Ku70 and Ku80 participate in LPS-induced pro-inflammatory cytokines production in human macrophages and monocytes

In human macrophages and monocytes, lipopolysaccharide (LPS) induces nuclear factor kappa B (NFκB) activation and pro-inflammatory cytokines production. We tested the possible involvement of Ku70 and Ku80 in the process. In THP-1 macrophages and primary human peripheral blood mononuclear cells (PBMCs), shRNA-induced double knockdown of Ku70 and Ku80 potently inhibited LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). Additionally, we developed CRISPR/Cas-9 gene-editing methods to knockout both Ku70 and Ku80 in THP-1 cells and PBMCs. Double knockout (DKO) largely inhibited LPS-induced pro-inflammatory cytokines production. Conversely, in THP-1 cells exogenous overexpression of both Ku70 and Ku80 enhanced the pro-inflammatory cytokines production by LPS. Ku70 and Ku80 co-immunoprecipitated with p65-p52 NFκB complex in the nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced NFκB activation was inhibited by Ku70 plus Ku80 double knockdown or DKO. It was however enhanced with Ku70 and Ku80 overexpression. Together, Ku70 and Ku80 promote LPS-induced NFκB activation and pro-inflammatory response in THP-1 cells and human PBMCs.

The heterodimeric protein Ku is composed of two subunits: Ku70 and Ku80. The two were originally identified as possible auto-antigens associated with multiple autoimmune diseases, including systemic lupus erythematosus (SLE), scleroderma, polymyositis, and possible others [11,12]. Ku70 and Ku80 are abundant in eucaryote cells. Both are ubiquitously expressed in cell nuclei [11,12]. The two recognize and bind ends of DNA AGING double-strand break (DSB), essential for non-homologous end-joining (NHEJ) repair [13][14][15]. Three structural domains for Ku70/Ku80 proteins have been identified, including the N-terminal domain, the DNA binding domain and the C-terminal domain [16][17][18]. In the present study, we showed that Ku70 and Ku80 together promoted LPS-induced NFκB activation and pro-inflammatory response in monocytes and macrophages.

In human macrophages Ku70 plus Ku80 double knockdown inhibits LPS-induced production of proinflammatory cytokines
In order to study the potential effect of Ku70 and Ku80 in LPS-induced pro-inflammatory response, shRNA strategy was applied. Ku70 shRNA lentivirus and/or Ku80 shRNA lentivirus were transduced to THP-1 human macrophages. Via selection by puromycin stable THP-1 cell lines were established. The qPCR assay results, Figure 1A, confirmed that each of the applied lentiviral shRNA led to dramatic downregulation of target mRNA in THP-1 cells. Cells with both shRNAs presented with significant knockdown of both Ku70 mRNA and Ku80 mRNA ( Figure 1A). Western blotting analyses of Ku70 and Ku80 protein expression demonstrated similar results as the qPCR results ( Figure  1B). TLR4 expression was not altered by knockdown of Ku70 and/or Ku80 ( Figure 1B).

