Introduction

Tuberculosis (TB) is a common chronic infectious disease of the respiratory tract, caused by Mycobacterium tuberculosis (M.tb) infection, causing great harm to human health. According to the Global TB report 2024 released by the World Health Organization, TB is currently the leading cause of death globally from a single infectious disease1. So far, the pathogenesis of TB, especially the immune mechanism of the body to TB infection, has not been fully elucidated. Neutrophils are the most abundant innate immune effector cells in the human immune system. They have powerful phagocytic and bactericidal functions and play an important role in the body’s immune response. As the first line of defense against pathogens, neutrophils engulf pathogens, produce sterilization medium, proinflammatory factors, and form neutrophil extracellular traps (NETs), to remove the pathogen, and are involved in inflammation and immune response2,3,4. Many studies have confirmed that neutrophils are involved in the pathogenesis of TB infection, but the role is controversial. Some scholars believe that neutrophils act as a key factor in mediating immunity against TB infection. After M.tb invade the body, neutrophils rapidly gather to the infection site, surround the invading bacteria in the local area, phagocytize and kill the bacteria, and then undergo apoptosis or necrosis. They are then recognized and removed by macrophages to prevent the spread of pathogenic microorganisms in the body resulting in control of the acute M.tb infection, reduction in occurrence of inflammation, and damage to the surrounding tissues5,6,7,8,9. It has also been suggested that neutrophils can have opposite role in late stage of TB host defense, where they were found to be more pathogenic, and the increased number of neutrophils was associated with lung injury and more severe disease10,11. Therefore, the role of neutrophils in M.tb infection needs to be further studied and explored.

Reactive oxygen species (ROS) produced by neutrophils is one of the important defensive substances in the process of anti-tuberculosis infection8,12which mainly depends on NADPH oxidase (NOX). NADPH oxidase complex (NOX2) is activated after neutrophils are stimulated. The production of ROS is released into the phagosome and extracellular, and plays a key role in phagocytosis, killing microorganisms or mediating inflammatory response13,14,15,16,17,18. Lipopolysaccharide (LPS) is a natural adjuvant synthesized from the outer membrane of Gram-negative bacteria. It is an important molecule that stimulates the body’s non-specific immune function and can cause a variety of inflammatory reactions. After LPS stimulation, neutrophils have increased levels of intracellular inflammatory factors, enhanced degranulation ability, and overexpression of chemokine receptors on their surface. These series of changes prepare neutrophils for reaching the lesion and timely elimination of pathogenic bacteria19,20. Whether LPS can activate neutrophils to regulate the production of ROS to promote the killing activity of cells against M.tb in the process of infection, and the specific mechanism involved in the regulation of neutrophil inflammatory response are still unclear.

In this study, we used neutrophils infected with M.tb to detect the killing function of neutrophils against M.tb, to explore the effect of neutrophils activated by LPS on the production of ROS, and to further explore the involved signaling pathways, so as to provide experimental basis for clarifying the role of neutrophils in anti-tuberculosis infection and theoretical basis for clinical treatment of TB.

Results

The killing rate of neutrophils to M.tb was detected by flow cytometry

M.tb and Fluorescein diacetate (FDA) were incubated together for 30 min to test the positive rate of FDA in bacteria, and the results showed that the survival rate was above 95% (Fig. 1A). To observe the killing effect of neutrophils on M.tb, the PMN was incubated with FDA-labeled M.tb for 20 min at 37 °C and then placed in a 37 °C water bath for 0 to 60 min. The bacterial survival rate was detected by flow cytometry. The results showed that the survival rate of M.tb gradually decreased with the increase of time (p < 0.05), indicating that the killing function of PMN to M.tb gradually increased with the increase of time (Fig. 1B and C).

Fig. 1
figure 1

Flow cytometric analysis of the killing function of neutrophils against M.tb. (A) Flow cytometry analysis of FDA-labeled M.tb. (B) Flow cytometric analysis of the killing function of neutrophils against M.tb for 0 to 60 min. (C) Box-plot showed the killing rate of neutrophils against M.tb for 0 to 60 min.

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The killing of M.tb by PMN was observed by fluorescence microscopy

Fluorescence microscopy showed that the M.tb was green rod-shaped after FDA staining, and the neutrophils were red round with intact cell membrane after CD16-PE staining. From 30 min to 60 min, the green rod-shaped bacteria in the red cells were gradually reduced, indicating that the survival rate of M.tb was gradually reduced, as, the killing rate of neutrophils to M.tb increased, the survival rate of M.tb gradually reduced (Fig. 2). .

