TLR2 and NLRP3 Orchestrate Regulatory Roles in Escherichia coli Infection-Induced Septicemia in Mouse Models

Abstract

Introduction

Escherichia coli (E. coli) is a significant commensal gram-negative bacterium that can give rise to various diseases. The roles of Toll-like receptor 2 (TLR2) and the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome in sepsis induced by E. coli infection remain unclear.

Methods

In vivo, we investigated differences in mortality, production of inflammatory mediators, organ damage, neutrophil count, and bacterial load during E. coli infection in C57BL/6J mice, as well as in mice deficient in TLR2 or NLRP3. In vitro, we investigated the impact of E. coli on the activation of TLR2 and NLRP3 in macrophages and the influence of TLR2 and NLRP3 on the activation of inflammatory signaling pathways and the secretion of inflammatory mediators in macrophages induced by E. coli infection.

Results

TLR2-deficient (TLR2^−/−) and NLRP3-deficient (NLRP3^−/−) mice exhibit significantly increased mortality and organ damage after E. coli infection. These mice also show elevated levels of TNF-α and IL-10 in serum and peritoneal lavage fluid. Additionally, TLR2^−/− and NLRP3^−/− mice display heightened neutrophil recruitment and increased bacterial load in the blood. Furthermore, macrophages from these mice demonstrate a significant reduction in the activation of the MAPK signaling pathway.

Conclusion

TLR2 and NLRP3 play crucial roles in modulating inflammatory mediator expression, immune cell recruitment, and bactericidal activity, thereby preventing excessive tissue damage and reducing mortality in E. coli-induced sepsis.

Introduction

Bacteria have intricately evolved interactions with mammals, leading to a range of effects, both beneficial and detrimental, on the host [1]. Within the host’s milieu, the molecular sensing apparatus of the innate immune system proficiently recognizes nonhost components and products, mainly encompassing conserved structural elements derived from microbes such as peptidoglycan, lipopolysaccharides (LPSs), and lipoteichoic acids [2]. These microbial-associated molecular patterns prompt the activation of receptors situated on and within host cells, commonly referred to as pattern-recognition receptors (PRRs) [3]. This activation creates a cascade of signal transduction events that produce immune mediators and antimicrobial peptides [4].

The PRRs, including Toll-like receptors (TLRs) and NOD-like receptors (NLRs), constitute the host’s initial line of defense against microbial infections [5]. Toll-like receptor 2 (TLR2) and TLR4 emerge as the principal transmembrane TLRs, attracting extensive attention for their pivotal role in controlling bacterial infections and hindering the dissemination of pathogens [6]. Upon binding with ligands, the TLR2 heterodimer initiates an intracellular signaling cascade, culminating in NF-κB activation, inflammasome assembly, and subsequent production of key inflammatory cytokines [7]. This early response to bacterial infection is pivotal in shaping the downstream immune response, carrying critical implications for the disease course and host outcomes [7–9]. Consequently, TLR2 frequently plays a dual role during infection. On the one hand, TLR2 activation induces beneficial proinflammatory responses essential for controlling infection and facilitating bacterial clearance [10–12]. On the other hand, an excess of TLR2 signaling can contribute to tissue damage and disease progression, stemming from the disproportionate levels of inflammation generated [13, 14]. Achieving a delicate balance in TLR2-mediated responses is critical to harness its protective effects while mitigating the potential for collateral damage to host tissues [15]. The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome, belonging to the NLRs family, is a cytosolic protein characterized by three domains: a C-terminal leucine-rich repeat domain, a central nucleotide-binding and oligomerization domain (NOD or NACHT), and an N-terminal pyrin domain (PYD) [16]. Activation of the NLRP3 inflammasome is a two-step process [17]. In the priming step, cells can recognize pathogen-associated molecular patterns or damage-associated molecular patterns through TLRs, NLRs, or other members of the PRR family [18, 19]. This recognition leads to the translational upregulation of NLRP3 and IL-1β by activating the NF-κB pathway [17]. The second step involves the assembly of the NLRP3 inflammasome after exposure to microbial toxins, ionophores, or endogenous danger molecules [20]. While contributing to the clearance of pathogens, the secretion of inflammatory cytokines and cell pyroptosis during NLRP3 inflammasome activation can simultaneously lead to tissue damage [21]. The NLRP3 inflammasome plays a critical role in maintaining homeostasis against pathogenic infections and has a dual role.

Escherichia coli (E. coli) is a significant commensal Gram-negative bacterium that can give rise to various diseases, including urinary tract infections, bloodstream infections, and sepsis [22, 23]. As understood, LPS, a prototypical pathogen-associated molecular pattern derived from the cell walls of E. coli, is recognized by TLR4 in conjunction with CD14 and MD2, initiating TLR4-mediated signaling cascades [24]. Braun lipoprotein, one of the most abundant components of the outer membrane of E. coli, can induce an inflammatory response through TLR2 [25]. Furthermore, E. coli RNA and enterotoxin have been shown to activate the NLRP3 inflammasome [26]. In response, E. coli induces mice and human cells to produce chemokines and proinflammatory mediators, and this process is dependent on NLRP3 [27]. As detailed research on TLR4-mediated inflammation and sepsis during E. coli infection is currently available, our study will specifically elucidate the regulatory roles of TLR2 and NLRP3 in E. coli-induced sepsis.