Ku70 plus Ku80 double overexpression enhances LPS-induced production of pro-inflammatory cytokines in human macrophages
Based on the results above, Ku70 plus Ku80 overexpression could possibly enhance LPS-induced pro-inflammatory response. Therefore, the Ku70expressing adeno-associated virus (AAV) plus the Ku80-expressing AAV were co-transfected to THP-1 macrophages. Stable cells were established by puromycin selection. Analyzing mRNA expression, by qPCR, confirmed that Ku70 mRNA and Ku80 mRNA levels increased over 6-8 folds in the stable cells ("Ku70/Ku80 D-OE" cells, Figure 3A). Ku70 and Ku80 proteins were upregulated as well in Ku70/Ku80 D-OE cells ( Figure 3B). Ku70 plus Ku80 overexpression did AGING Figure 1. In human macrophages Ku70 plus Ku80 double knockdown inhibits LPS-induced production of pro-inflammatory cytokines. THP-1 human macrophages were transduced with Ku70 shRNA lentivirus ("sh-Ku70") and/or Ku80 shRNA lentivirus ("sh-Ku80"), control cells were treated with scramble control shRNA lentivirus ("sh-c"), stable cells were established following puromycin selection, mRNA and protein expression of listed genes were tested by qPCR (A) and Western blotting (B); Cells were treated with LPS (500 ng/mL) or vehicle control ("C") for indicated time, mRNA expression (C-E) and protein contents in the culture medium (F-H) of listed pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were tested by qRT-PCR and ELISA assays; Cell survival and death were tested by MTT (I) and Trypan blue staining (J), respectively. Expression of listed proteins was quantified, normalized to the loading control (B). Data were expressed as mean ± standard deviation (SD, n=5). *p<0.05 vs. "C" treatment of "sh-c" cells. # p<0.05. LPS treatment of "sh-c" cells. Experiments in this figure were repeated five times, and similar results were obtained.
In THP-1 cells ectopic overexpression of Ku70 or Ku80 (single overexpression) (Supplementary Figure 2A  construct, control cells were transfected with CRISPR/Cas9 control vector ("Cas9-C"), stable cells were established following puromycin selection, mRNA and protein expression of listed genes were tested by qPCR (A) and Western blotting (B); Cells were treated with LPS (500 ng/mL) or vehicle control ("C") for indicated time, cell viability and death were tested by MTT (C) and Trypan blue staining (D), respectively; mRNA expression (E-G) and protein contents in the culture medium (H-J) of listed pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were tested by qRT-PCR and ELISA assays; Expression of listed proteins was quantified, normalized to the loading control (B). Data were expressed as mean ± standard deviation (SD, n=5). *p<0.05 vs. "C" treatment of "Cas9-C" cells. # p<0.05. LPS treatment of "Cas9-C" cells. Experiments in this figure were repeated three times, and similar results were obtained. AGING Thus, single Ku70 or Ku80 overexpression of failed to promote LPS-induced production of pro-inflammatory cytokines in THP-1 cells.

cells and primary human PBMCs
NFκB activation is essential for LPS-induced proinflammatory cytokines production in macrophages and monocytes [19,20]. Performing a co-immunoprecipitation (Co-IP) experiments of nuclear lysates in THP-1 cells, we demonstrated Ku70 and Ku80 coimmunoprecipitated with NFκB proteins p52 and p65 in response to LPS stimulation ( Figure 4F). "INPUT" results confirmed p52/p65 protein nuclear translocation with LPS stimulation ( Figure 4F). Nuclear Ku70 and Ku80 expression was however unchanged ( Figure 4F). The NFκB (p65) DNA-binding activity assay results showed that LPS-induced NFκB activation was largely inhibited by shRNA-mediated double knockdown of Ku70 and Ku80 in THP-1 macrophages ( Figure 4G). Whereas Ku70 or Ku80 single knockdown was completely ineffective ( Figure 4G).

