Lipopolysaccharide-induced bacterial infection model: microRNA-370-3p participates in the anti-infection response by targeting the macrophage TLR4-NLRP3 caspase-1 cellular pyroptosis pathway

Abstract

Objective

To explore the effect of miR-370-3p on LPS triggering, in particular its involvement in disease progression by targeting the TLR4-NLRP3-caspase-1 cellular pyroptosis pathway in macrophages.

Methods

Human macrophage RAW264.7 was divided into 6 groups: control, LPS, LPS + inhibitor-NC, LPS + miR-370-3p inhibitor, LPS + mimics-NC and LPS + miR-370-3p mimics. RT-qPCR was used to detect the expression level of miR-370-3p and analyzed comparatively. CCK-8 and flow cytometry assays were used to detect cell viability and apoptosis. ELISA assay was used to detect the levels of IL-1β and TNF-α in the supernatant of the cells. The WB assay was used to detect TLR4, NLRP3, Caspase-1 and GSDMD levels.

Results

After LPS induction, macrophage miR-370-3p levels decreased, cell viability decreased, and apoptosis increased. At the same time, the levels of TLR4, NLRP3, Caspase-1 and GSDMD increased in the cells, and the levels of IL-1β and TNF-α increased in the cell supernatant. Compared with the LPS group, the significantly higher expression level of miR-370-3p in the cells of the LPS + miR-370-3p mimics group was accompanied by significantly higher cell viability, significantly lower apoptosis rate, significantly lower levels of TLR4, NLRP3, Caspase-1, and GSDMD in the cells, and significantly lower levels of IL-1β and TNF-α in the cell supernatant.

Conclusion

MiR-370-3p may be involved in anti-infective immune responses by targeting and inhibiting the macrophage TLR4-NLRP3-caspase-1 cellular pyroptosis pathway.

Introduction

Bacterial infections are complex biological struggles between hosts and pathogens. ¹ Lipopolysaccharide (LPS), as a major component of the bacterial outer membrane, has a potent immunostimulatory capacity, which initiates and regulates the host’s anti-infection immune response by interacting with host immune cells - especially macrophages. ² In particular, LPS activates phagocytosis and bactericidal functions of macrophages and induces pro-inflammatory responses by binding to Toll-like receptor 4 (TLR4) on the macrophage surface, initiating a series of signaling pathways. ³ These responses not only directly combat invading pathogens, but also coordinate and enhance the defenses of the entire immune system by releasing inflammatory mediators and cytokines. ⁴ However, bacteria that cause long-term infections in humans, such as Mycobacterium tuberculosis (MTB), are able to survive and multiply in the host for long periods of time, leading to a state of chronic infection. ⁵ Macrophages play an equally important role during chronic infection by recognizing the causative bacterium and attempting to destroy it. ⁶ Such pathogenic bacteria have complex escape mechanisms that allow them to survive in the host for long periods of time and trigger a chronic inflammatory response, the exact mechanisms of which are not yet understood.

MicroRNA (miRNA) is a single-stranded RNA of about 22 nucleotides in length, which can negatively regulate gene expression after transcription. ⁷ Recently, miRNAs have been found to play an important role in the development of infectious diseases. ⁸ The study of anti-infective immune mechanisms and the search for potential biomarkers from the perspective of miRNAs are the current hot directions in the study of bacterial infectious diseases. ⁹ miR- 370-3p is one of the newly discovered miRNAs associated with immune responses to bacterial infections, ¹⁰ but the mechanism of miR-370-3p’s role in anti-infection immunity has not yet been thoroughly investigated.

In the present study, we used LPS stimulation to mimic the effect of bacterial infection on miR-370-3p expression and found that LPS similarly mediated the downregulation of miR-370-3p expression in macrophages. To explore the underlying molecular mechanisms, the targeting relationship between miR-370-3p and TLR4 was discovered through TargetScan website prediction. In addition, it has been demonstrated that TLR4 is a target gene of miR-370-3p, suggesting that miR-370-3p may exert its biological function by targeting TLR4. ¹¹ TLR4 is the earliest TLR discovered to mediate the body’s response to pathogens, and it has been demonstrated that TLR4 is able to recognize bacterial antigens to activate inflammatory signaling pathways. ¹² TLR4 has been shown to activate the NLRP3 inflammasome pathway. ¹³ Activation of inflammasome can mediate the onset of cellular pyroptosis. ¹⁴ Thus, it has been suggested that bacteria activate NLRP3 inflammasome-mediated cellular sepsis by being recognized by macrophage TLR4.