In this study, we aimed to ascertain the involvement of TLR2 and NLRP3 in sepsis induced by E. coli infection. In vivo, we investigated differences in mortality, production of inflammatory mediators, organ damage, neutrophil count, and bacterial load during E. coli infection in C57BL/6J mice, as well as in mice deficient in TLR2 or NLRP3. In vitro, we investigated the impact of E. coli on the activation of TLR2 and NLRP3 in macrophages and the influence of TLR2 and NLRP3 on the activation of inflammatory signaling pathways and the secretion of inflammatory mediators in macrophages induced by E. coli infection.

Materials and Methods

Bacterial Strains and Animals

The E. coli CFT073 (ATCC 700928) strain was maintained in our laboratory. A 1 mL bacterial suspension containing 1 × 10⁷ colony-forming units (CFU) was inoculated into 100 mL of Luria-Bertani broth and incubated at 37°C with shaking for 12 h, reaching an optical density (OD 600) of approximately 0.9 in the log phase. The cultured suspensions were subsequently serially diluted and plated on Luria-Bertani agar plates, followed by incubation at 37°C for 12 h. The colony counting technique was employed to determine the total CFU, which was approximately 1 × 10⁸ CFU/mL. This process was conducted in at least 8 independent experiments, and consistent CFU count results were obtained. For this study, C57BL/6J and TLR2^−/− mice were provided by the Model Animal Research Center of Nanjing University (China), while NLRP3^−/− mice were supplied by The Jackson Laboratory (USA).

Experimental Infection and Treatment in vitro

C57BL/6J, TLR2^−/−, and NLRP3^−/− mice received intraperitoneal injections of thioglycolate medium (2 mL 3%, BD Biosciences, USA). After 3 days of intraperitoneal injection, the mice were euthanized, and endotoxin-free phosphate-buffered saline (PBS, Hyclone, USA) was used to lavage the peritoneal cavity to collect peritoneal macrophages. Subsequently, peritoneal macrophages were plated in 6-well culture plates (2 × 10⁶/well) with 1 mL of fresh culture medium (RPMI 1640 medium supplemented with 10% fetal bovine serum, Hyclone, USA) and incubated at 37°C in 5% CO₂. Before stimulation, the peritoneal macrophages were washed three times with PBS. For E. coli stimulation, peritoneal macrophages were exposed to E. coli (1 × 10⁷ CFU/well, multiplicity of infection [MOI] 5:1). After a 1 h infection, extracellular E. coli was eliminated by washing with fresh medium containing 20 U/mL interferon gamma and 10 mg/mL ovalbumin [24].

Experimental Infection and Treatment in vivo

In each experimental group, mice, either C57BL/6J, TLR2^−/−, or NLRP3^−/−, 8 weeks of age, were intraperitoneally injected with E. coli (1 × 10⁸ CFU). Serum and peritoneal lavage fluid (PLF) samples were collected at 3 h and 6 h postinfection, while kidneys and livers were harvested at 12 and 24 h postinfection. The mouse serum was incubated at 37°C for 1 h and overnight at 4°C. Subsequently, the serum was centrifuged at 1,000 g for 15 min at 25°C, and the supernatants were collected for subsequent tests. For PLF collection, the abdominal skin was cleaned with 75% ethanol, and the abdominal wall was exposed by cutting the skin. Sterile physiological saline (3 mL) was slowly injected into the peritoneal cavity and aspirated three times. The recovered PLF was then placed on ice until processed for further examination.

Western Blot Analysis

Macrophages were infected with E. coli (MOI 5:1) for the indicated durations (15 min, 30 min, and 60 min). Total cellular protein from macrophages was extracted using the mammalian protein extraction reagent (Thermo Fisher Scientific, USA), and protein concentrations were determined with the BCA Assay Kit (Thermo Fisher Scientific, USA). For Western blotting analysis, antibodies used included rabbit anti-phospho-ERK, anti-ERK, anti-phospho-p38, anti-p38 (1:1,000, Cell Signaling Technology, MA), and rabbit anti-GAPDH (1:10,000, Abcam, UK) for protein detection. Proteins were visualized using a secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (1:10,000, Cell Signaling Technology, Beverly, MA) and Pierce SuperSignal West Femto chemiluminescent substrate (Thermo Scientific, IL). Grayscale values of bands generated by Western blotting were measured using ImageJ software (version 1.48v, National Institutes of Health, Bethesda, MD).

Enzyme-Linked Immunosorbent Assay Analysis

The supernatants from cultured macrophages were centrifuged at 300 g for 8 min at 4°C and then stored at −80°C. The serum and PLF collected from mice were centrifuged at 1,000 g for 15 min, and the resulting supernatants were collected for subsequent tests. The concentrations of cytokines and chemokines, including TNF-α and IL-1β (BioLegend, USA), IL-10 (Thermo Fisher Scientific, USA), CXCL1, and CXCL2 (PeproTech, USA), were measured using mouse ELISA kits following the instructions. Three biological replicates were analyzed.

