Macrophages, mouse Lewis lung cancer (LLC) cells, and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, USA) at 37°C with 5% CO₂.

Male C57BL/6 mice were purchased from Biocytogen Co., Ltd (Beijing, China). The TLR4^−/− and myeloid differentiation factor 88 (MyD88)^−/− C57BL/6 mice were provided by Professor Qingwu Yang. Animal experiments were conducted according to the experimental animal guidelines of the Army Medical University of China.

Recombinant PcrV protein was expressed from the expression strain Escherichia coli JM109/pQE31-PcrV as described previously (13). Protein purification was performed using His-Trap HP affinity columns (GE Healthcare, Sweden), and endotoxin was removed using Detoxi-Gel endotoxin removing gel (Thermo Fisher, USA), according to the manufacturer’s instructions.

Mouse bone marrow cells were isolated from the tibia and femur of C57BL/6 mice. Bone marrow-derived macrophages (BMDMs) were induced by the administration of mouse macrophage colony-stimulating factor (M-CSF; 30 ng/ml) in DMEM supplemented with 10% FBS and 100 U/ml of penicillin for 7 days at 37°C with 5% CO₂. For in vitro TAM induction, BMDMs were cultured in DMEM/FBS (10%) containing 20% (v/v) LLC cell culture supernatant for 24 h (14).

For PcrV administration, C57BL/6 mice were subcutaneously inoculated with 1 × 10⁶ LLC cells. On day 9, the animals were randomly divided into two groups and were intratumorally injected with either phosphate-buffered saline (PBS; control group) or PcrV (0.5 mg/kg, every 4 days). For the BMDM inoculation experiment, mice were subcutaneously inoculated with 1 × 10⁶ LLC cells (day 0). On days 0 and 7, the animals were peritoneally injected with 200 μl of clodronate liposomes (Liposoma BV, Netherlands) to deplete endogenous macrophages. PBS or PcrV (0.5 mg/kg) was intratumorally injected at days 12, 14, 16, and 18, while the wild type (WT), TLR4^−/−, or MyD88^−/− BMDMs (2 × 10⁶ cells) primed or not with PcrV were intratumorally inoculated at days 16 and 18. At the end of the experiment, the mice were euthanized with pentobarbital, and tumor tissues were harvested for subsequent analyses.

Tumor tissues were minced and incubated with collagenase type II (1 mg/ml, Thermo Fisher) at 37°C for 40 min to obtain a single-cell suspension. For the staining of extracellular target proteins, 1 × 10⁶ cells were first incubated in a mixture of PBS, 1% FBS, and anti-CD16/32 antibody (BioLegend, #101301) to block non-specific binding and then labeled with the indicated antibodies at room temperature for 30 min. Fluorescence-activated cell sorting (FACS) was performed using a Beckman Coulter Gallios flow cytometer (USA), and the results were analyzed using FlowJo software version 10.0.7 (TreeStar). The following fluorochrome-coupled antibodies were used in the experiment: anti-CD45 (#103115), anti-CD206 (#141711), anti-CD3 (#100235), anti-CD4 (#100203), anti-CD8 (#100733), anti-NK1.1 (#108709), and anti-Gr1 (#108425) (all from BioLegend); and anti-CD11b (#12-0112), anti-F4/80 (#11-4801), anti-CD11c (25–0114), anti-MHCII (#17-5321), and anti-CD86 (#17-0862) (all from eBioscience) antibodies; the fixable viability dye eFluor 506 was from eBioscience (#65-0866).

Tissue apoptosis was detected using the TdT-mediated dUTP nick end labeling (TUNEL) assay by staining the tissue sections using an In Situ Cell Death Detection Kit, POD (Roche, USA), at 37°C for 30 min. Nuclei were counterstained with Hoechst 33342, and images were obtained by laser scanning confocal microscopy (Leica TCS SP5, Germany).

