Quaking Deficiency Amplifies Inflammation in Experimental Endotoxemia via the Aryl Hydrocarbon Receptor/Signal Transducer and Activator of Transcription 1–NF-κB Pathway

Macrophages, characterized by considerable diversity and plasticity, play a crucial role in a broad spectrum of biological processes, including inflammation. However, the molecular mechanisms underlying the diverse phenotypes of macrophages are not well defined. Here, we show that the RNA-binding protein, quaking (QKI), dynamically modulates macrophage polarization states. After lipopolysaccharide (LPS) stimulation, QKI-silenced RAW 264.7 cells displayed a pro-inflammatory M1 phenotype characterized by increased expression of iNOS, TNF-α, and IL-6 and decreased expression of anti-inflammatory factors, such as IL-10, found in inflammatory zone (Fizz1), and chitinase-like 3 (Chil3 or Ym1). By contrast, QKI5 overexpression led to a suppressive phenotype resembling M2 macrophages, even under M1 differentiation conditions. Moreover, myeloid-specific QKI-deficient mice tended to be more susceptible to LPS-induced endotoxic shock, while the exogenous transfer of macrophages overexpressing QKI5 exerted a significant improving effect. This improvement corresponded to a higher proportion of M2 macrophages, in line with elevated levels of IL-10, and a decrease in levels of pro-inflammatory mediators, such as IL-6, TNF-α, and IL-1β. Further mechanistic studies disclosed that QKI was a potent inhibitor of the nuclear factor-kappa B (NF-κB) pathway, suppressing p65 expression and phosphorylation. Strikingly, reduced expression of the aryl hydrocarbon receptor (Ahr) and reduced phosphorylation of signal transducer and activator of transcription 1 in QKI-deficient cells failed to restrain the transcriptional activity of NF-κB and NRL pyrin domain containing 3 (NLRP3) activation, while restoring QKI expression skewed the above M1-like response toward an anti-inflammatory M2 state. Taken together, these findings suggest a role for QKI in restraining overt innate immune responses by regulating the Ahr/STAT1–NF-κB pathway.

Macrophages, characterized by considerable diversity and plasticity, play a crucial role in a broad spectrum of biological processes, including inflammation. However, the molecular mechanisms underlying the diverse phenotypes of macrophages are not well defined. Here, we show that the RNA-binding protein, quaking (QKI), dynamically modulates macrophage polarization states. After lipopolysaccharide (LPS) stimulation, QKI-silenced RAW 264.7 cells displayed a pro-inflammatory M1 phenotype characterized by increased expression of iNOS, TNF-α, and IL-6 and decreased expression of anti-inflammatory factors, such as IL-10, found in inflammatory zone (Fizz1), and chitinase-like 3 (Chil3 or Ym1). By contrast, QKI5 overexpression led to a suppressive phenotype resembling M2 macrophages, even under M1 differentiation conditions. Moreover, myeloid-specific QKI-deficient mice tended to be more susceptible to LPS-induced endotoxic shock, while the exogenous transfer of macrophages overexpressing QKI5 exerted a significant improving effect. This improvement corresponded to a higher proportion of M2 macrophages, in line with elevated levels of IL-10, and a decrease in levels of pro-inflammatory mediators, such as IL-6, TNF-α, and IL-1β. Further mechanistic studies disclosed that QKI was a potent inhibitor of the nuclear factor-kappa B (NF-κB) pathway, suppressing p65 expression and phosphorylation. Strikingly, reduced expression of the aryl hydrocarbon receptor (Ahr) and reduced phosphorylation of signal transducer and activator of transcription 1 in QKI-deficient cells failed to restrain the transcriptional activity of NF-κB and NRL pyrin domain containing 3 (NLRP3) activation, while restoring QKI expression skewed the above M1-like response toward an anti-inflammatory M2 state. Taken together, these findings suggest a role for QKI in restraining overt innate immune responses by regulating the Ahr/STAT1-NF-κB pathway. inTrODUcTiOn Lipopolysaccharide (LPS), a cell wall component of Gramnegative bacteria, is one of the most powerful macrophage activators, inducing an innate immune response via toll-like receptor 4 (TLR4) and intracellular pathways. Overwhelming inflammatory responses are the leading cause of death in infected patients. Therefore, disclosing intracellular pathways to curb inflammatory responses against LPS stimuli is required.