Bacterial Counts

Bacterial load was determined in blood. Samples were serially diluted in sterile saline and incubated at 37°C for 12 h on eosin-methylene blue agar plates. CFUs were counted to determine bacterial load and expressed as CFU/mL.

Flow Cytometry Analysis

For flow cytometry analysis, C57BL/6J, TLR2^−/−, and NLRP3^−/− macrophages were infected with E. coli (1 × 10⁷ CFU, MOI 5:1) for 12 h. The cells were suspended using pancreatic digestion, washed twice with PBS, and collected. Following washing, the cells were fixed and permeabilized using the Fixation/Permeabilization Buffer set for 45 min. Subsequently, the cells were stained with PE-conjugated anti-TLR2 (mT2.7, eBioscience, San Diego, CA, USA) or Alexa Fluor 488-conjugated anti-NLRP3 (768319, R&D Systems, Minneapolis, MN, USA) antibodies. For blood sample testing, 100 μL of the sample was mixed with 20 μL of fluorescent antibodies each (PE-conjugated anti-mouse CD11b and Alexa Fluor 488-conjugated anti-mouse Gr-1) and incubated at 4°C for 30 min. The red blood cells were lysed using 3 mL of red blood cell lysis solution at room temperature in the dark for 10 min, followed by centrifugation at 1,200 rpm for 10 min. The supernatant was discarded, and 0.5 mL of fixing solution was added and mixed well for subsequent machine detection. Data were analyzed using FlowJo 10.0 software (TreeStar, Ashland, OR, USA). At least three biological replicates were performed.

Immunofluorescence Assay and Morphological Observations of Livers and Kidneys

The mice were intraperitoneally injected with E. coli and subsequently euthanized to obtain liver and kidney samples. The liver and kidney sections, 6 μm thick, were prepared at −20°C and fixed in cold acetone for 10 min at room temperature. After washing the glass slides with cold PBS containing 0.25% Tween, the slides were blocked for 1 h with 3% bovine serum albumin. A rabbit anti-high-mobility group box protein 1 (HMGB1) polyclonal antibody (diluted 1:200, Novus Biologicals, USA) and anti-hyaluronic acid-binding protein 2 (HABP2) monoclonal antibody (1:100, Abcam, Cambridge, UK) was applied, and the sections were incubated overnight at 4°C in the dark. Subsequently, the slides were washed three times in PBS containing 0.25% Tween and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H&L) secondary antibody (diluted 1:1,000, Abcam) for 1 h at room temperature. Fluorescence images were captured using a confocal microscope (LSM 800 from Zeiss, Germany) at a magnification ×100. One randomly selected field from the three captured images was used for fluorescence intensity analysis. The images were captured under identical conditions for each of the different samples. The livers and kidneys from the mice were collected to prepare paraffin sections, followed by hematoxylin and eosin staining. The observed pathological changes in liver and kidney tissues were examined using a light microscope (DP 72, Olympus, Japan). The histological liver damage was scored based on hepatocellular necrosis, bleeding, and inflammatory cell infiltration in the liver [28] as shown in Table 1. The histological kidney injury was scored based on the injured renal tubules and shrunk glomerulus [28] as shown in Table 2.

Table 1.

Liver injury score parameters

Index	0	1	2	3	Maximum
Necrosis	None	Focal piecemeal	Continuous	Continuous	3
			<50%	>50%
Bleeding	None	<30%	30–50%	>50%	3
Infiltration	None	2- to 3-fold	3- to 10-fold	>10-fold	3

Table 2.

Kidney injury score parameters

Injured renal tubules and shrunk glomerulus	Index
None	0
<10%	1
11–25%	2
26–45%	3
46–75%	4
>76%	5

Data Analysis

All data were analyzed using GraphPad Prism 6 (GraphPad InStat Software, USA) and expressed as means ± standard deviations (SDs). Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparisons test or two-way ANOVA with Bonferroni’s post hoc test, as appropriate. Differences with p values ≤0.05 were considered statistically significant.

Results

TLR2 and NLRP3 Deficiencies Enhance Lethality in E. coli-Infected Mice

To analyze the effects of TLR2 and NLRP3 on E. coli-induced mouse mortality, E. coli was used to infect C57BL/6J, TLR2^−/−, and NLRP3^−/− mice. The survival of the mice was then observed. After intraperitoneal injection of E. coli (1 × 10⁸ CFU) for 14 h, C57BL/6J mice exhibited a 100% survival rate compared to the uninfected control. In contrast, TLR2^−/− mice under the same conditions showed an 80% survival rate, while NLRP3^−/− mice demonstrated 0% survival (Fig. 1). All 3 groups of mice experienced complete mortality 24 h after E. coli infection. The results suggest that TLR2 and NLRP3 regulate the mortality rate in mice infected with E. coli. However, neither of them can reverse the overall outcome.