Murine macrophages (Raw264.7 cells) cultured on coverslips were treated with PcrV (10 μg/ml) for 24 h, fixed in 4% paraformaldehyde at room temperature for 15 min, and stained with antibodies targeting TLR4 (ProteinTech, #19811-1-AP) and PcrV (generated in our laboratory). For tissue staining, paraffin-embedded tumor tissues were sectioned and stained first with antibodies against inducible NO synthase (iNOS) (ProteinTech, # 18985-1-AP), ARG1 (ProteinTech, # 16001-1-AP), and/or F4/80 (Abcam, #ab6640) and then with the corresponding secondary antibodies (labeled with fluorescein isothiocyanate (FITC) or Alexa Fluor 647). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, China). Images were acquired by laser scanning confocal microscopy (Leica).

BMDMs were seeded at 5 × 10⁵ cells/well in a six-well plate and treated or not with PcrV (10 μg/ml) for 24 h. After the supernatants were discarded, the macrophages (lower chamber of a Transwell plate [0.4-μm pore; Corning, USA]) were co-cultured with LLC cells (upper chamber) in DMEM/FBS (10%) at a ratio of 1:1 for another 24 h. LLC cells were harvested and stained using an Annexin V/7-AAD apoptosis detection kit (BD Biosciences, USA) according to the manufacturer’s instructions. Apoptosis was determined by FACS.

Protein extraction and concentration determination were performed following the manufacturer’s instructions (Beyotime). Primary antibodies targeting iNOS (BioLegend, #696802), p-AKT (Ser473) (CST, #4060), AKT (Epitomics, #1081), p-mTOR (Ser2448) (CST, #5536), mTOR (CST, #2983), COX2 (ProteinTech, #12375-1-AP), vimentin (CST, #5741), HA tag (CST, #3724), His tag (ProteinTech, #10001-0-AP), and GAPDH (ProteinTech, #60004-1-Ig) were used in this study. The relative optical densities of the protein bands were quantified using ImageJ software.

Total RNA extraction, reverse transcription, and RT-qPCR were performed according to the manufacturer’s protocols. Primers were provided in the Supplementary Material (Table S1).

The levels of TNFA and IL12 p40/70 in the cell culture supernatants were quantified by ELISA kits (BD Biosciences, USA) according to the manufacturer’s instructions.

NO level in the culture supernatants was measured by Griess reagent (Beyotime, China) according to the manufacturer’s instruction.

Cells were incubated with 2′7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Santa Cruz, USA) dye in DMEM at a final concentration of 5 μM at room temperature for 30 min. Intracellular ROS was measured by FACS.

Lactic acid level in cell supernatant was measured according to the manufacturer’s instruction (Dojindo Laboratories, Japan).

Cells were cultured in a Seahorse XFp cell culture microplate (Agilent, USA) at 5 × 10⁴ cells/well. After pretreatment with the corresponding inhibitors or PcrV, the cells were washed with Seahorse XF Base Medium (103193-100, Agilent) supplemented with 2 mM of glutamine (103579, Agilent). The extracellular acidification rate (ECAR) was measured using a Seahorse XFp Glycolysis Stress Test Kit (103020-100, Agilent) with a Seahorse XFp Analyzer (Agilent) according to the manufacturer’s instructions.

LLC cells (3 × 10⁴ cells in serum-free DMEM) that had been serum-starved overnight were seeded in the upper chamber of a Transwell insert (8-μm pore, 24-well, Corning). DMEM supplemented with 20% FBS and with or without PcrV (20 μg/ml) was added to the lower chamber as a chemoattractant. After incubation for 24 or 48 h, the non-migrated LLC cells in the upper chambers were removed. Migrated cells in the bottom chamber were fixed and then stained with 0.5% crystal violet solution for 5 min. The number of migrated cells was counted under an optical microscope (Olympus, Tokyo, Japan).

Tumor cells and TAMs were treated with various concentrations of PcrV for 48 h. Cytotoxicity was assessed using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan) according to the manufacturer’s protocol.

Cells were lysed in lysis buffer, and the ATP concentration was tested using an enhanced ATP assay kit (Beyotime) according to the manufacturer’s protocol. The luminescence data as determined using a microplate reader (Varioskan Flash, Thermo Fisher) were normalized against protein concentration.

HEK293T cells were transfected with the pCDNA3.1-TLR4 vector using Lipofectamine 2000 (Invitrogen, USA) and cultured in an incubator at 37°C with 5% CO₂ for 48 h. The obtained cell lysates were incubated with purified PcrV (5 μg) at 4°C for 4 h before the addition of 20 μl of His Mag Sepharose™ Ni (GE Healthcare) and incubation at 4°C for 1 h. PcrV and TLR4 were detected by Western blotting using anti-His and anti-HA-tag antibodies, respectively.