Fig. 2
figure 2

Fluorescence microscopy analysis of the neutrophil killing function against M.tb.

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Temporal kinetics of M.tb killing by PMN by ROS production

Neutrophils were infected with M.tb and treated with DCFH-DA. The activation rate of PMN and the production of reactive oxygen species (ROS) were detected by flow cytometry. After incubation of PMN with M.tb in 37 °C water bath for 0 to 30 min, the activation rate of neutrophils increased with time (p < 0.05), and the amount of ROS produced by activated neutrophils also increased with time (p < 0.05) (Fig. 3).

Fig. 3
figure 3

Flow cytometric analysis of the activation rate of neutrophils and the amount of ROS. (A) Flow cytometric analysis of the activation rate of neutrophils for 0 to 60 min. (B) Box-plot showed the activation rate of neutrophils for 0 to 60 min. (C) Box-plot showed the amount of ROS produced by activated neutrophils for 0 to 60 min.

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LPS affects the signaling pathway of neutrophils producing ROS to kill M.tb

After human neutrophils were pretreated with different concentrations of LPS, its activation rate and ROS production at 5 min was higher than those of the control group, and the activation rate and ROS production increased with the increase of LPS concentration (P < 0.05) (Fig. 4A and B). When neutrophils were pretreated with TLR4 monoclonal antibody or NADPH oxidase inhibitor DPI, and then treated with LPS, the activation rate and ROS production of neutrophils were significantly lower than those of the control group (P < 0.05) (Fig. 4C, D and E, and 4F).

Fig. 4
figure 4

Analysis of the signaling pathway of LPS affecting the production of ROS by neutrophils to kill M.tb. (A) Effect of LPS on the activation rate of PMN after M.tb infection. (B) Effect of LPS on ROS production after PMN infection with M.tb. (C) Effect of LPS on the activation rate of PMN after M.tb infection on pretreatment with a TLR4 mAb (D) Effect of LPS on ROS production after M.tb infection on pretreatment with a TLR4 mAb. (E) Effect of LPS on ROS production after M.tb infection on pretreatment with DPI. (F) Effect of LPS on ROS production after M.tb infection on pretreatment with DPI.

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Discussion

Neutrophils are the most abundant innate immune cells in humans and are an important part of the first line of defense against pathogenic microbial invasion21. In the resistance to external microbial infection, mature neutrophils respond rapidly and become activated by phagocytosis, chemotaxis, and production of reactive oxygen species. Previous studies have confirmed that neutrophils can kill M.tb. When M.tb invades into the body, neutrophils first infiltrates the TB infection site and plays a protective role against M.tb through the host defense mechanism in the early stage22,23,24. Neutrophil’s phagocytosis and killing is the first barrier against M.tb. The traditional method to detect the killing activity of neutrophils is to take peripheral blood and mix with bacteria, mixed culture, and observe the results under microscope. It takes about 2 to 3 weeks, which is complicated, time-consuming, and affected by many factors. The study established a new method to detect the drug susceptibility of M.tb, which used fluorescein diacetate (FDA) staining to detect the activity of anti-M.tb drugs by flow cytometry. The method was easy to operate, accurate, reproducible and especially rapid, and the results could be obtained within 24 h25. FDA is a non-fluorescent esterase substrate. After being taken up by living cells, FDA is hydrolyzed by intracellular esterases to a charged fluorescein, which can be retained in cells as a strong green fluorescence. However, in apoptotic cells and necrotic cells, fluorescein decreased or was absent. The study used flow cytometry to detect the killing function of neutrophils against BCECF-AM labeled Candida albicans in the whole blood of normal people and thalassemia patients, which was quick and simple and only required a small amount of blood samples26. M.tb is able to survive within host cells. In order to elucidate the role of neutrophils in anti-tuberculosis immunity, we established a method for detecting the cytolytic activity of M.tb by combining the advantages of the two methods.In this study, cultured M.tb was stained with FDA to detect the survival rate of M.tb, and M.tb with a survival rate of more than 95% was treated with human peripheral blood neutrophils for 20 min, and the killing function was detected at different times. The results showed a bactericidal function that the killing rate of neutrophils to M.tb gradually increased with time. This method is simple, convenient, accurate and with a small sample size. It also avoids cell activation that may complicate the assay and lead to the changes in the bactericidal function. The cytolytic activity of neutrophils against M.tb was further verified by fluorescence microscopy.