Fig. 1.

TLR2 and NLRP3 deficiencies enhance lethality in E. coli-infected mice. C57BL/6J (n = 30), TLR2-deficient (n = 30), and NLRP3-deficient (n = 30) mice were injected intraperitoneally with E. coli (1 × 10⁸ CFU). Differences in survival between the experimental groups were compared by the log-rank test. Data are representative of at least three independent experiments.

TLR2 and NLRP3 Deficiencies Exacerbate Organ Damage in E. coli-Infected Mice

Liver and kidney damage in mice exposed to E. coli (1 × 10⁸ CFU, 12 h and 24 h postinfection) was measured via morphological observations. After 12 h of E. coli infection, the liver tissues of C57BL/6J mice exhibited granular degeneration and scattered necrosis in hepatocytes around the periportal vein. In TLR2^−/− mice, granular degeneration, and sporadic or focal cell necrosis. NLRP3^−/− mice displayed mild granular degeneration in a few hepatocytes, scattered or focal cell necrosis, and sporadic infiltration of neutrophils in the liver tissue (Fig. 2). C57BL/6J mice livers showed granular degeneration, and scattered hepatocyte necrosis around the periportal vein in E. coli-infected for 24 h. In TLR2^−/− mice, hepatocytes were generally faintly stained, displaying pronounced granular degeneration, mild lipid vacuolation, widespread scattered or focal neutrophil infiltration, and sporadic hepatocyte necrosis. NLRP3^−/− mice demonstrated mild to severe vacuolar steatosis and occasional small areas of neutrophil infiltration in the liver tissue (Fig. 2).

Fig. 2.

TLR2 and NLRP3 deficiencies exacerbate organ damage in E. coli-infected mice. C57BL/6J (n = 18), TLR2-deficient (n = 18), and NLRP3-deficient (n = 18) mice were injected intraperitoneally with E. coli (1 × 10⁸ CFU). Tissue sections were stained with hematoxylin and eosin staining (12 h and 24 h postinfection). Three independent experiments were performed. Scale label of the liver and kidney = 20 and 10 μm. Red arrow: hepatocellular necrosis; black arrow: inflammatory cell.

After 12 h of E. coli infection, C57BL/6J mice exhibited mild swelling and granular degeneration in renal tubular epithelial cells. In TLR2^−/− mice, renal tubular epithelial cells appeared faint, with granular degeneration, and some displayed evident vacuolar degeneration. NLRP3^−/− mice showed experiencing mild to severe granular degeneration, and some cells exhibited vacuolar degeneration. After 24 h of E. coli infection, C57BL/6J mice displayed mild pallor in renal tissues, with generally mild swelling, granular degeneration, and mild vacuolar degeneration in renal tubular epithelial cells. TLR2^−/− and NLRP3^−/− mice exhibited renal tissues with pallor, swelling of renal tubular epithelial cells, marked granular degeneration, and mild vacuolar changes. The above results indicate that TLR2 and NLRP3 protect liver and kidney tissue damage induced by E. coli infection in mice.

TLR2 and NLRP3 Deficiencies Increase the Expression of HMGB1 and HABP2 in E. coli-Infected Mice

HABP2 is implicated in various disease processes, particularly in inflammation, and may contribute to developing conditions characterized by heightened inflammatory states [29]. HMGB1, recognized as a biomarker indicative of tissue damage, plays a pivotal role in recruiting mononuclear cells to infection sites [30]. C57BL/6J, TLR2^−/−, and NLRP3^−/− mice were intraperitoneally injected with E. coli (1 × 10⁸ CFU) to investigate the regulatory roles of TLR2 and NLRP3 in the expression of HABP2 and HMGB1. Results demonstrated that during E. coli infection, the expression of HABP2 in the liver and kidney of TLR2^−/− and NLRP3^−/− mice was significantly higher than that in C57BL/6J mice at 12 h postinfection (p < 0.05) (Fig. 3a, c). Additionally, a similar trend was observed for the expression of HMGB1 in E. coli-infected mice, with higher levels in TLR2^−/− and NLRP3^−/− mice compared to C57BL/6J mice (p < 0.01) (Fig. 3b, d). These results further indicated that organ damage might be more serious in mice with TLR2 and NLRP3 deficiency.

Fig. 3.

TLR2 and NLRP3 deficiencies increase the expression of HMGB1 and HABP2 in E. coli-infected mice. C57BL/6J (n = 9), TLR2-deficient (n = 9), and NLRP3-deficient (n = 9) mice were intraperitoneally injected with E. coli (1 × 10⁸ CFU) or uninfected. The protein expression level of HABP2 and HMGB1 (green) in the livers and kidneys were determined with microscopy (12 h after E. coli infection, ×100 magnification, a–d). The arithmetic mean intensities of HABP2 and HMGB1 expression were analyzed by Zen software (Zeiss, a. u., arbitrary unit). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences between two experimental groups.