DISCUSSION
The results of the present study suggested that Ku70 and Ku80 both participated in LPS-induced proinflammatory response in THP-1 macrophages and primary human PBMCs. It is possible that Ku70 and Ku80 can compensate each other. shRNA-mediated Ku70 and Ku80 double knockdown (DKD) potently inhibited LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). While each single knockdown of Ku70 or Ku80 was complete ineffective. Further, CRISPR/Cas-9-mediated double knockout (DKO) of Ku70 and Ku80 largely inhibited LPSinduced pro-inflammatory cytokines production in THP-1 cells and human PBMCs. While single knockout failed to attenuate LPS-induced actions. On the contrary, in THP-1 cells exogenous overexpression of both Ku70 and Ku80 (D-OE) enhanced proinflammatory cytokines production by LPS, with Ku70 or Ku80 single OE completely ineffective. These results convincingly showed that Ku70 and Ku80 are both important in regulating LPS-induced pro-inflammatory response in human macrophages and monocytes.
One important finding of the current study is that Ku70 and Ku80 are important regulators of LPS-induced NFκB activation. We demonstrated that Ku70 and Ku80 co-immunoprecipitated with p65-p52 NFκB complex in nuclei of LPS-treated THP-1 cells, required for full NFκB activation. In THP-1 human macrophages and primary human PBMCs, Ku70 plus Ku80 double knockdown or DKO potently inhibited LPS-induced NFκB activation. Yet, single shRNA or KO did not alter LPS-induced pro-inflammatory responses. On the contrary, forced overexpression of Ku70 plus Ku80 (D-OE) in THP-1 cells potentiated NFκB activation by LPS. Thus, Ku70 plus Ku80 are key regulators of NFκB activation by LPS.
It has been shown that depleting Ku70/Ku80 could reduce cell viability and induce cytotoxicity in certain human cells [23,24]. Here we found that in THP-1 and PBMCs cells Ku70 and/or Ku80 silencing or KO did not change cell viability nor inducing cell death. Furthermore, forced overexpression of Ku70 and/or Ku80 did not alter cell viability in these cells. Therefore Ku70/Ku80 appear to have a cell type specific effect. Indeed, Ma et al., found that Ku70 silencing using different shRNAs did not alter cell viability in human pancreatic cancer cells [25].
In murine cells the lack of Ku70 could lead to depleted expression of Ku80 [26]. In human cells, however, silencing of Ku70 may not result in depletion of Ku80 [25,27]. In the current study we showed that Ku70 silencing (by targeted shRNA) did not alter Ku80 mRNA and protein expression in THP-1 cells. Neither did Ku80 silencing inhibit Ku70 expression. Furthermore, ectopic overexpression of Ku70 did not alter Ku80 mRNA expression in THP-1 cells. Follow up studies will be needed to further explore the relationship between expression of Ku70 and Ku80 in human macrophages and monocytes.

THP-1 cell culture
From the Cell Bank of Shanghai Institute of Biological Science (Shanghai, China) THP-1 human macrophages AGING were purchased. Cells were cultured in RPMI-1640 medium plus 10% FBS.

Human peripheral blood mononuclear cells (PBMCs) primary culture
As previously described [28,29], by using the lymphocyte separation medium (Sigma, C-44010) PBMCs were collected from healthy donors (all male, 25-35 years old) with the written-informed consent. PBMCs were cultured in DMEM with 10% FBS and necessary supplements [30]. The protocols of this study were approved by the Ethics Committee of Wenzhou Medical University.

Quantitative real-time reverse transcriptase polymerase chain reaction (qPCR)
At a density of 1.5 ×10 5 cells/well THP-1 cells or the primary human PBMCs were seeded into 12-well plates. Following the applied LPS treatment (500 ng/mL, 8h), TRIzol reagent was utilized to extract total cellular RNA [31,32]. For qPCR, we utilized an ABI Prism 7600 Fast Real-Time PCR system with the SYBR Green Real-Time Master Mix kit (A46109, Thermo-Fisher, Shanghai, China). The product melting temperatures were calculated by the melt curve analysis. A 2 −ΔΔCt method was utilized for the quantification of target mRNA [33], and normalized to the endogenous reference gene GAPDH. mRNA primers were listed in Table 1.

Western blotting
At a density of 3 ×10 5 cells/well THP-1 cells or the primary human PBMCs were seeded into six-well plates. Following the applied LPS treatment, 20-30 μg of protein lysates (from each treatment in each lane) were separated by a 10% SDS-PAGE gel, and transferred to a polyvinylidene difluoride (PVDF) blot. After blocking in 10% non-fat milk in PBST, the blot was incubated with applied primary and secondary antibodies. Enhanced chemiluminescence (ECL) reagents (Amersham, Piscataway, NJ) were applied to visualize the immuno-reactive proteins through autoradiography [34]. The quantification of the protein band was through the NIH ImageJ software.

MTT assay
Briefly, at a density of 1 ×10 4 cells/well THP-1 human macrophage or PBMCs were plated into 96-well plates. After the applied LPS treatment, MTT dye (5 mg/mL, 20 μL in each well) was added. After incubation for another 2-3h, The optical density (OD) of MTT at 490 nm was recorded.

Cell death assay
At a density of 1 ×10 5 cells/well THP-1 cells or the primary human PBMCs were seeded into the 12-well plates. After the applied LPS treatment, trypan blue staining was performed to quantify cell death. Trypan blue ratio was recorded [35].