Macrophages are the sentinels of the innate immune system, safeguarding against infection or tissue damage. In response to cytokines and microbial signals, they reprogram their dynamic gene expression profiles in order to produce corresponding phenotypes, referred to as M1 (classically) or M2 (alternatively) polarized macrophages. M1 macrophages are the pro-inflammatory subtype, secreting high levels of inflammatory cytokines, which are responsible for host defense against foreign pathogens, while M2 macrophages are more often associated with tissue repair and remodeling by producing anti-inflammatory mediators in order to maintain immune homeostasis (1,2).
In fact, the two states of macrophage activation often coexist in a pathogen infection. Microbial LPS induces the activation of downstream transcription factors, such as NF-κB and signal transducer and activator of transcription 1 (STAT1), resulting in the upregulation of pro-inflammatory factors, such as TNF-α and IL-6, in macrophages (3)(4)(5)(6). Although adequate inflammatory cytokines are essential for pathogen clearance, overproduction leads to a harmful result, such as LPS-induced shock. In order to maintain the immune balance, various mechanisms negatively regulate TLR signaling (7). Among the cellular negative regulators, the aryl hydrocarbon receptor (Ahr), acts as a transcription factor (8) and adjusts the various toxicological effects and provides immune tolerance (9,10). Ahr is also responsible for inducing anti-inflammatory effects by negatively modulating NF-κB-dependent inflammatory responses as well as inflammasome activation in LPS-stimulated macrophages (11,12). In addition, Ahr is involved in changing cell fate in adaptive T cell immune responses, such as the differentiation into regulatory (Treg) or T helper lineages (Th17 or Th1) (13)(14)(15). At the cytokine level, IL-10, as one of the typical alternative macrophage cytokines is able to dampen the inflammatory responses by restraining both the expression and function of TNF and IL-1 (16). Mechanistic analysis suggested that STAT3 activation is mainly involved in transcriptional regulation of various suppressive factors (17,18). Thus, defining the intracellular molecular mechanism underlying the two macrophage polarization states will be valuable in inflammation-related disease treatments.
RNA-binding proteins are a group of proteins responsible for gene regulation at the posttranscriptional level by modulating mRNA splicing, stability, and translation (19,20). Several proteins are known to exert key regulatory roles in immune responses. For example, myeloid cell tristetraprolin protected mice against LPS-induced septic shock through posttranscriptional regulation of TNF mRNA stability (21). In response to virus infection, a human RNA-binding protein called embryonic lethal abnormal vision drosophila-like 1 or Hu antigen R could stabilize IFN-β mRNA by binding with AU-rich sequences at its 3′ untranslated regions (22). Moreover, the RNA-binding protein, quaking (QKI), belonging to the signal transduction and activation of RNA family, displayed various critical functions in early embryonic development (23), as well as additional cellular processes (24)(25)(26). The QKI gene mainly encodes three protein isoforms called QKI5, QKI6, and QKI7 (27,28). All of them contain the featured KH RNA-binding domain, but possess various lengths in their C-terminus (27,28). QKI5, which harbors a nuclear localization signal, is the most abundant isoform and is predominantly located in the nucleus. QKI6 and QKI7 are dynamically transported between the nucleus and cytoplasmic regions and form homo or hetero-dimers with QKI5 to regulate target gene expression by specifically binding to the conserved QKI response element (QRE) on target mRNAs (29).
Recently, van der Veer et al. reported that QKI promoted monocyte differentiation toward the pro-atherosclerotic macrophage lineage, significantly altering more than 1,000 QKI-dependent mRNA levels (19). Previous findings have suggested that QKI itself precisely regulates the maturation process of monocytes to macrophages via negative modulation of colony-stimulating factor receptor stability (30). However, its role in the regulation of macrophage polarization, especially in host defense against microbial LPS, has yet to be documented.