TLR2 and NLRP3 Are Involved in Proinflammatory Cytokine and Anti-Inflammatory Cytokine Production in E. coli-Infected Mice

Elevated levels of proinflammatory and anti-inflammatory cytokines in circulation contribute to heightened immune system activity [31]. In extreme cases, this heightened activity can lead to organ damage and, in severe instances, result in death [32]. In this study, in vivo experiments were conducted to further elucidate the roles of TLR2 and NLRP3 in the systemic inflammatory response of mice to bacterial infection. The inflammatory response induced by E. coli infection was characterized by analyzing cytokines and chemokines, serving as biomarkers. The production of TNF-α, IL-1β, and IL-10 in the serum and PLF of mice (C57BL/6J, TLR2^−/−, or NLRP3^−/−) was assessed following intraperitoneal injection of E. coli. Serum from E. coli-infected TLR2^−/− and NLRP3^−/− mice displayed heightened secretion of TNF-α and IL-1β compared to serum from C57BL/6J mice. Additionally, an increased secretion of IL-10 was observed in the serum of TLR2^−/− and NLRP3^−/− mice (p < 0.001) (Fig. 4a). Furthermore, TNF-α, IL-1β, and IL-10 production were detected in mice PLF. The results revealed elevated levels of TNF-α and IL-10 secretion in TLR2^−/− and NLRP3^−/− mice, whereas E. coli-infected TLR2^−/− and NLRP3^−/− mice exhibited impaired IL-1β secretion in PLF (p < 0.001) (Fig. 4b). The presented results underscore that, in addition to their crucial role in pathogen recognition, both TLR2 and NLRP3 actively participate in the intricate regulation of transitional inflammatory mediators. This dual functionality extends beyond mere recognition, encompassing a regulatory aspect that has the potential to alleviate cytokine storms and decelerate the progression of organ damage.

Fig. 4.

TLR2 and NLRP3 are involved in proinflammatory cytokine and anti-inflammatory cytokine production in E. coli-infected mice. C57BL/6J (n = 18), TLR2-deficient (n = 18), and NLRP3-deficient (n = 18) mice were intraperitoneally injected with E. coli (1 × 10⁸ CFU) or uninfected. a, b TNF-α, IL-1β, and IL-10 production in mouse serum and peritoneal lavage fluid (PLF) were analyzed by ELISA at 3 h and 6 h postinfection. Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to the respective control group. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences between two experimental groups.

TLR2 and NLRP3 Orchestrate Neutrophil Recruitment and Bacterial Load in E. coli-Infected Mice

The phagocytosis of bacteria by innate immune cells, such as neutrophils, is pivotal in eliminating invading pathogens [33]. C57BL/6J, TLR2^−/−, and NLRP3^−/− mice received intraperitoneal injections of E. coli (1 × 10⁸ CFU) to examine the regulatory influence of TLR2 and NLRP3 on neutrophil recruitment in mice. The results revealed a significant increase in neutrophil recruitment in C57BL/6J, TLR2^−/−, and NLRP3^−/− mice following E. coli infection compared to uninfected mice (Fig. 5a). Neutrophil recruitment was elevated in E. coli-infected TLR2^−/− and NLRP3^−/− mice compared to C57BL/6J mice with E. coli infection. Chemokines from the CXC family, specifically CXCL1 and CXCL2, play a crucial role in the early recruitment of neutrophils after injury or during inflammation [34]. In mice infected with E. coli and lacking TLR2 and NLRP3, elevated levels of CXCL1 and CXCL2 in the serum were observed, aligning seamlessly with the heightened trend of neutrophil recruitment (p < 0.001) (Fig. 5b). Interestingly, the bacterial load of E. coli in the bloodstream of TLR2^−/− and NLRP3^−/− mice exhibited a significant increase compared to the C57BL/6J mice group, with this trend consistently paralleling the observed pattern of neutrophil recruitment (p < 0.05) (Fig. 5c, d). These results suggest that TLR2 and NLRP3 are crucial in regulating the recognition and elimination of E. coli by immune cells, underscoring the essential involvement of CXCL1 and CXCL2 in orchestrating the recruitment of neutrophils during the inflammatory response induced by E. coli infection.

Fig. 5.

TLR2 and NLRP3 orchestrate neutrophil recruitment and bacterial load in E. coli-infected mice. C57BL/6J, TLR2-deficient and NLRP3-deficient mice were intraperitoneally injected with E. coli (1 × 10⁸ CFU) or uninfected. Neutrophil recruitment at 12 h after E. coli infection C57BL/6J (n = 9), TLR2-deficient (n = 9) and NLRP3-deficient (n = 9) mice was examined by blood. a The cells isolated from mice blood were stained with FITC-CD11b and PE-Gr-1 and subjected to sort purification by using a flow cytometry-based cell sorting system. b CXCL1 and CXCL2 production in C57BL/6J (n = 18), TLR2-deficient (n = 18) and NLRP3-deficient (n = 18) mice serum were analyzed by ELISA at 3 h and 6 h postinfection. c, d C57BL/6J (n = 9), TLR2-deficient (n = 9), and NLRP3-deficient (n = 9) mice were intraperitoneally injected with E. coli (1 × 10⁸ CFU) for 3 h and 6 h, collected blood, and cultured on eosin-methylene blue (EMB) agar plates to determine the bacterial load and colony counting in mice after E. coli infection. Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to the respective control group. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences between two experimental groups.