Enzyme-linked immunosorbent assay (ELISA)
Briefly, at a density of 1 ×10 4 cells/well THP-1 human macrophage or PBMCs were plated into 96-well plates. Following LPS treatment, supernatants were collected and cytokines were determined by using the commercial available ELISA kits.

shRNA
The verified Ku70 lentiviral shRNA (-a) and Ku80 lentiviral shRNA (-a) were provided by Dr. Xiang at Shanghai Jiao Tong University School of Medicine [25]. At a density of 3 ×10 5 cells/well THP-1 cells or the primary human PBMCs were seeded into the sixwell plates. The shRNA lentivirus was added to cultured cells (in polybrene-containing medium) for 24h. Afterwards, cells were cultured in fresh complete medium. Puromycin (5.0 μg/mL) was added to select resistant stable cells for four more passages. Expression of Ku70 and Ku80 was tested by qPCR and Western blotting. The non-sense control lentiviral shRNA ("sh-c", Santa Cruz Biotech) was transfected to the control cells.

Exogenous Ku70 and Ku80 overexpression
The full-length human Ku70 cDNA and Ku80 cDNA were provided by Dr. Xiang at Shanghai Jiao Tong University School of Medicine [25], individually sub-cloned into pSuper-flag-puro construct. The construct and the adeno-associated virus (AAV) package plasmids (Genechem) were co-transfected to HEK293 cells to generate Ku70-or Ku80-expressing AAV [36]. At a density of 3 ×10 5 cells/well THP-1 cells were seeded into six-well plates. The virus was enriched, filtered and added to THP-1 cells. Afterwards, cells were cultured in fresh complete medium. Puromycin (5.0 μg/mL) was added to select resistant stable cells for four more passages. Expression of Ku70 and Ku80 was verified by qPCR and Western blotting. Control cells were transfected with AAV with empty vector.

Ku70 and Ku80 knockout
The lentiviral CRISPR/Cas-9 PX458-green fluorescent protein (GFP) [34] plasmid with sgRNA targeting AGING To obtain monoclonal cells, GFP-positive cells were further sorted by fluorescence activated cell sorting (FACS), and cultured for another two weeks. In the stable cells Ku70 and Ku80 double knockout (DKO) was verified by qPCR and Western blotting. Control cells were transfected with CRISPR/Cas-9 PX458-GFP control plasmid ("Cas9-C").

NFκB (p65) DNA-binding activity
Following the applied treatment, nuclear proteins were extracted. By using a TransAM™ ELISA kit (Active Motif, Carlsbad, CA, 43296) the NFκB (p65) DNAbinding activity was tested. For each treatment, 1.0 μg of nuclear extracts were subjected to the binding of p65 to an immobilized consensus sequence in a 96-well plate (1 ×10 4 cells/well). After the colorimetric reaction, OD value was measured in an ELISA reader at the test wavelength of 450 nm.

Co-Immunoprecipitation (Co-IP) assay of nuclear proteins
As previously described [10], the nuclear protein lysates (500 μg proteins in each treatment treatment) were pre-cleared using protein A/G Sepharose (Sigma, Shanghai, China). The pre-cleared nuclear lysates were incubated with anti-Ku70/ anti-Ku80 antibody (Cell Signaling Tech, Shanghai, China) for 12h. Afterwards, the protein A/G Sepharose (30 µL for each treatment, Sigma) was added back to the lysates. Ku70/Ku80 coimmunoprecipitation with NFκB protein complex (p52-p65) was subjected.

Statistics analysis
Data were expressed as mean ± standard deviation (SD). Statistics analyses were performed by using the SPSS software (SPSS Inc., Chicago, IL), with p < 0.05 considered as statistical significant [31]. For comparisons among multiple groups, two-way ANOVA with the Bonferroni post hoc testing was performed. A two-tailed unpaired T test (Excel 2007) was applied to test significance between two treatment groups.

Editorial note
& This corresponding author has a verified history of publications using a personal email address for correspondence.

CONFLICTS OF INTEREST
The authors listed no conflicts of interest.

FUNDING
This work was generously supported by grants from the Wenzhou Science and Technology Bureau (Y20130260), and by foundation of the Science and Technology Project of Wujin (WS201607), the Scientific research project of Changzhou Health Commission (QN201829). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.