We developed a myeloid-specific QKI knockout mouse model. The in vitro and in vivo data indicated that QKI deficiency made mice more susceptible to endotoxic shock, which was rescued by overexpression of QKI5 in peritoneal macrophages, favoring M2 polarization, with lower levels of pro-inflammatory cytokines. The QKI-mediated Ahr/STAT1-NF-κB pathway was involved. Therefore, targeting QKI-related signals in macrophages is a novel method to turn off the exaggerated "inflammatory storm" against LPS.
MaTerials anD MeThODs cells RAW 264.7 cells (American Type Culture Collection) were cultured in RPMI-1640 with 10% fetal bovine serum (Gibco by Life Technologies). Mouse peritoneal macrophages were obtained as described previously (31). Three days before collecting peritoneal cells, 8-to 12-week-old mice were injected with 1 ml of 3% thioglycollate medium. Cells were further enriched by discarding the culture medium containing nonadhesive cells after seeding into 6-well plates for 2 h. Bone marrow-derived macrophages were harvested as previously described (32). In brief, bone marrow cells were collected from femurs of male QKI fl/fl and LysMCre QKI fl/fl mice. These cells were seeded into Plastic petri dishes in complete RPMI 1640 medium that was supplemented with 20 ng/ml recombinant murine macrophage colony-stimulating factor (M-CSF, Peprotech, Rock Hill, USA) for 7 days. Adherent cells were washed with PBS and M1 macrophages were induced by 1 µg/ ml LPS from Escherichia coli O111:B4 (Sigma-Aldrich), while M2 macrophages were obtained by adding 20 ng/ml IL-4 plus 20 ng/ml IL-13 (PEPROTECH, Rocky Hill, NJ, USA).  cytokine and alanine aminotransferase (alT) assay The concentrations of cytokines were measured by ELISA kits (Dakewe Biotech Co. Ltd, Shenzhen, China), according to the manufacturer's instructions. The concentration of ALT was measured using the ALT assay kit (Nanjing JianCheng Bioengineering Institute).

Flow cytometry
For surface cell staining, 5 × 10 5 cells were incubated for 15-20 min with anti-mouse CD16/CD32 to block non-specific binding of immunoglobulins to Fc receptors expressed on monocytes, macrophages, and granulocytes (Biolegend, clone 93). Labeling was performed for 30 min with specific antibodies. The following antibodies were used: PE anti-mouse CD11b (Biolegend, clone M1/70), FITC anti-mouse F4/80 (eBioscience, clone BM8), and FITC anti-mouse Ly6G (Biolegend, clone 1A8). All staining was done on ice in PBS supplemented with 2% BSA followed by washing once in cold PBS. The FACS Vantage system (Becton-Dickinson, San Jose, CA, USA) was used to analyze samples.

Western Blot
Cells were lysed and centrifuged at 12,000 rpm for 15 min. After quantification of the protein concentration using a BCA kit, the same amounts of proteins were resolved on SDS polyacrylamide gels and transferred onto a PVDF membrane (Millipore). The membrane was blocked in 5% non-fat milk with TBST with 0.05% Tween for 2 h at room temperature and then incubations were performed with the indicated primary antibodies for QKI (Sigma), Ahr (R&D Systems), STAT1 and phosphor-STAT1 Tyr701 (Sangon Biotech), NF-κB p65 and phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, clones D14E12 and 93H1), NLRP3 (Abcam), procaspase-1 and active caspase-1 (Abcam, clone EPR16883), and β-actin (Abcam) overnight at 4°C. After washing with TBST three times for 10 min each, the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Next, the membrane was washed in TBST and detected by the UVP ChemiDoc-It 510 imaging system.