TLR2 and NLRP3 Participate in the Regulation of MAPK Signaling Pathway Activation and the Secretion of Inflammatory Mediators in Macrophages during E. coli Infection

In vitro experiments explored the potential activation of TLR2 and NLRP3 in macrophages following E. coli infection. The expression levels of TLR2 and NLRP3 in macrophages from C57BL/6J mice were assessed after infection with E. coli (MOI 5:1). The results indicated that E. coli infection significantly upregulated the expression of TLR2 and NLRP3 in macrophages compared to the control group (p < 0.01) (Fig. 6a). The above results suggest that E. coli could activate the TLR2 and NLRP3 in macrophages. Subsequently, we will explore the impact of E. coli infection on the activation of the MAPK signaling pathway and the secretion of inflammatory mediators in the absence of TLR2 and NLRP3. The result showed that only NLRP3-deficient macrophages have lower ratios of P-p38/P38 and of P-ERK/ERK, TLR2-deficient macrophages display only lower P-ERK/ERK ratios (p < 0.05) (Fig. 6b). After the deletion of TLR2 and NLRP3, macrophages exhibited significantly elevated levels of TNF-α secretion in comparison to the C57BL/6J group following E. coli infection (postinfection 3 h, 6 h, 9 h, 12 h and 24 h, p < 0.001) (Fig. 6c). Furthermore, following E. coli infection for 3 h, 6 h, and 9 h, TLR2^−/− and NLRP3^−/− macrophages showed impaired secretion of IL-1β compared to C57BL/6J macrophages. Interestingly, after E. coli infection for 12 h and 24 h, this phenomenon was reversed in TLR2^−/− mice (p < 0.01) (Fig. 6c). The levels of IL-10 secreted by TLR2^−/− and NLRP3^−/− macrophages were significantly higher than those of C57BL/6J macrophages at 6 h and 9 h after E. coli infection. Conversely, the levels of IL-10 secreted by C57BL/6J macrophages after 12 h and 24 h of E. coli infection were significantly higher than those of TLR2^−/− and NLRP3^−/− macrophages (p < 0.001) (Fig. 6c). These findings suggest that TLR2 and NLRP3 play crucial roles in the secretion of cytokines and the activation of the MAPK signaling pathway during the macrophage response to E. coli infection.

Fig. 6.

TLR2 and NLRP3 participate in the regulation of MAPK signaling pathway activation and the secretion of inflammatory mediators in macrophages during E. coli infection. C57BL/6J, TLR2^−/−, and NLRP3^−/− macrophages were infected with E. coli (1 × 10⁷ CFU, MOI 5:1) for 12 h. a Cells were stained with PE-conjugated anti-TLR2, or Alexa Fluor 488-conjugated anti-NLRP3 antibodies. Macrophages were infected with E. coli (1 × 10⁷ CFU, MOI 5:1) for the indicated durations (15 min, 30 min, or 60 min), or were not stimulated. b Activation of the MAPK (P-ERK and P-p38) pathways was evaluated by Western blotting; the expression of GAPDH was detected as a loading control. Grayscale values were measured using ImageJ software. c TNF-α, IL-1β, and IL-10 secretion in macrophages were analyzed by ELISA at 3 h, 6 h, 9 h, 12 h, and 24 h postinfection. Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to the respective control group. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences between two experimental groups.

Discussion

Sepsis, a life-threatening condition characterized by organ dysfunction resulting from dysregulated host responses to infection, stands as the primary cause of inhospital mortality worldwide [35]. According to the latest global estimates from the Global Burden of Disease Study 2017, sepsis-related deaths reached 11.0 million, accounting for 19.7% of total deaths that year [36, 37]. LPS, an endotoxin derived from the membranes of Gram-negative bacteria such as E. coli, plays a significant role in sepsis and septic shock [38]. Several studies have shown the critical role of PRRs and inflammasome signaling in the E. coli-induced cytokine storm observed during sepsis [39]. The initial immune response, activated by TLR, acts as the primary defense against invading pathogens, playing a crucial role in maintaining overall bodily homeostasis. However, the detailed mechanism of TLR2 and NLRP3 in regulating the host inflammatory response and sepsis in E. coli-infected mice remains unclear.