For surface plasmon resonance (SPR) analysis, 30 μg/ml of human recombinant TLR4 protein containing a 6×His tag (Abcam, #ab233665) was fixed on the NH2 sensor chip (Nicoya, Canada) using Amine Coupling Kit (Nicoya). Then, PcrV at the indicated concentrations was sequentially injected into the chamber in PBS running buffer. TLR4-PcrV interaction was detected using OpenSPR (Nicoya). The parameters of the binding reactions were calculated and analyzed using Trace Drawer software (Nicoya).

Data were analyzed using unpaired Student’s t-tests or one-/two-way ANOVA in GraphPad Prism version 7.0. Tumor volume data were reported as means ± SEM. Other data were expressed as means ± SD.

As PcrV has been reported to activate the host immune response (11, 12), we investigated whether PcrV exerts similar effects in the immunosuppressive TME. First, we assessed the efficacy of PcrV in vivo by subcutaneously inoculating LLC cells into the right flanks of C57BL/6 mice followed by intratumoral injection of PcrV ( Figure 1A ). Compared with the control group, PcrV treatment decreased tumor growth ( Figure 1B ) and weight ( Figure 1C ). We previously demonstrated that PcrV significantly increased NO production in normal BMDMs (13). Considering that NO-mediated cytotoxicity is associated with tissue apoptosis and the inhibition of tumor growth, we then evaluated the levels of tumor cell apoptosis and the expression of the NO-generating enzyme—iNOS—in PcrV-treated tumor tissues. The results showed that PcrV treatment increased the levels of apoptosis ( Figure 1D ) and iNOS expression ( Figure 1E ) in the tumor tissues, indicating that PcrV-induced NO generation is associated with the suppression of tumor growth. In addition, we also treated LLC cells and TAMs with PcrV to assess whether PcrV administration might directly cause cytotoxicity or affect tumor cell growth and metastasis. We found that at the concentrations tested, PcrV did not cause significant cytotoxicity against either tumor cells or TAMs ( Supplementary Figure 1A ). Moreover, PcrV treatment did not affect the expression levels of genes (e.g., Cox2, Mmp9, Vegfa, and Hif1a; Supplementary Figure 1B ) or proteins (e.g., COX2 and vimentin; Supplementary Figure 1C ) related to tumor growth and metastasis, or the metastatic ability of LLC cells ( Supplementary Figure 1D, E ).

Given that immune cells infiltrated in the TME, such as TAMs, CD4⁺ and CD8⁺ T cells, NK cells, and myeloid-derived suppressor cells (MDSCs), play a critical role in modulating antitumoral immune responses (15), we further analyzed the ratios of these cells in the TME following PcrV treatment. The results showed that exposure to PcrV did not significantly affect the percentages of CD4⁺ and CD8⁺ T cells, NK cells, or MDSCs in tumor tissues ( Supplementary Figure 2A – F ). However, even though PcrV treatment did not alter the percentage of TAMs infiltrated in the TME ( Supplementary Figure 2G, H ), the ratios of M1-polarized TAMs (F4/80⁺CD11c⁺CD206⁻, F4/80⁺MHCII⁺, and F4/80⁺CD86⁺ macrophages) were increased in PcrV-treated tumor tissues ( Figures 2A – D ), whereas those of M2-polarized TAMs (F4/80⁺CD11c⁻CD206⁺ macrophages) were decreased ( Figures 2A, B ). Subsequent analysis of iNOS⁺ TAMs by immunofluorescence (IF) staining indicated that PcrV treatment increased the percentage of F4/80⁺iNOS⁺ M1 TAMs ( Figure 2E ), whereas that of F4/80⁺ARG1⁺ M2 TAMs was decreased ( Figure 2F ). Collectively, these results suggested that PcrV reeducates TAMs to a tumoricidal M1 phenotype in vivo.