chromatin immunoprecipitation assay
Chromatin immunoprecipitation analysis was accomplished using the Magna ChIP™ A kit (Millipore). In brief, QKI5silenced RAW 264.7 cells and control cells were incubated with medium or LPS (1 µg/ml) for 4 h, crosslinked in 1% formaldehyde for 10 min at room temperature and quenched with unreacted formaldehyde by the addition of 0.125 M glycine. Cells were washed twice with PBS and suspended in cell lysis buffer containing protease inhibitors, incubated on ice for 15 min, and centrifuged at 800 × g for 5 min. The pellet was homogenized in nuclei lysis buffer containing protease inhibitors and subjected to sonication on ice, followed by incubation overnight at 4°C with anti-Ahr (Thermo Fisher, clone RPT9), anti-p50 (Abcam), anti-p65 (Merck), anti-STAT1 (CST), normal mouse IgG, or normal rabbit IgG in the presence of protein A magnetic beads. The reactions were incubated overnight at 65°C to reverse the cross-links. DNA was purified by spin column and analyzed by PCR with the following primers: 5′-CGATGCTAAACGACGTCACATTGTGCA-3′ and 5′-CTCCAGAGCAGAATGAGCTACAGACAT-3′, specific for the κB site in the IL-6 promoter.

lPs-induced endotoxin shock
For the LPS-induced endotoxin shock model, LPS (50 mg/kg or 40 mg/kg, Sigma) was intraperitoneally (i.p.) injected into the mice. Body temperatures were monitored using a rectal thermometer at various times following LPS injection. Death of mice was recorded and the data were analyzed for statistical significance of differences between the groups. For rescue experiments, 1.2 × 10 8 peritoneal macrophages were isolated from 20 male, C57BL/6 mice (6-8 weeks old) as previously described and divided into cell culture dishes (6.0 cm). The cells were transduced by either QKI5 overexpression plasmid or by lentiviral particles with empty vector and incubated at 37°C, in a 5% CO2 incubator for 8 h. Polybrene (8 µg/ml) was supplemented to enhance the infection efficiency. Cells were washed and maintained in complete medium followed by blasticidin (4 µg/ml) selection for 3 days. Peritoneal macrophages overexpressing QKI5 (2.8 × 10 7 cells) were collected and i.p. injected into mice (2 × 10 6 cells per mouse) 0.5 h after LPS injection (40 mg/kg). The survival was recorded and the serum was collected.

statistical analysis
Statistical analyses were performed using the Student's t-test (two comparisons, or two-tailed) and one-way ANOVA (multiple comparisons). For the survival studies, the log-rank test was used to determine significance. p < 0.05 was deemed significant.

Dynamic changes in QKi expression in Differently Polarized Macrophages
To determine whether QKI is involved in macrophage polarization, we first analyzed QKI protein expression in mouse peritoneal macrophages at 16 h after stimulation with M1 (LPS) or M2 (IL-4 plus IL-13) polarizing agents. As shown in Figure 1A, QKI protein expression was reduced by LPS and increased by IL-4 plus IL-13 treatment. QKI mRNA expression was determined using primers specific for a common sequence that can detect QKI5, QKI6, and QKI7. In RAW 264.7 cells, mRNA expression showed biphasic patterns in M1 macrophages, showing a decrease by 2.5-fold within 8 h, while beyond 24 h, transcripts progressively returned to their initial level and then increased again at 48 h. However, under M2 polarization conditions, QKI expression increased above twofold at 48 h ( Figure 1B). These results suggest that QKI expression is closely related to the macrophage polarization state, potentially participating in the establishment of macrophage plasticity.
higher QKi levels causes a shift of Macrophages from M1 Toward M2 Following the above observations, we continued to explore whether QKI could directly affect the expression of M1/M2 macrophage phenotypic markers. QKI5 silencing or overexpression in RAW 264.7 cells was achieved. Western blot detection confirmed the efficacy of QKI5 dysregulation (Figure 2A). Interestingly, we observed that silencing QKI5 could also interfere with the expression of QKI6 and QKI7 in macrophages (unpublished data). After LPS stimulation, significant mRNA expression of iNOS, TNF-α, and IL-6 was observed, while reduced levels of anti-inflammatory factors such as IL-10, Fizz1, and Ym1 were seen in QKI5-silenced cells ( Figure 2B). ELISA further confirmed the above changes at the protein level ( Figure 2D). On the contrary, QKI5 overexpression led to decreased levels of TNF-α and IL-6 but increased levels of IL-10, Fizz1, and Ym1 ( Figure 2C). Secretions of TNF-α and IL-6 were also reduced ( Figure 2E). Production of anti-inflammatory IL-10 by RAW 264.7 cells overexpressing QKI5 was enhanced, even under M1 polarization conditions ( Figure 2E). Together, these results indicate that QKI5 is able to regulate macrophage polarization and skew the differentiation of macrophages toward an anti-inflammatory M2 phenotype.