Neutrophil activation is accompanied by oxidative burst, exocytosis (degranulation) of various granule types, and a delay in spontaneous apoptosis. After engulfing invading M.tb, neutrophils kill bacteria by directly secreting bactericidal substances and releasing ROS. These bactericidal substances include cathepsin G, elastase, defensins, and antimicrobial peptides. By engulfing the NADPH oxidase complex on the lysosomal membrane, neutrophils produce ROS such as superoxide and hydrogen peroxide. Phagocytosis of bacterial complement of M.tb or antibody by neutrophils triggers phagocytosis induced-cell death (PICD), leading to their rapid apoptosis, recognition and engulfment by macrophages27. Since the study reported in 1983 that a fluorescent substance DCFH-DA was utilized to detect the production of reactive oxygen species by flow cytometry, this method has been widely used as an index to evaluate the oxidative status and bactericidal function of neutrophils28. In this study, DCFH-DA was used to detect the oxidation status of neutrophils infected with M.tb. The results showed that the activation rate and the amount of ROS produced by neutrophils increased gradually with the extension of time, indicating that LPS could be beneficial to neutrophil-ROS mediated killing of M.tb. Lipopolysaccharide (LPS) is the most potent bioactive substance in the immune system, which can activate airway epithelial cells and alveolar macrophages to release a variety of inflammatory mediators, such as nitric oxide (NO), superoxide anion, cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Overproduction of these inflammatory mediators can be involved in damage to the alveolar capillary barrier and increase capillary permeability, leading to many inflammation-related diseases29,30. Studies have confirmed that LPS can directly activate macrophages and promote the production of ROS to exert direct bactericidal effect31. Therefore, can lipopolysaccharide (LPS) also activate neutrophils to recruit to the infection site and promote the anti-infection immune function in the process of M.tb infection? In this study, neutrophils were treated with different concentrations of LPS, and neutrophils activation and ROS production were detected by flow cytometry. The results showed that the activation rate of neutrophils and the amount of ROS produced by neutrophils increased gradually with the extension of time, indicating that LPS was beneficial to neutrophils in killing M.tb.

LPS can activate a variety of immune cells including neutrophils and macrophages through Toll-like receptors (TLRs)32. Studies have shown that in the regulation of anti-tuberculosis immune response, TLRs, are one of the key receptors, that induce the secretion of cytokines by recognizing the components of the cell wall of M.tb, to initiate an inflammatory33,34,35. TLR4 is the classical receptor of lipopolysaccharide (LPS) of Gram-negative bacteria36which is expressed on the cell surface and is conducive to the recognition of bacterial components when bacteria invade. The combination of LPS and TLR4 can activate the TLR4 signal transduction pathway, produce immune response, and then affect the occurrence and development of inflammatory diseases37. In order to further investigate how LPS affects the neutrophil mediated killing, we used TLR4 receptor blocker to treat neutrophils, and observed the activation rate and ROS production of neutrophils. The results showed that pretreatment of neutrophils with anti-TLR4 monoclonal antibody reduced their activation rate and ROS production as compared with the control group, suggesting that TLR4 could be involved in the killing function of neutrophils against M.tb. Cells produce reactive oxygen species (ROS) to eliminate phagocytosis microorganisms. ROS in the body is mainly derived from NADPH oxidase and mitochondrial respiratory chain complex38. NADPH oxidase, also known as NOX, is a multi-enzyme complex composed of plasma membrane-bound components and cytoplasmic components. A variety of exogenous factors can induce its phosphorylation and translocation from the cytoplasm to the plasma membrane, and then assemble into a fully functional NADPH oxidase. Subsequently, NADPH oxidase can catalyze the generation of ROS, including O2-, H2O2, ·HO, ·NO, etc., through a series of electron transfer to exert the killing function39. In this study, NADPH oxidase inhibitor DPI was used to observe the changes in the activation rate and ROS production of neutrophils. The results showed that the activation rate and ROS production of neutrophils were decreased after pretreatment with DPI compared with the control group. These suggests that NADPH oxidase was involved in the process of ROS production.

In summary, the present study confirmed that LPS-TLR4 pathway is involved in neutrophils induced ROS mediated killing of M.tb. LPS promotes neutrophils mediated killing of M.tb, through ROS mediated production of NADPH oxidase which provides a theoretical basis for testing the role of neutrophils in clearance of M.tb in humans, and reducing the M.tb pathogenesis. Further studies should test the role of neutrophils as a target in clinical treatment of TB.