To determine whether PcrV directly affects TAM polarization in vitro, we generated in vitro-trained TAMs by incubating BMDMs with LLC cell culture supernatant. These cells showed increased expression of genes related to tumor growth (Cox2 and Vegfa), metastasis (Cox2, Mmp2, and Mmp9), and immune suppression (Ido2) ( Supplementary Figure 3A ), indicating that TAM generation in vitro had been successful. The levels of tumoricidal M1 polarization markers, including iNos, Cd11c, MhcI, MhcII, Cd86 ( Supplementary Figure 3B ), iNOS ( Supplementary Figure 3C ), NO ( Supplementary Figure 3D ), IL12 p40/70, TNFA ( Supplementary Figure 3E ), MHCII, and CD86 ( Supplementary Figure 3F, G ), were found to be higher in PcrV-primed than mock-treated TAMs, whereas the levels of the protumoral M2 polarization markers c-Myc, Egr2, Fn1, Arg1 (13), and Cd206 were lower ( Supplementary Figure 3B ), demonstrating that PcrV directly reprograms TAMs to an antitumoral M1 profile in vitro.

To further determine whether the PcrV-mediated M1 polarization of TAMs is responsible for tumor growth inhibition, LLC tumor-bearing mice were peritoneally injected with clodronate liposomes to deplete endogenous macrophages, after which the mice were intratumorally injected with PBS, PcrV, or BMDMs primed or not with PcrV ( Figure 3A ). The results showed that clodronate liposome treatment reduced macrophage infiltration in tumor tissues ( Figure 3B ). The treatment with PcrV-primed BMDMs decreased tumor growth ( Figure 3C ) and weight ( Figure 3D ) and increased the levels of apoptosis ( Figure 3E ) and iNOS expression ( Figure 3F ), and the percentage of iNOS⁺F4/80⁺ TAMs ( Figure 3F ) in tumor tissues; however, PcrV treatment did not affect either tumor growth ( Figure 3C ) or weight ( Figure 3D ) in mice depleted of endogenous macrophages due to clodronate liposome administration. Collectively, these results demonstrated that the PcrV-mediated tumoricidal effect is associated with the reprogramming of TAMs to an M1 phenotype.

Based on the above in vivo and in vitro results, we next investigated the direct cytotoxic effect of PcrV-primed BMDMs on tumor cells by co-culturing these cells with LLC cells. The co-culture of BMDMs with tumor cells polarized normal BMDMs into TAMs. Hence, BMDMs are referred to as TAMs after their co-culture with tumor cells. We found that co-culture increased the rate of apoptosis among the tumor cells ( Figures 4A, B ). Considering that ROS and NO are critical mediators of cancer cell cytotoxicity, we also examined the levels of these factors in the co-culture system. Unexpectedly, PcrV treatment did not affect the levels of ROS either in TAMs or LLC cells ( Supplementary Figure 4 ). In contrast, NO production was found to be higher in the PcrV/TAM-LLC cell co-culture medium than in that of the TAM-LLC cell group ( Figure 4C ). The levels of iNos ( Figure 4D ) and iNOS ( Figures 4E, F ) were also higher in both TAMs and LLC cells. The inhibition of NO production in PcrV/TAMs through S-methyl thiourea (SMT) treatment decreased apoptosis ( Figures 4A, B ) in LLC cells. The addition of the NO donor DETA-NONOate (DETA-NO) into a co-culture system in which TAMs had been pretreated with SMT led to an increase in NO levels in the culture medium ( Figure 4C ) while also promoting the apoptosis of LLC cells ( Figures 4G, H ) in the PcrV/TAM-LLC cell co-culture group, thus providing further evidence that PcrV-primed TAMs display enhanced NO-associated cytotoxicity against tumor cells.

Even though the levels of NO were similar among the three groups [LLC cells ( Supplementary Figure 5A ), TAM-LLC cell co-culture group ( Figure 4C ), and PcrV/TAM-LLC cell co-culture group ( Figure 4C )] following the addition of DETA-NO to the culture medium, the increased concentrations of NO did not enhance the apoptosis rate of LLC cells cultured individually ( Supplementary Figure 5B ) or with BMDMs ( Figures 4G, H ) compared with that of the PcrV/TAM-LLC cell co-culture system, suggesting that the PcrV-mediated augmentation of TAM-associated cytotoxicity against cancer cells relies on a synergistic effect of NO and other factors generated by PcrV-primed TAMs.