QKi inhibits nF-κB-Mediated inflammatory cytokine Production in M1 Macrophages
We next sought to identify the molecular mechanism for QKImediated regulation of macrophage plasticity. NF-κB is the key transcriptional factor in mediating production of inflammatory cytokines, such as TNF-α and IL-6 in response to LPS stimulation. It was speculated that QKI deficiency might enhance the activation of NF-κB. While LPS-treated RAW 264.7 cells induced nuclear translocation of p65, this was significantly enhanced in siQKI5 cells (Figures 3A,B). Western blot results revealed that the phosphorylation of p65 (Ser 536) and IΚKβ and total p65 expression were significantly increased in QKI5silenced cells compared with those in silenced control cells (siNC) after LPS treatment. By contrast, QKI5 overexpression significantly suppressed these effects ( Figure 3C). Therefore, our data disclose a suppressive role for QKI5 in the LPS-induced production of pro-inflammatory cytokines by inhibiting NF-κB activation.
inhibition of nF-κB Transcriptional activity and inflammasome activation by QKi is ahr-Dependent In order to validate the signaling pathway, we investigated how QKI regulates NF-κB-mediated pro-inflammatory signaling. Bioinformatics analysis predicted that the 3′UTR region of Ahr mRNA contained one QRE motif ( Figure 4A). The ablation of QKI reduced both mRNA and protein expression of Ahr, as expected (Figures 4B,C). Notably, QKI5 silencing could decrease Ahr mRNA expression in untreated RAW 264.7 cells. QKI loss led to faster degradation of Ahr mRNA, with a half-life of approximately 2.5 h (Figure 4D). To further clarify if QKI modulated Ahr expression, RIP analysis was performed to determine if QKI binds directly to Ahr mRNA. Our results revealed that the anti-QKI antibody specifically interacted with Ahr mRNA compared to control IgG (Figure 4E), indicating that Ahr mRNA stability in macrophages was directly regulated by QKI. A previous study reported that Ahr interacted with STAT1 to negatively regulate NF-κB-dependent pro-inflammatory cytokine production in response to LPS (12). Interestingly, we found that silencing QKI5 repressed the expression and phosphorylation of STAT1 ( Figure 4F). Since STAT3 is closely related to LPS-induced macrophage polarization, its expression was also detected. As shown in Figure 4G, silencing QKI5 significantly promoted STAT3 activation compared with that in siNC cells, while there was no difference between QKI5-silenced and -overexpressed cells. The CHIP assay further showed that Ahr and STAT1 were recruited to the IL-6 promoter after LPS stimulation in siNC cells, but not in siQKI5 cells (Figure 4H). These results support that QKI represses LPS-induced pro-inflammatory cytokine secretion in macrophages by enhancing Ahr expression and STAT1 expression and phosphorylation, leading to the inhibition of NF-κB transcriptional activity through their promoter regions. The inflammasome was recently reported to play a crucial role in inflammation in response to pathogens and Ahr is known to negatively regulate NLRP3 inflammasome activity, and subsequent IL-1β secretion (11). Here, we further analyzed

generation of Macrophage-specific QKI-Deficient Mice
To explore the in vivo functions of QKI, we employed a conditional gene targeting strategy to generate QKI-floxed mice by introducing two loxP sites flanking exon 2 of the quaking gene (QKI fl/fl ) (shown in Figure 5A). The QKI-floxed mice were mated to mice expressing the Flp recombinase to delete the neo gene. Then these mice were crossed with LysM Cre mice.