After infecting mice with pathogenic E. coli, we discovered a fascinating phenomenon regarding mouse mortality. Specifically, TLR2^−/−, NLRP3^−/− mice exhibited a significantly faster death rate than C57BL/6J mice (Fig. 1). Previous studies have shown that total inhibition of PRRs can be detrimental, as demonstrated by increased mortality in TLR2- and TLR4-deficient mice in response to Gram-negative or Gram-positive bacteria, respectively [40, 41]. Furthermore, the examination of tissue and organ pathology in mice revealed that the extent of liver and kidney pathological damage in TLR2 and NLRP3 deletion mice was significantly higher than that observed in C57BL/6J mice (Fig. 2). This finding was consistent with the mortality trend among the mice. HABP2 and HMGB1, as signals of tissue damage, are implicated in various disease processes, including renal injury, liver fibrosis, and inflammatory responses. In alignment with pathological damage, the expression levels of tissue damage-related proteins HABP2 and HMGB1 were significantly elevated following the deletion of TLR2 and NLRP3 (Fig. 3). Based on this, we deduce that during the process of E. coli infection, TLR2 and NLRP3 play a crucial role in regulating mouse mortality induced by transitional tissue damage. When infected with pathogenic microorganisms, the body secretes proinflammatory cytokines, and immune cells clear these microorganisms by inducing an inflammatory response [42]. However, a cytokine storm driven by an exaggerated inflammatory response in the initial phases of infection can potentially result in tissue damage and organ dysfunction [43]. Our research findings indicate that, following E. coli infection, there is a significant elevation in the expression levels of inflammatory cytokines in the bodies of C57BL/6J mice compared to the uninfected control group. However, in mice lacking TLR2 and NLRP3, the levels of inflammatory cytokines are markedly higher than those in C57BL/6J mice (Fig. 4). Noteworthy, TNF-α and IL-1β serum levels differ considerably between TLR2-deficient and NLRP3-deficient mice; nevertheless, their organ damage score differs only slightly. Previous studies showed that the inflammatory factors are generated, cytotoxic substances are released, enzymes and immune cells are implicated in tissue injury [44]. So inflammatory cytokines are only one of the regulate factors in organ damage. We posit that the heightened levels of inflammatory mediators in TLR2- and NLRP3-deficient mice are aberrant and detrimental to the organism rather than beneficial. Furthermore, we note that the production of IL-10 also is high in E. coli-infected TLR2- and NLRP3-deficient mice (Fig. 4). IL-10 plays essential roles in maintaining tissue homeostasis during infection and inflammation by restricting excessive inflammatory responses [45]. However, some study showed that high levels of IL-10 in patients have been correlated with poor prognosis, such as septic shock cancer [46–48]. Based on our study, the production of proinflammatory cytokines in serum and PLF of mice showed an upward trend and did not decrease due to the increase of IL-10. Therefore, we believe that the high level of IL-10 in this study indicates poor prognosis and more serious tissue damage. Of note, we observed a significant downregulation in the expression levels of IL-1β in PLF from TLR2- and NLRP3-deficient mice. NLRP3 activation follows a two-step process, where engaged TLRs or cytokine receptors deliver an initial signal, inducing transcriptional upregulation of NLRP3 inflammasome components. Subsequently, a “second signal” is provided by intracellular stress pathways, leading to the formation of the NLRP3-ASC complex, activation of caspase-1, and the release of active IL-1β. Therefore, during E. coli infection, TLR2 may provide an activation signal for NLRP3, thereby enabling IL-1β release. Interestingly, the overshooting IL-1β levels in TLR2^−/− and NLRP3^−/− mice are remarkable in the serum and cells at some time (Fig. 4b and Fig. 6c). The previous study showed that other inflammasomes, such as NLRC4 or AIM2, could compensate for the loss of NLRP3, leading to increased IL-1β production [49, 50]. Additionally, gasdermin D also mediates IL-1β and IL-18 secretion and can occur dependently or independently of NLRP3 signaling [51]. Furthermore, in its absence, a dysregulated or hyperactive immune response might lead to increased production of proinflammatory cytokines, including IL-1β [52, 53]. TLR2-deficient mice may have an altered ability to control the E. coli infection, resulting in a higher bacterial load or prolonged infection. This could lead to sustained or elevated production of IL-1β as the immune system attempts to combat the infection [54–56].