In addition, to observe whether PcrV-primed TAMs can affect tumor growth and metastasis, BMDMs treated or not with PcrV were co-cultured with LLC cells, and the levels of related genes were analyzed. No differences in the expression levels of genes related to tumor growth and metastasis, such as Cox2, Mmp9, Vegfa, and Hif1a, were found in LLC cells that were co-cultured with PcrV-primed TAMs ( Figure 4D ).

M1 macrophages are reported to rely on aerobic glycolysis for their energy supply (16), suggesting that the PcrV-mediated increase in TAM cytotoxic activity against cancer cells might involve the regulation of glycolysis. Similar to that seen with LPS/IFNG-primed M1-like TAMs, PcrV treatment increased the glycolysis-related ECAR ( Figure 5A ), lactic acid production ( Figure 5B ), and ATP generation ( Figure 5C ) in TAMs. The inhibition of glycolysis in TAMs by the application of 2-deoxy-d-glucose (2-DG) suppressed the PcrV-induced production of lactic acid ( Figure 5B ) and ATP ( Figure 5C ), as well as the levels of NO ( Figure 5D ), IL12 p40/70, and TNFA ( Figure 5E ), indicating that PcrV reprograms TAMs toward an M1 profile through increasing glycolysis. In addition, to examine the role of glycolysis in PcrV-induced, TAM-mediated cytotoxicity against cancer cells, BMDMs pretreated with 2-DG and PcrV were co-cultured with LLC cells. We found that 2-DG treatment decreased the PcrV-induced production of NO ( Figure 5F ) and the rate of apoptosis in LLC cells ( Figures 5G, H ), demonstrating that the enhancement of glycolysis by PcrV promotes the NO-associated tumoricidal activity of TAMs.

Studies have shown that the PI3K/AKT/mTOR pathway is closely related to macrophage activation (17) as well as glycolysis in pulmonary fibrosis (18). To observe whether the PcrV-mediated regulation of TAM repolarization might involve this signaling pathway, we examined AKT and mTOR phosphorylation levels in TAMs treated or not with PcrV. The results revealed that PcrV treatment increased the phosphorylation levels of both proteins ( Figure 6A ) in TAMs. The treatment of PI3K, AKT, or mTOR with the corresponding inhibitor suppressed the levels of M1 markers in PcrV-treated TAMs, including that of iNos ( Supplementary Figure 6A ), iNOS ( Figure 6B ), NO ( Figure 6C ), IL12 p40/70, and TNFA ( Supplementary Figure 6B ) while increasing the levels of the M2 markers Fn1, c-Myc, and Egr2 ( Supplementary Figure 6A ), suggesting that PcrV repolarizes TAMs into an M1 phenotype through the activation of the PI3K/AKT/mTOR signaling pathway. To further investigate whether the PI3K/AKT/mTOR signaling pathway is involved in the enhancement of the tumoricidal effect of TAMs elicited by PcrV, TAMs pretreated with the AKT or mTOR inhibitor plus PcrV were co-cultured with LLC cells. The suppression of AKT or mTOR activation reduced NO production in the co-culture medium ( Figure 6D ) and also decreased the levels of PcrV/TAM-induced apoptosis in LLC cells ( Figures 6E, F ).

We also investigated the cross-talk among PI3K/AKT/mTOR, glycolysis, and NO. The inhibition of PI3K, AKT, or mTOR led to impaired glycolysis-related ECAR and lactic acid production in PcrV-primed TAMs ( Figures 6G, H ). Inversely, the suppression of glycolysis by 2-DG led to impaired AKT and mTOR phosphorylation and iNOS expression ( Figure 6I ), suggesting that glycolysis positively regulates AKT/mTOR activation and NO generation in PcrV-primed TAMs. Given that NO promotes glycolysis in neurons (19) and also activates the PI3K/AKT axis in cancer cells (20, 21), we next examined the role of NO in triggering AKT/mTOR activation and glycolysis in PcrV-primed TAMs. Inhibiting NO generation in PcrV-primed TAMs with SMT treatment reduced AKT and mTOR phosphorylation ( Figure 6J ) as well as the ECAR ( Figure 6G ) and lactic acid levels ( Figure 6H ). Intriguingly, the exogenous administration of DETA-NO in combination with PcrV, but not DETA-NO alone, promoted AKT/mTOR activation in TAMs ( Figure 6K ) and upregulated lactic acid production ( Figure 6L ), indicating that NO-associated regulation of glycolysis and the AKT/mTOR signaling pathway in TAMs requires the involvement of other factors activated by PcrV. Collectively, these results indicated that the PcrV-mediated increase in the antitumoral effects of TAMs is associated with the activation of a PI3K/AKT/mTOR-glycolysis-NO feedback loop.