In the resulting mice, quaking gene expression was specifically inactivated in myeloid cells, referred to as LysMCre QKI fl/fl mice. The genotypes of mice were identified by PCR using three different types of PCR primers and mouse tail DNA as the template. Consequently, the 700-bp amplicon was indicated as Cre allele-positive mice, the 410-bp product indicated the presence of the floxed allele, and the 370-bp product indicated the presence of the WT allele ( Figure 5B). Myeloid cell-specific  Real-time PCR was performed for the analysis of Ahr mRNA expression. *p < 0.05 versus scramble control. (e) RNA immunoprecipitation analysis using QKI antibody followed by real-time PCR to measure the mRNA levels of Ahr in RAW 264.7 macrophages treated by LPS for 6 h, with β-actin used as a normalization control. (F,g) Cells were stimulated with 1 µg/ml LPS for the time indicated and cell lysates were analyzed by immunoblotting with specific antibodies. One representative western blot of p-STAT1, total STAT1 (F), STAT3 and p-STAT3 (g) is shown. Graphs present the quantification of band intensity for immunoblotting from three independent experiments. (h) Cells were treated with 1 µg/ml LPS for 4 h and the ChIP assay was performed using anti-p65, anti-p50, anti-STAT1, and anti-Ahr antibodies. Purified DNA fragments were amplified using primers specific for the IL-6 promoter. One representative result from three independent experiments is shown. (i) Cells were pretreated with dimethyl sulfoxide or FICZ for 40 min and stimulated with LPS for 8 h and ATP for the last 30 min. Cell lysates were subjected to western blot analysis. One representative immunoblot of cleaved caspase-1 (p10), procaspase-1, and NLRP3 is shown. (J) Cells were treated as described in (i) and the levels of IL-1β in the culture supernatants were determined by enzyme-linked immunosorbent assay. All the bars represent the mean of measurements from three independent experiments, and the error bars indicate ±SEM. *p < 0.05, **p < 0.01. deficiency of QKI in LysMCre QKI fl/fl mice was verified by the reduced expression of QKI at both mRNA and protein levels in peritoneal macrophage cells, compared to that in spleen lymphocytes (Figures 5C,D, p < 0.001). The WT and QKI fl/fl mice were used as controls. For all experiments, mice were heterozygous for LyM Cre. QKi-Deficient Mice are hyper-responsive to lPs-induced endotoxic shock Using the above mice, we observed the in vivo function of myeloid QKI in the LPS-induced endotoxic shock model. LPS was intraperitoneally injected into mice and the survival status was observed for 72 h. As shown in Figure 6A, 70% of the QKI fl/fl mice were still alive, while 100% of the LysMCre QKI fl/fl mice were dead. In addition, the LysMCre QKI fl/fl mice showed hypothermia, with body temperatures decreasing more rapidly than the control mice ( Figure 6B, p < 0.01). Compared with QKI fl/fl , the serum levels of TNF-α, IL-1β, and IL-6 were increased significantly in LysMCre QKI fl/fl mice ( Figure 6C, p < 0.05). Since the liver is the most sensitive to infections, the levels of ALT were measured to reflect the extent of liver injury. LPS-treated mice displayed higher levels of ALT in the serum compared with serum from healthy C57BL/6 mice (about 20 U/L) (34). Moreover, as shown in Figure 6D, LysMCre QKI fl/fl mice displayed much higher levels of ALT in the serum than QKI fl/fl mice, indicating more severe liver injury. Cell type differentials were determined in peritoneal lavage fluid (total live cells, macrophages, and neutrophils) using Wright-Giemsa staining and were similar in the two groups of mice ( Figure 6E).