In response to inflammation or microbial invasion, neutrophils undergo swift activation, stimulating the hematopoietic system to release numerous neutrophils into the bloodstream to engage in phagocytic and bactericidal functions [57]. This study demonstrates a notable elevation in neutrophil count in the bloodstream of mice post-E. coli infection, with TLR2- and NLRP3-deficient mice showing significantly higher levels compared to C57BL/6J mice (Fig. 5a). Chemokines such as CXCL1, CXCL2, and CXCL8 play a pivotal role as crucial chemoattractant, guiding neutrophils to the sites of inflammation [58]. Consistent with the neutrophil count in the bloodstream, the deficiency of TLR2 and NLRP3 results in a significant elevation in the expression levels of CXCL1 and CXCL2 in the serum of mice (Fig. 5b). Research indicates that the TLR2 ligand, lipoteichoic acid, could downregulate the expression of neutrophil CXCR2 in septic mice, reducing the chemotaxis of neutrophils and inhibiting their migratory functions [59]. Furthermore, following stimulation with the TLR2 ligand (Pam3CSK4), although the surface expression of both CXCR1 and CXCR2 decreases, the remaining chemokine receptors can still maintain their functionality, ensuring that neutrophils’ migration and aggregation capacity in response to chemokines like CXCL8 remains unaffected [60]. These findings suggest that TLR2 and NLRP3 play a regulatory role in the migration and recruitment of neutrophils in sepsis induced by E. coli infection. Additionally, we further examined the bacterial load in the blood of mice, and the results indicated a significant increase in E. coli load in the bloodstream following TLR2 and NLRP3 deficiency (Fig. 4c, d). An interesting thing that TLR2^−/− and NLRP3^−/− mice have higher bacterial numbers in their blood compared to wild-type mice. However, gene-deficient mice appear to recruit also higher numbers of neutrophils. The effectiveness of neutrophils is not solely dependent on their numbers but also on their ability to phagocytose and kill bacteria. TLR2-deficient neutrophils might have impaired phagocytosis because TLR2 plays a role in recognizing bacterial components and facilitating the phagocytic process [61, 62]. Furthermore, research indicates that in polymicrobial sepsis induced by cecal ligation and puncture, the deficiency of NLRP3 significantly inhibits the recruitment of peritoneal neutrophils, cytokine secretion, and weakens the ability to clear bacteria [63]. The phagocytic activity of innate immune cells, particularly neutrophils, is essential in eliminating invading bacteria, and any functional impairment in the phagocytic immune system renders the host more susceptible to bacterial infections [33]. In a word, if the phagocytic activity or killing efficiency of neutrophils was impaired in TLR2−/− and NLRP3−/− mice, the body tends to recruit more neutrophils to compensate the impaired phagocytic activity or killing efficiency of signal neutrophil. The observations above indicate that, during E. coli infection, the deficiency of TLR2 and NLRP3, while increasing the recruitment of neutrophils, diminishes the host’s bactericidal activity, thereby elevating bacterial burden. This could be a contributing factor to the increased mortality rate in mice.

In immune cells, TLR2- and NLRP3-mediated MAPK signaling activation can induce the secretion of inflammatory cytokines. As anticipated, the results reveal that E. coli infection can also induce the activation of TLR2 and NLRP3 in macrophages (Fig. 6a). Additionally, impairment in the activation of MAPK (ERK and p38) signaling is observed in macrophages from and NLRP3-deficient mice (Fig. 6b). However, the expression of inflammatory mediators excites us because the expression trends of different inflammatory mediators vary at different time points during E. coli infection. Specifically, the secretion of IL-1β and IL-10 reversed 9 h after E. coli infection. The secretion of TNF-α was at a high level, while the ratio of P-p38/P38 and/or P-ERK/ERK is lower in infected NLRP3^−/− and TLR2^−/− mice, respectively. We think that TLR2 or NLRP3 signaling is impaired, and other pathways may compensate to maintain an immune response. This can result in elevated TNF-α production despite reduced MAPK pathway activation (lower P-p38/P38 and P-ERK/ERK ratios). Furthermore, the signaling pathways involved in immune responses are highly interconnected. The downregulation of one pathway (e.g., MAPK) can lead to the upregulation of other pathways or molecules (e.g., TNF-α) as a compensatory response to ensure the host can still mount an effective defense against the infection. The intricate mechanisms involved in this process require further investigation.

In summary, based on our current research findings, we infer that during E. coli infection, TLR2 and NLRP3 may maintain a balanced expression of inflammatory mediators through the MAPK signaling pathway, thereby preventing excessive tissue damage and regulating mouse mortality. Additionally, TLR2 and NLRP3 play a role in modulating immune cells’ recruitment and bactericidal activity (Fig. 7).

Fig. 7.

TLR2 and NLRP3 play regulatory roles in E. coli infection-induced inflammatory response and sepsis. During E. coli infection, the TLR2 and NLRP3 maintain a balanced expression of inflammatory mediators through the MAPK signaling pathway, preventing excessive tissue damage and regulating mouse mortality. Furthermore, TLR2 and NLRP3 also influence the recruitment of immune cells and enhance bactericidal activity. The red arrows indicate upregulation, and the blue arrows indicate regulatory effect.

Statement of Ethics

All animal experiments were conducted following the regulations of the Chinese Experimental Animal Management Committee. The experimental protocol was approved by the Animal Welfare and Research Ethics Committee of Inner Mongolia Agricultural University (Approval ID. NND2021013).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding Sources

This work was supported by the Basic Scientific Research Fund Project of Universities Directly under the Inner Mongolia Autonomous Region (BR220125) and the National Natural Science Foundation of China (Grant No. 32260903, 32060815).

Author Contributions

Zhiguo Gong: conceived and designed research, performed experiments, analyzed data, interpreted results of experiments, prepared figures, and drafted manuscript. Wei Mao, Jiamin Zhao, and Peipei Ren: performed experiments and analyzed data. Zhuoya Yu: analyzed data. Yunjie Bai, Chao Wang, and Yuze Liu: performed experiments. Shuang Feng and Surong Hasi: conceived and designed research, drafted manuscript, edited and revised the manuscript, and approved the final version of the manuscript.

Funding Statement

This work was supported by the Basic Scientific Research Fund Project of Universities Directly under the Inner Mongolia Autonomous Region (BR220125) and the National Natural Science Foundation of China (Grant No. 32260903, 32060815).

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.