In this study, LPS stimulation was utilized to mimic the low expression of miR-370-3p mediated by bacterial infection. Subsequently, the effects of miR-370-3p on RAW264.7 cell viability, IL-1β and TNF-α secretion levels, TLR4-NLRP3-caspase-1 pathway and cellular pyrolysis were verified by interfering with and overexpressing miR-370-3p. The aim was to provide a reference for the mechanism of miR-370-3p in bacterial infection.

Materials and methods

Prediction of gene interactions by bioinformatics

The STRING11.0 online database was used to explore the protein-protein interaction (PPI) network between TLR4, NLRP3, caspase-1, IL-1β, TNF-α and GSDMD. The TargetScan website was used to predict whether there is a targeting relationship between miR-370-3p and TLR4.

Experimental grouping and treatment

Control group was normal cultured macrophages (RAW264.7). Model group was 100 ng/mL LPS treated RAW264.7 cells for 24 h. Inhibitor-NC group was pre-addition of LPS, 1 h later transfected inhibitor-NC. Inhibitor group was pre-addition of LPS, 1h later transfected miR-370-3p inhibitor. Mimics-NC group was pre-addition of LPS, 1 h later transfected mimics-NC. Mimics group was pre-addition of LPS, 1 h later transfected miR-370-3p mimic.

Cell culture

The macrophage cell line (RAW264.7) was cultured in DMEM medium containing 10% fetal bovine serum (Gibco, USA) in an incubator at 37°C with 5% CO₂ concentration.

RT-qPCR

Total cellular RNA was extracted by the conventional Trizol method and reverse transcribed into cDNA using the reverse transcription kit SYBR® FAST Universal qPCR Kit [KAPA Biosystems]. RT-qPCR was used to detect the level of miR-370-3p. MiR-370-3p primer sequences were: forward primer 5′-GGC​CTG​CTG​GGG​GTG​GAA-3′, reverse primer 5′- AGT​GCA​GGG​TCC​GAG​GTA​TT-3′. U6 primer sequences were: Forward primer 5′- CTC​GCT​TCG​GCA​GGT​ATT-3′, reverse primer 5′- CTC​GCT​TCG​GGC​AGG​TAT​T-3′.

CCK8 assay

Take 100 μL of cell suspension and add it to the 96-well plate, after incubation for 48 h, add a volume of 10 μL of CCK-8 solution to each well and incubate for 4 h. Detect the absorbance (A450) value of each well with an enzyme meter.

Apoptosis assay

Collect the cells of each group, take 1 × 10⁶ cells re-suspended in culture medium, 300 g, centrifuge at 4°C for 5 min, and discard the supernatant. Add 1 mL of pre-cooled PBS, gently blow to mix the cells, 300 g, centrifuge at 4°C for 5 min, discard the supernatant. Cells were re-suspended in 500 μL of binding buffer followed by the addition of 5 μL of AnnexinV-APC staining solution and 5 μL of 7-AAD staining solution (100 μg/mL) and incubated for 20 min away from light. Flow-through assay was performed immediately. Analysis was performed using NovoExpress analysis software.

ELISA experiment

40 μL of sample was added to the ELISA plate, followed by 10 μL of biotin-labeled antibody. 50 μL of ELISA reagent was added to each well, then the plate was sealed with a plate sealing film and incubated for 30 min at 37°C in an incubator. Each well was incubated at 37°C for 10 min after addition of color developer, followed by addition of 50 μL of termination solution. The enzyme marker was set at 450 nm, and the contents of IL-1β and TNF-α in each group of samples were calculated according to the standard curves obtained in turn.

Western blot

Cells were removed from the incubator and lysed by adding 200 μL of lysate per 1*106 cells. After boiling the samples in a metal bath for 10 min to denature the proteins, the samples were centrifuged and the supernatant was taken. After electrophoresis, membrane transfer and sealing, the primary antibody was diluted according to the instructions and incubated for 1 h. The HRP-labeled secondary antibody was incubated for 1 h. The ECL luminescent solution was taken and added on the front side of the membrane, and then detected by the fully automated chemiluminescence analyzer.

Results

Analysis of miR-370-3p, TLR4, NLRP3, caspase-1, GSDMD, IL-1β, TNF-α interaction networks

The study route is shown in Figure 1. PPI networks for TLR4, NLRP3, caspase-1, GSDMD, IL-1β and TNF-α were constructed from the STRING database (Figure 2A). Analysis of the TargetScan web page showed that miR-370-3p was able to target bind TLR4 (Figure 2B).

Figure 1.

Research design and process of this study.

Figure 2.