TLR4, a key pattern recognition receptor (PPR), is abundantly expressed on immune cells, where it functions in the recognition of pathogen-associated molecular patterns (PAMPs). Following pathogen recognition, TLR4 activates host immune response through both MyD88-dependent and MyD88-independent mechanisms (22). Given that PcrV is a needle tip protein component of the T3SS of P. aeruginosa, it seems likely that it can interact with host PPRs and thus activate immune responses. To address this possibility, we investigated the role of the TLR4/MyD88 signaling pathway in PcrV-mediated regulation of TAM polarization. Compared with WT PcrV-primed TAMs, TLR4^−/− or MyD88^−/− PcrV-primed TAMs showed reduced levels of NO ( Figure 7A ), iNos ( Figure 7B ), iNOS ( Figure 7C ), IL12 p40/70, and TNFA ( Supplementary Figure 7A ) but higher levels of c-Myc, Egr2, and Fn1 ( Supplementary Figure 7B ), suggesting that PcrV skews the TAM phenotype toward an M1 profile through the TLR4-MyD88 signaling pathway.

As it has been reported that the PI3K/AKT/mTOR pathway is under the control of the TLR4/MyD88 signaling axis (23), we next examined PI3K/AKT/mTOR pathway activation in TLR4^−/− or MyD88^−/− PcrV-primed TAMs. The results showed that the PcrV-induced activation of AKT and mTOR was impaired in TLR4^−/− or MyD88^−/− TAMs, respectively ( Figure 7C ). Analyses of glycolysis-related factors demonstrated that the PcrV-induced increase in the ECAR ( Figure 7D ) and lactic acid production ( Figure 7E ) was reduced in TLR4^−/− and MyD88^−/− TAMs.

We further analyzed the role of the TLR4/MyD88 axis in the PcrV-mediated regulation of the tumoricidal effect of TAMs. The co-culture of LLC cells with PcrV-primed TLR4^−/− or MyD88^−/− TAMs, but not WT TAMs, reduced NO production in the culture medium ( Figure 7F ) as well as the levels of apoptosis in LLC cells ( Figures 7G, H ). In addition, the LLC tumor-bearing mice in which endogenous macrophages were depleted by clodronate liposomes were intratumorally injected with WT, TLR4^−/−, or MyD88^−/− BMDMs primed or not with PcrV ( Supplementary Figure 8A ). The result showed that treatment with PcrV-primed WT BMDMs, but not TLR4^−/− or MyD88^−/− BMDMs, decreased tumor growth ( Supplementary Figure 8B ) and weight ( Supplementary Figure 8C ). Collectively, these results indicated that the TLR4/MyD88 signaling axis participates in the PcrV-mediated modulation of the PI3K/AKT/mTOR-glycolysis-NO circuit and the tumoricidal effect of TAMs.

To further explore whether PcrV alters TAM polarization through interaction with TLR4, we performed immunoprecipitation by incubating purified PcrV protein with the lysate of HEK293T cells overexpressing recombinant human TLR4 protein. The result showed a successful pull-down of TLR4 protein by PcrV ( Figure 7I ). SPR analysis using purified protein further revealed a direct interaction between TLR4 and PcrV ( Figure 7J ). Moreover, IF staining result showed that PcrV colocalized with TLR4 in Raw264.7 macrophages ( Figure 7K ). In addition, to examine the effect of PcrV–TLR4 interaction on PcrV-mediated regulation of TAMs, we blocked TLR4 using an antibody, and we found that the PcrV-induced production of IL12 p40/70, TNFA ( Supplementary Figure 7C ), and NO ( Supplementary Figure 7D ) in TAMs was reduced, indicating that PcrV-mediated regulation of TAM repolarization involves direct interaction between PcrV and TLR4.