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The therapeutic potential for targeting CSE/H2S signaling in macrophages against Escherichia coli infection

Veterinary Research volume 54, Article number: 71 (2023) Cite this article

Abstract

Macrophages play a pivotal role in the inflammatory response to the zoonotic pathogen E. coli, responsible for causing enteric infections. While considerable research has been conducted to comprehend the pathogenesis of this disease, scant attention devoted to host-derived H2S. Herein, we reported that E. coli infection enhanced the expression of CSE in macrophages, accompanied by a significantly increased inflammatory response. This process may be mediated by the involvement of excessive autophagy. Inhibition of AMPK or autophagy with pharmacological inhibitors could alleviate the inflammation. Additionally, cell model showed that the mRNA expression of classic inflammatory factors (Il-1β, Il-6), macrophage polarization markers (iNOS, Arg1) and ROS production was significantly down-regulated after employing CSE specific inhibitor PAG. And PAG is capable of inhibiting excessive autophagy through the LKB1-AMPK-ULK1 axis. Interestingly, exogenous H2S could suppress inflammation response. Our study emphasizes the importance of CSE in regulating the macrophage-mediated response to E. coli. Increased CSE in macrophages leads to excessive inflammation, which should be considered a new target for drug development to treat intestinal infection.

Introduction

Escherichia coli (E. coli) are present in both humans and farm animals, is a leading cause of acute diarrhea worldwide [1]. Emerging drug-resistant bacteria and the cliff-like reduction of new antibacterial drugs sharpened the difficulty in the treatment of E. coli infections, giving rise to significant morbidity and mortality [2]. In developing regions, diarrheagenic E. coli can cause up to 40% of diarrhea in children under five [3]. Apart from being an anaerobic bioreactor programmed with a huge population of bacteria, the intestinal lumen ultimately serves as a natural reservoir for antibiotic resistance genes (ARGs), making it easy for plasmid-mediated horizontal transfer of ARGs occurs during antibiotic therapy [4]. In China, the number of ARGs per isolate animal-derived E. coli samples collected from the 1970s to 2019 has doubled and poses a significant threat to public health [5]. Therefore, it is urgent to develop safer and more effective strategies to combat E. coli infections.

Macrophages residing in intestinal tissues are crucial for maintaining intestinal homeostasis and preventing disease development [6]. These cells play a vital role in inflammation resolution and can be classified into two subtypes based on microenvironmental cues: the classically activated M1 macrophages and the alternatively activated M2 macrophages. While M1 macrophages secrete proinflammatory cytokines like interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α), exacerbating inflammation, M2 macrophages produce anti-inflammatory cytokines like interleukin-10 (IL-10), promoting tissue regeneration and alleviating inflammation [7]. However, excessive activation of M2 macrophages can impair intestinal protective function and worsen intestinal damage [8]. Therefore, it is crucial to examine how the chemotaxis, proliferation, differentiation, and activation of macrophages are regulated during intestinal infection. Such understanding can provide insights into the mechanisms and new treatment options for intestinal inflammatory diseases.

Hydrogen sulfide (hereafter referred to as H2S) the latest identified endogenous gaseous mediator, was once considered a foul-smelling and hazardous gas found in the environment [9]. Recently, H2S has gained increasing attention as an important player in modulating a wide range of physiological and pathological conditions [10, 11]. In mammals, similar to NO and CO, three enzymes including cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and the 3-mercaptopyruvate sulphur transferase (3-MPST) are responsible for H2S generation. Specifically, CSE and CBS promote de-sulphydration of l-cysteine to generate H2S, while 3-MPST induces H2S production by controlling the enzymatic activity of cysteine aminotransferase [12]. In fact, the effects of H2S are often bell-shaped: beneficial at normal physiological concentrations but toxic at high doses due to its inhibition of mitochondrial complex IV. Aberrations of CBS and CSE can lead to different outcomes, as H2S has both anti-inflammatory and pro-inflammatory effects [13]. Autophagy is a fundamental process involving the delivery of cellular material to lysosomes for degradation and recycling that contributes to cellular and tissue homeostasis, physiology, and development. However, abnormalities in autophagy may contribute to many different pathophysiological conditions [14]. There is an increasing amount of evidence suggesting that both endogenous and exogenous H2S could exhibit two evidently opposite effects on autophagy [15]. Therefore, it is urgent and essential to elucidate the mechanism of action of H2S in the autophagy process.

In this study, we have observed that an E. coli infection in the intestine can trigger an immune response that results in enhanced macrophage activity and recruitment to the site of infection in vivo. Additionally, in vitro experiments have shown that an increase in CSE expression of macrophages occurs in response to E. coli, accompanied by a significant rise in inflammation that may be associated with excessive autophagy. Both inhibiting CSE expression or autophagy can reduce inflammation. These findings shed light on the molecular mechanisms involved in antimicrobial defense and regulation of host sulfur metabolism, providing a potential strategy to address the growing issue of enteric infectious diseases.