Transfer of Peritoneal Macrophages
Overexpressing QKi5 confers resistance to lPs-induced endotoxic shock in lysMcre QKi fl/fl Mice Since the conditional QKI-deficient mice were more sensitive to endotoxin challenge, we further validated this by restoring QKI5 expression in peritoneal macrophages to determine if this would rescue the lethal phenotype. Peritoneal macrophages, with a purity of >90% and isolated from C57BL/6 mice, were used to construct macrophages either overexpressing QKI5 or control virus. The efficacy of QKI5 overexpression in peritoneal macrophages was then verified by western blot analysis. Our results showed that the QKI5 protein expression increased twofold in LV-QKI5 cells compared with that in wild-type (WT) or LV-Cherry cells (data not shown). These cells were transferred into LPS-challenged LysMCre QKI fl/fl mice, leading to significantly improved survival, from 30 to 80% (Figure 7A, p < 0.05). This corresponded to a decrease in pro-inflammatory mediators in the serum, including TNF-α, IL-6, and IL-1β ( Figure 7B, p < 0.05), while there was an increase in anti-inflammatory cytokines, such as IL-10 ( Figure 7C, p < 0.01). Given the reduced pro-inflammatory responses in LysMCre QKI fl/fl mice after transferring peritoneal macrophages that overexpress QKI5 in the LPS-induced mice model, alternatively activated macrophages (M2) were anticipated. Thus, we measured changes in the proportion of peritoneal M1 macrophages (F4/80 int CD11b int ) and M2 macrophages (F4/80 hi CD11b hi ) (35). Compared to macrophages in QKI fl/fl mice, an increase in M1 macrophages and a decrease in M2 macrophages were shown in LPS-treated LysMCre QKI fl/fl mice transferred with control cells. However, the proportion of M2 macrophages in the peritoneal cavity of LysMCre QKI fl/fl mice after transfer of peritoneal macrophages overexpressing QKI5 was significantly elevated, with a parallel reduction in M1 macrophages (Figures 7D,E). Interestingly, transferring peritoneal macrophages that were derived from WT mice and transfected with LV-cherry did not improve the lethal state of QKI-floxed mice after LPS treatment. We anticipated that endogenous QKI expression would be inhibited after transferring into LPS-stimulated mice under a state of M1 polarization. However, QKI was still overexpressed in exogenous macrophages that were still present, exerting an anti-inflammatory effect.

DiscUssiOn
In response to the invasion of bacteria and other types of pathogens, macrophages, the main innate immune cells, phagocytize pathogens and secrete a variety of pro-inflammatory cytokines, which evoke the host defense responses (36). During this process, macrophage activation is dynamically regulated and diverse phenotypes often coexist (35).
Two extreme states of macrophage activation, M1 versus M2, are often described. Briefly, M1 macrophages are referred to as the pro-inflammatory subtype, secreting more inflammatory factors, such as TNF-α, IL-6, and IL-1β, exerting pathogen killing effects by enhanced phagocytosis and intracellular killing, as well as other paracrine cytokines effects (37). M2 macrophages are more anti-inflammatory and work to shut down the overt inflammatory response (32). Homeostasis of these two polarized states in acute immune responses is particularly beneficial for the body's defense system. In clinics, either an overwhelming inflammatory state or immune suppression related to chronic inflammation is the etiologies that lead to lethal conditions (38). Therefore, understanding the intrinsic mechanisms underlying macrophage polarization will help us to gain control of innate immune responses during the development of sepsis.
It has been reported that in the LPS-stimulated THP-1 promonocyte activation model, there is a sequential reprogramming of metabolic and inflammatory-related signals, resulting in a switch from M1 macrophages to an M2 phenotype. The dynamic conversions between the two phenotypes are known to be mediated by several critical factors. Generally, M1 macrophages are dependent on glycolysis and hypoxia-inducible factor 1 (HIF-1α) expression, resulting in higher transcription of TNF-α and Rel-B. Subsequent skirting 1 (SirT1) elevation is responsible for promoting the M2 state by enhancing fatty acid uptake and oxidation (39). Thus, clarifying the underlying mechanisms by which the M2 state was established after M1 suppression will be valuable for the control of sepsis.
