, and TNF-α were suppressed in REPELL-microglia similarly to data of past reports with a high-dose LPS model, REPELL-microglia did not exhibit global downregulation of pro-inflammatory molecules, but instead showed high expression of Nos2, Ccl1, IL-12B, and CD86 (Fig. 1). The mRNA expression of IL-12B was slightly suppressed in REPELL-microglia, whereas the protein level was as high as that of SINGLL-microglia, and this may be associated with a protein stabilization mechanism at the posttranscriptional level [23][24][25] . REPELL-microglia expression of anti-inflammatory molecules was partially consistent with previous reports of upregulated IL-10, Arg1, and Mrc1 by repetitive high-dose LPS 8,26,27 . The co-expression of pro-inflammatory and anti-inflammatory molecules is one of the unique characteristics of REPELL-microglia. Because the combination of autologous inactivated tumor cells expressing IL-12 and IL-10 was reported to induce synergic tumor remission by controlling local inflammation 28 , it is suggested that REPELL-microglia expressing both IL-12 and IL-10 have the potential for regulating inflammation. In addition, REPELL-microglia highly express neuroprotective molecules Ntf5, Gipr, and Ccl7 (Fig. 3). NTF5 29 and GIPR [30][31][32] have neuroprotective effects via their anti-oxidant and anti-apoptosis qualities during encephalitis, and CCL7 associated with neuron differentiation 33 , suggesting the neuroprotective potential of REPELL-microglia through these factors. It has been reported that microglia "memorize" repetitive LPS stimulation and transform into neuroprotective cells 7 , and the neuroprotective molecules identified in this study may be involved in this mechanism. Meanwhile, microRNA (miRNA) is one of the mechanisms by which synthesis of protein from mRNA is suppressed. It has been reported that miRNA-146a, miRNA-155, miRNA-221, miRNA-125b, miRNA-132, miRNA-579, and miRNA-21 are involved in LPS tolerance [34][35][36] . In this context, it was confirmed by miRNA database (microRNA.org) that molecules with high mRNA expression in REPELL-microglia such as Nos2, Ccl1 (Fig. 1b), Arg1, Il13ra2 (Fig. 2a,b), Ntf5, Ccl7, and Gipr (Fig. 3a-c) were not targets of the miRNAs induced during LPS tolerances as described above. Therefore, it is suggested that translation of mRNA of Nos2, Ccl1, Arg1, Il13ra2, Ntf5, Ccl7, and Gipr into protein is not likely to be suppressed under the conditions of our study at least by this miRNA mechanism.
REPELL-microglia furthermore showed high phagocytic activity (Fig. 4). It was reported that microglia improved Alzheimer's disease-related pathologies by promoting the phagocytosis of Aβ 7 , suggesting that REPELL-microglia with high phagocytic activity contributes to the clearance of brain xenobiotics that cause neuropathy. TREM2 is one of the molecules involved in promotion of phagocytosis and suppression of pro-inflammatory molecules such as TNF-α and NOS2 in microglia 37,38 . In REPELL-microglia, however, the expression of Trem2 was unchanged, whereas Nos2 expression and phagocytosis were promoted. Therefore, it is suggested that phagocytosis of REPELL-microglia is promoted by a TREM2-independent mechanism. Indeed, phagocytosis is a remarkably complex process involving various phagocytic receptors and actin remodeling 39 . Because IL-12B and TNF-α promote NOS2 induction 40,41 , and NOS2 has a pivotal role not only in intracellular killing but also in phagocytic activity 42 , it is suggested that these molecules are associated with high phagocytic activity of REPELL-microglia.
Taken together, the present study has demonstrated for the first time that REPELL-microglia are characterized by high phagocytic activity and high expression of pro-inflammatory, anti-inflammatory, and neuroprotective molecules. Interestingly, the unique characteristics of REPELL-microglia are suggested to contribute to maintaining brain homeostasis, almost as if they are "repelling disease" both in name and reality. In addition, it was reported that mild oxygen-glucose deprivation induced microglia to show an anti-inflammatory and protective phenotype in vitro 43 , which is a phenomenon similar to that of REPELL-microglia in our study. Therefore, these data suggest that microglial protective phenotype can be induced by "mild" stimulation whether LPS or oxygen-glucose deprivation.
Importantly, the characteristics of REPELL-microglia are consistent with those of microglia induced by orally administered LPS in an Alzheimer's disease mouse model in vivo 16 in terms of high phagocytic activity and high IL-10 expression. In this context, another study has reported that microglia transformed by intraperitoneal administration of repetitive low-dose LPS also exhibit the characteristics of enhanced Aβ phagocytosis and high IL-10 expression, again consistent with the characteristics of REPELL-microglia 7 . Therefore, characteristics of REPELL-microglia as described above may partially reflect those of microglia transformed by oral administration of LPS in vivo. Because it was reported that intraperitoneal administration of IL-10 can induce microglia to express high levels of IL-10 as in the case of intraperitoneal administration of repetitive low-dose LPS 7 , IL-10 also may be a key mediator common to the induction mechanisms of both REPELL-microglia in vitro and microglia transformed by oral administration of LPS in vivo.