Materials and methods

Blood specimen

Blood samples were collected from 24 healthy staff and students of Bengbu Medical University, including 12 males and 12 females, aged 18–55 years old, all of whom signed the informed consent voluntarily. This study involving human blood specimens was approved by the Ethics Committee of Bengbu Medical University (No. 2020LK70), and was conducted following the ethical guidelines of the Declaration of Helsinki.

Bacterial culture

M.tb strain was cultured in Roche medium at 37 °C for 1 month. Bacteria were collected, centrifuged at 10,000 rpm for 2 min, and resuspended in 0.9% sodium chloride solution at a density of 5 × 108 cells/ml for use.

Detection of killing rate

30 µl of bacterial solution was added to 4 µl of FDA (Fluorescein diacetate) solution (1 µg/ml) (Shanghai yuanye Bio-Technology Co., Item No.: S19128), and then placed at 37 °C for 30 min, centrifuged at 10,000 r/min for 2 min. The supernatant was discarded, washed twice with PBS, and the rate of FDA labeling of M.tb, the survival rate of M.tb, was detected by flow cytometry. Fresh anticoagulant blood was added into a flow tube, placed on ice for 10 min, to which 30 µl labeled bacteria solution wad added, mixed, and placed in a 37 °C water bath for 20 min. It was then washed with ice-cold PBS, supernatant was discarded. The sample was then hemolyzed, centrifuged, washed, and 10 µl autologous serum was added, placed in a 37 °C water bath. After 0 to 60 min, 200 µl of ice-cold triple-distilled water was immediately added, incubated for 2 min, and sample was run on flow cytometer. Unlabeled bacteria group and 0 °Ccontrol group were set up at the same time. The positive rate and negative rate of FDA were detected by flow cytometry. The killing rate was calculated using the following formula: killing rate = FDA negative rate/ (FDA positive rate + FDA negative rate)×100%.

Fluorescence microscope observation

Fresh anticoagulant blood from healthy adults was added into a flow tube, pre-added with CD16-PE antibody (Becton, Dickinson and Company, Item No.:550868) for 30 min, incubated for 30 min, and then FDA-labeled M.tb was added for 30 and 60 min at 37 °C. Fluorescence microscopy was utilized to observe the killing effect of neutrophils on M.tb. At the same time, a 0 °C control was also set up.

Detection of ROS production

2.5 µl/ tube of DCFH-DA (2’, 7’-dichlorofluorescein diacetate; Abbkine Bio Inc, Item No: KTB1910) was added to the control and 30 µl of unlabeled bacteria solution, mixed, placed in a 37 °C water bath, and quickly removed after 0 to 30 min. The PMN was washed twice with ice-cold PBS, and the supernatant was discarded. The PMN was then hemolyzed and placed at room temperature for 15 min. The PMN was centrifuged, the supernatant was discarded, and the neutrophils were washed twice with PBS. At the same time, whole blood control alone, whole blood plus DCFH-DA for 30 min, and 0 °C control at each time point were set.

Effect of LPS on ROS production

30 µl of fresh anticoagulant whole blood was collected and divided into control group and LPS (Langeke Biotechnology Co., LTD, Item No.:BS904) group. The final concentration of LPS group was 50ng /ml, 100ng /ml, 200ng /ml, 400ng /ml and 800 ng /ml, respectively. The blank control group was added with 30 µl of unlabeled bacteria, the negative control group and LPS group were added with 30 µl of labeled bacteria, respectively. After incubation at 37 °C for 5 min, the activation rate and ROS production of neutrophils were detected by flow cytometry.

Signaling pathway analysis

Neutrophils were pretreated with TLR4 monoclonal antibody (Jiangsu Pro-biological Research Center Co. LTD, Item No.: AF7017) or NADPH oxidase inhibitor (MCE, Item No.: HY100965) for 30 min, and then incubated with LPS (at a concentration of 400 ng/ml) for 30 min before addition of labeled M.tb. The activation rate and ROS production of neutrophils were detected.

Statistical analysis

Statistical analyses were performed with the use of R software, version 4.0.3. Data are presented as mean ± standard deviation. The neutrophil killing rate, activation rate and ROS production were compared by Kruskal-Wallis test among multiple groups, and Dunn’s test was used for pairwise comparison between groups. The effects of LPS on ROS, TLR4 blocker effects and DPI effects were compared using Wilcoxon rank sum test and T test, and P < 0.05 was considered statistically significant.