Interaction network between miR-370-3p, TLR4, NLRP3, caspase-1, GSDMD, IL-1β, and TNF-α. (A) Interaction network between TLR4, NLRP3, caspase-1, GSDMD, IL-1β, and TNF-α. (B) The binding site of miR-370-3p and the 3′ untranslated region of TLR4 mRNA.

Infection model construction and effect of miR-370-3p on cell proliferation

The level of miR-370-3p was significantly decreased after LPS induction (p < .05). The level of miR-370-3p was significantly decreased after transfection with miR-370-3p inhibitor (p < .05). The level of miR-370-3p was significantly increased transfection with miR-370-3p mimics (p < .05) (Figure 3A). Cell viability was significantly reduced after LPS induction (p < .05). Transfection of miR-370-3p inhibitor significantly decreased cell viability (p < .05). After transfection of miR-370-3p mimics, cell viability was significantly increased (p < .05) (Figure 3B).

Figure 3.

Infection model construction effect and macrophage cell viability detection. (A) Verification of infection model construction and transfection effect. (B) Effect of miR-370-3p on cell viability in a bacterial infection model. *Indicates statistical significance compared with the control group. #indicates statistically significant compared with the Model group. Δindicates statistical significance compared with the Model group.

Effect of miR-370-3p on apoptosis in a bacterial infection model

The apoptosis rate was significantly higher after LPS induction compared with the control group (p < .05). Transfection of miR-370-3p inhibitor significantly increased the apoptosis rate (p < .05). Transfection of miR-370-3p mimics significantly decreased the apoptosis rate (p < .05) (Figure 4).

Figure 4.

Detection of apoptosis in a bacterial infection model by flow cytometry. (A) Apoptosis rate in the bacterial infection model and each treatment group. (B) Statistical analysis of flow cytometry results. *Indicates statistical significance compared with the control group. #indicates statistically significant compared with the Model group. Δindicates statistical significance compared with the Model group.

Effect of miR-370-3p on inflammatory factor production in a bacterial infection model

After LPS treatment, the levels of IL-1β and TNF-α in cell supernatants were significantly increased (p < .05). The levels of IL-1β and TNF-α in cell supernatants were further increased by transfection with miR-370-3p inhibitor (p < .05). After transfection of miR-370-3p mimics, the levels of IL-1β and TNF-α in the cell supernatant were significantly reduced (p < .05) (Figure 5).

Figure 5.

The production levels of inflammatory factors in the bacterial infection model were detected by ELISA. (A) Levels of IL-1β production in cell supernatants of each treatment group. (B) Levels of TNF-α production in cell supernatants of each treatment group. (n = 3, each repeated three times). *Statistically significant compared to the control group. # Statistically significant compared to the Model group. △ Statistically significant compared to the Model group.

Effect of miR-370-3p on pathway proteins in a bacterial infection model

LPS treatment significantly elevated TLR4, NLRP3, Caspase-1 and GSDMD levels (p < .05). TLR4, NLRP3, Caspase-1 and GSDMD levels were further elevated after transfection with miR-370-3p inhibitor (p < .05). The levels of TLR4, NLRP3, Caspase-1 and GSDMD were significantly reduced after transfection of miR-370-3p mimics (p < .05) (Figure 6).

Figure 6.

WB assay for detection of pathway proteins in a bacterial infection model. (A) Changes in pathway proteins in the bacterial infection model and in each treatment group. (B) Statistical analysis of the differences in TLR4 expression in each treatment group. (C) Statistical analysis of the differences in NLRP3 expression in each treatment group. (D) Statistical analysis of the differences in caspase-1 expression in each treatment group. (E) Statistical analysis of the differences in GSDMD expression in each treatment group. (n = 3, each repeated three times). *Statistically significant compared with the control group. #Statistically significant compared with the Model group. △Statistically significant compared with the Model group.

Discussion

Bacteria are one of the major threats to human health, and the host immune system plays a key role in defense against bacterial attack. ¹⁵ Therefore, an in-depth understanding of the interactions between bacterial infections and host immune responses is important for the prevention and treatment of diseases caused by infectious pathogens. The host immune system recognizes pathogenic microbe-associated molecular patterns (PAMPs), a critical initial step in the process of bacterial infection, through a variety of pattern-recognition receptors (PRRs), which include Toll-like receptors (TLRs) and NOD-like receptors (NLRs), among others. ¹⁶ These receptors recognize bacterial components such as lipopolysaccharides (LPS), peptidoglycans, lipoproteins, and nucleic acids. ¹⁷ miRs have been a hot topic of research in recent years, which recognize and bind mRNAs by base-pairing and thus inhibit mRNA translation. ¹⁸ miR-370-3p is one of the newly identified miRNAs associated with immune response to bacterial infections. ¹⁰ In this study, it was found that miR-370-3p has a targeting effect on TLR4 as Toll like receptors (TLRs). In an LPS-induced bacterial infection model, adjusting the expression level of miR-370-3p significantly affected the macrophage response capacity, suggesting that miR-370-3p has a potential function in regulating host immune responses.