Moreover, the present study demonstrates that REPELL-microglia are not simply a miniature version of microglia transformed by repetitive high-dose LPS 10 but exhibit fundamentally distinct characteristics (Fig. 5). The results suggest that the condition of low-dose LPS is an important molecule to induce transformation into REPELL-microglia. Microglia play an important role in innate immunity for homeostatic maintenance by diverse transformation adapted to various microenvironment conditions [44][45][46] , and it has been recently reported that the Scientific RepoRtS | (2020) 10:8945 | https://doi.org/10.1038/s41598-020-65998-8 www.nature.com/scientificreports www.nature.com/scientificreports/ diversity of microglia can be transformed with different ligands, diseases, and time courses [45][46][47][48][49] . However, little has been recognized about the diversity of microglia transformation by different doses of LPS, and the phenotype of microglia transformed by low-dose LPS often has been considered to be simply a reduced version of that of high-dose LPS 10 . Therefore, the present study provides a new perspective of the diversity of microglia transformation by different doses of LPS. This concept is also supported by a recent report that pre-treatment with low-dose LPS (1 fg/mL) primes microglia to enhance pro-inflammatory cytokine production in response to subsequent stimulation with high-dose LPS (100 ng/mL) mediated by PI3Kγ 50 . In addition, other report showed in macrophages that pre-stimulation of low-dose LPS (5 pg/mL) induces RelB degradation in contrast to RelB induction that occurs during pre-stimulation of high-dose LPS (100 ng/mL) 51 , suggesting that different intracellular signal transductions operate in microglia stimulated with low dose versus high-dose LPS.
In conclusion, the present study demonstrates that REPELL-microglia are uniquely characterized by high phagocytic activity and high expression of pro-inflammatory, anti-inflammatory, and neuroprotective molecules (Fig. 6), suggesting the potential of REPELL-microglia to "repel disease" and to promote the maintenance of brain homeostasis. Moreover, this study revealed that low-dose LPS is a key factor for inducing REPELL-microglia, indicating the diversity of microglia transformation by different doses of LPS. Appropriate LPS treatment with repetitive low-dose administration may be expected to become a new therapeutic or preventive target for repelling various neurological disorders by priming the teleological transformation of microglia.

Methods
Cell culture and LPS treatment. The murine microglial cell line C8-B4 was purchased from the American Type Culture Collection (Manassas, VA, USA). A minimum sample size for one-way ANOVA was determined using power analysis in a prior examination with the statistical analysis software R (R Core Team (2019). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.). C8-B4 microglia (2 × 10 5 cells/mL) were seeded in 12-well tissue culture plates (n = 3, in triplicate) and cultured in Dulbecco's modified Eagle's medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA, USA), at 37 °C in 5% CO 2 . C8-B4 microglia were treated with or without LPS (1 or 100 ng/mL, purified LPS derived from Pantoea agglomerans, Macrophi Inc., Kagawa, Japan) (Fig. 1a). The concentration of LPS was set as low as 1 ng/mL because previous in vivo studies suggest that serum LPS levels were estimated to be at most 1 ng/ mL after oral administration of LPS at a concentration of 1 mg/kg/day 52 . For repetitive treatment with LPS, cells received fresh medium containing LPS three times every 24 h. For single treatment with LPS, cells received fresh medium without LPS for the first 48 h and then received a one-time dose of LPS in fresh medium. Samples were collected at the indicated time points.
Quantitative RT-PCR. Four hours after the last LPS treatment, RNA was extracted from cells by RNeasy mini kit (QIAGEN, Hilden, Germany) and cDNA was synthesized by reverse transcription using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturers' instructions. Real-time PCR assay was carried out using 2 μL of cDNA as the template and 10 μL of Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) on the Stratagene Mx 3005 P QPCR System (Agilent Technologies, Santa Clara, CA, USA). Primers used in this study are listed in Supplementary Table 2. Data were analyzed by the 2 −∆∆Ct method and normalized to GADPH. The thermal cycling conditions for PCR were 95 °C Figure 6. Model of a unique hybrid characteristic having both pro-and anti-inflammatory phenotype transformed by repetitive low-dose lipopolysaccharide in C8-B4 microglia. REPELL-microglia is characterized by high phagocytic activity and mixed expression of pro-inflammatory molecules (NOS2, IL-12B, CCL1, and CD86), anti-inflammatory molecules (IL-10, Arg1, IL-13RA2, and Mrc1), and neuroprotective molecules (NTF5, CCL7, and GIPR). Gene expression patterns of REPELL-microglia are distinct from that of microglia transformed by repetitive high-dose LPS, indicating the diverse transformation of microglia by different LPS concentrations.