Here, we first defined a dynamic expression pattern of QKI in macrophages treated with M1 or M2 stimuli. They showed a reduced M1 state, but an enhanced M2 state, suggesting the potential role of QKI in the M2-related phenotype. Further mechanistic characterization revealed that silencing QKI led to increased expression of M1-related pro-inflammatory genes by enhancing p65 expression and phosphorylation, with decreased STAT1 phosphorylation. Whereas elevated QKI5 expression exerted an anti-inflammatory effect by upregulating the levels of IL-10 and other M2-related genes, such as Arg-1, Fizz1, and Ym1. Unexpectedly, SirT1 expression had little change following QKI alterations (data not shown). Collectively, these in vitro data indicated that QKI plays a dual role in modulating M1/M2 polarized states, specifically promoting M2 polarization at the expense of the M1 state. In terms of the mechanism, QKI may mediate its inflammatory suppressive effect through influence on more than one factor.
Aryl hydrocarbon receptor, a ligand-activated transcriptional factor, displays an important regulatory function in various biological processes, including detoxification and immune responses. It is also involved in LPS-induced immune tolerance in macrophages (11) by suppressing NLRP3 inflammasome transcription, downstream caspase-1 activation, and subsequent IL-1 β secretion.
In our study, we defined a novel pathway to regulate Ahr expression at the posttranscriptional level. As an RNA-binding protein, QKI affects Ahr mRNA stability by directly binding to its 3′UTR. More importantly, p65-mediated transcriptional activity at the promoter region of target genes is also prohibited by QKI-mediated Ahr changes. In addition, the alterations in the NLRP3 inflammasome activation, caspase-1 cleavage, and IL-1β secretion are also alleviated by Ahr signaling in QKI-overexpressed cells. Therefore, these findings confirm the importance of Ahr signaling in mediating QKI-related antiinflammatory regulation.
It is worth mentioning that consistent with our observations, another group has also reported that miR-155-mediated downregulation of QKI in RAW 264.7 cells was important for enhanced responses to LPS, as well as for leukemogenesis (40). Moreover, they defined that the c-Jun N-terminal kinase was involved in QKI-induced anti-inflammatory responses, using the p38 MAPK pathway. However, they did not show the effects by using primary cells or in an in vivo mouse model.
In order to test the in vivo function of QKI, we established myeloid-specific QKI knockout mice. The overall inflammatory responses in the LPS-induced endotoxic shock model were more severe in conditional QKI-deficient mice, parallel with higher levels of TNF-α, IL-6, and IL-1β. The LPS-induced lethal phenotype in QKI-deficient mice could be largely alleviated by transferring peritoneal macrophages overexpressing QKI5, which was associated with decreased production of the systemic pro-inflammatory cytokines but increased numbers of M2 macrophages and higher levels of IL-10. Strikingly, the peritoneal macrophage subgroup analysis revealed that transfer of macrophages overexpressing QKI5 significantly induced a shift of M1 (F4/80 int CD11b int ) macrophages toward the M2 (F4/80 hi CD11b hi ) phenotype.
Compared to monocytes, neutrophils have a shorter lifespan and play a significant role in the rapid removal of microorganisms. Granulocyte homeostasis constitutes a subtle balance between being helpful or harmful in terms of damage to the tissues in response to microbes (41). Our current mouse model features QKI deficiency in the myeloid lineage. Following LPS treatment, the numbers of macrophages are mildly elevated whereas neutrophils are slightly decreased, with no significant differences in either cell type. Since we did not perform any relevant functional tests in QKI-deficient neutrophils, it is difficult to draw a definitive conclusion on whether the lethal phenotype with exaggerated inflammatory responses in LysMCre QKI fl/fl mice against LPS is related to neutrophils or not. Therefore, more experimental evidence is needed in this area.
On the whole, our results suggest a novel role for QKI in restraining LPS-induced overt innate immune responses in mice by favoring the anti-inflammatory M2-polarized macrophages rather than the pro-inflammatory M1-polarized macrophages. Therefore, modulating the QKI expression level may be a potential way to treat macrophage-mediated inflammatory diseases such as sepsis.

eThics sTaTeMenT
This study was carried out in accordance with the recommendations of the Laboratory Animal Center of Fourth Military Medical University.