To investigate the mechanism of miR-370-3p in bacterial infection, this study simulated bacterial infection by LPS stimulation. The level of miR-370-3p in macrophages was reduced after LPS induction, a result similar to the expression pattern of miR-370-3p after tuberculosis infection. ¹⁹ The down-regulation of miR-370-3p was accompanied by a decrease in macrophage viability, an increase in the level of apoptosis, and an increase in the secretion of inflammatory factors. The mechanism of action of miR-370-3p was subsequently investigated on the basis of an infection model. After LPS-induced reduction in miR-370-3p levels, macrophage viability was elevated and macrophage apoptosis was reduced after upregulation of miR-370-3p expression levels by miR-370-3p mimic. On the contrary, after down-regulation of miR-370-3p expression level by miR-370-3p inhibitor, macrophage viability was decreased and macrophage apoptosis level was increased. The above results suggest that bacterial infection-mediated down-regulation of miR-370-3p may have an effect on macrophage phenotype.

To investigate the specific mechanism by which miR-370-3p functions, we identified TLR4 as a miR-370-3p target gene. TLR4 protein is an infectious immune receptor protein that recognizes a variety of pathogens, promotes macrophage activation, and initiates the body’s immune response. ²⁰ It has been demonstrated that TLR4 can activate the NLRP3 inflammasome pathway. ¹³ NLRP3 is an immunomodulatory protein that forms inflammasome when NLRP3 is activated in macrophages. ²¹ Activated NLRP3 inflammasomes are capable of triggering the activation of cysteine protease-1 (caspase-1), which in turn leads to the precursor forms of IL-1β and TNF-α being sheared into active forms and released outside the cell. ²² The results of the present study confirmed that the decrease in miR-370-3p levels mediated the increase in TLR4 protein levels, suggesting that miR-370-3p has a regulatory role in the expression of TLR4. With the elevation of TLR4 protein levels, the protein levels of NLRP3, caspase-1, and GSDMD, which are key molecules of its downstream inflammasome pathway, were simultaneously increased, and the secretion of IL-1β and TNF-α was elevated. Conversely, upregulation of miR-370-3p levels inhibited TLR4, with reduced protein levels of its downstream key molecules of the inflammasome pathway, NLRP3, caspase-1, and GSDMD, and decreased secretion of IL-1β and TNF-α. Activation of inflammasome can mediate the onset of cellular pyroptosis. ²³ Activation of the inflammasome pathway in the study was accompanied by elevated cell viability and cell death. Thus, it is suggested that bacterial infection-mediated down-regulation of miR-370-3p promotes cellular pyroptosis through the TLR4/NLRP3/caspase-1 pathway. Interestingly, serum levels of miR-370-3p were reduced in tuberculosis patients. ²⁴ The activation of NLRP3 inflammatory vesicles in macrophages after MTB recognition by TLR4 has increased the secretion of inflammatory factors, suggesting that NLRP3-mediated cellular pyroptosis may be closely related to tuberculosis progression. ¹³ Therefore, the hypothesis was proposed that MTB infection-mediated down-regulation of miR-370-3p may promote cellular colocalization through the TLR4/NLRP3/caspase-1 pathway. However, this hypothesis is speculative based on the current study, and further construction of MTB infection models is needed to verify it.

Conclusion

The present study used a simplified model to simulate bacterial infection and interpreted that miR-370-3p promotes cellular pyroptosis as well as secretion of L-1β and TNF-α by regulating the TLR4-NLRP3-caspase-1 pathway, which leads to activation of the inflammatory response. The results of the present study provide valuable insights in understanding the role of miR-370-3p in bacterial infections, as well as a theoretical basis for the possibility that miR-370-3p may be a new target for antipathogenic bacterial therapy.

Appendix.

Abbreviations

miRNAs

MicroRNAs

3′-UTR

3′-untranslated region

PPI

protein protein interaction

TLR4

Toll-like receptor 4

caspase-1

cysteine protease-1

MTB

Mycobacterium tuberculosis.

Ethical statement

Ethical approval

The experiment was approved by the Ethics Committee of the First People’s Hospital of Kashi.

ORCID iD

Abdusemer Reyimu https://orcid.org/0000-0002-9323-2390