Materials and methods

Bacterial strains, cell culture, and treatment

Escherichia coli (strain ATCC 25922) was inoculated into Luria–Bertani (LB) culture and incubated at 37 °C in an orbital shaker to log-phase growth OD600 = 0.4–0.6. RAW264.7 cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (Evergreen, Huzhou, China) and plated at 80% confluence in a 6-well cell plates, the monolayers were treated with or without 500 µM PAG (inhibitor of CSE: Aladdin, Shanghai, China) for 4 h, 10 µM Choroquine (inhibitor of autophagy, TargetMol, Boston, USA) and Compound C (inhibitor of AMPK, Selleck Chemicals, Houston, USA) for 2 h, 200 µM GYY4137 (Sigma-Aldrich, MO, USA) for 24 h, all at 37 °C. The treated cells were infected with E. coli at a multiplicity of infection (MOI) of 10 at 37 ℃ according to different test conditions. The supernatant and cells were collected separately and stored at −80 ℃ until use. Other reagents and materials are listed in Additional file 1.

Mice, and establishment of infection model

All animal experiments were carried out in accordance with the animal welfare standards and complied with the guidelines of the Animal Welfare Council of China and was approved by the Ethical Committee for Animal Experiments of Nanjing Agricultural University (NJAU. No20220509099). Mice were randomly divided into two groups (control group and infected group). One hundred microliters of 5% NaHCO3 was inoculated by oral gavage to neutralize gastric acidity, and 30 min after this treatment, 2 × 1010 CFU in 100 μL E. coli ATCC 25922 was inoculated by oral gavage, and mice in control group were gavaged with equivalent volume of normal saline solution. Following the 24 h time point, mice were sacrificed, corresponding samples were collected and stored at −80 °C until use.

RNA extraction and quantitative real-time polymerase chain reaction (qPCR)

Total RNA was extracted by TRIzol reagent (AG, Changsha, China). The corresponding cDNA was obtained using reverse transcriptase (AG, Changsha, China). An aliquot of cDNA was mixed with 5 µL TB Green PCR Master Mix (AG, Changsha, China) and 0.2 µL of each specific forward and reverse primer. All mixed systems were analyzed in a Roche LightCycler 96. β-actin was set as reference gene, and fold changes were calculated as 2−ΔΔCt. All primer sequences (Additional file 2) were synthesized by Tsingke Biological Company (Nanjing, China).

Total protein extraction and Western blotting

Cells were washed three times on ice-cold phosphate buffered saline and lysed by incubation in RIPA Lysis buffer containing protease inhibitor phenylmethanesulfonyl fluoride (Beyotime, Nantong, China). After grinding, supernatants were collected by centrifugation (5000 × g for 10 min at 4 °C). Protein concentration was determined by bicinchoninic acid assay (BCA kit, Beyotime, Nantong, China). Extracts with equal amounts of protein were solubilized in SDS sample buffer, separated by SDS–PAGE, transferred to polyvinylidene difluoride membranes (Millipore), blocked with 5% nonfat milk diluted in Tris-buffered saline with Tween‐20 (TBST) for 2 h at room temperature, and hybridized with primary antibody at 4 ℃ overnight. Before and after incubation with the secondary antibodies for 2 h at room temperature, the membranes were washed three times for 15 min with TBST. Secondary antibody was HRP‐linked anti‐rabbit IgG (CST; 1:10 000). Signals were detected by an ECL Western Blot Analysis System (Tanon, Shanghai, China). Bands were quantified with ImageJ 1.8 software (NIH, Bethesda, MD, USA).

Immunohistochemical staining

The immunohistochemistry procedures were carried out following previously described with minor modifications [16]. First, sections were sliced from paraffin-embedded specimens, deparaffinized in xylene and hydrated in a graded series of ethanol, placed in 0.01 mol/L citrate buffer (pH 6.0), and heated in a microwave oven over medium–high heat for 10 min. Then, the slices were incubated with 3% H2O2-methanol solution at room temperature for 10 min. Following primary and HRP-conjugated secondary antibody incubation, diaminobenzidine was used as a chromogen and hematoxylin for counterstaining. Finally, the slices were observed under a microscope and photographed.

BODIPY staining and microscopy

Cells in twelve-well plate were washed three times with PBS and fixed with 4% formaldehyde for 15 min at room temperature. Then were again washed 3 times with PBS after incubation with 2 µM BODIPY in the dark for 15 min at 37 ℃. Next, they were then incubated with 20 µM DAPI in the dark for 10 min at room temperature and washed 3 times in PBS. Finally, the plate was photographed inverted fluorescence microscope (EVOS FL Auto 2, Invitrogen).

Assessment of biochemical parameters

The protein samples from cells were extracted using cold lysis bufer and assayed using a BCA Kit. The levels of total cholesterol (TC), triglycerides (TGs), lactate dehydrogenase (LDH) and endogenous H2S, in cell lysates were measured using commercial kits according to the manufacturer’s instructions (Jiancheng, Nanjing, China).

Statistical analyses

Statistical analyses were performed using GraphPad Prism 8 software. Differences were evaluated by unpaired t test or one-way ANOVA followed by Tukeys post hoc tests. All data are represented as the means ± standard error of the mean (SEM). The significance level was set as P < 0.05.

Results

E. coli infection alters CSE levels in macrophages

When a pathogen invades an organism, macrophages are recruited through migration and release cytokines to induce an inflammatory response. The infiltration of macrophages was identified by immunohistochemical staining for the macrophage marker CD68 (Figure 1A). In view of the significant increase of macrophage infiltration after E. coli infection, we were becoming inquisitive about the contributor to this. The sulfur metabolism pathway plays an important role in organisms, especially transsulfuration pathway (Figure 1B). Infection of RAW 264.7 macrophages with E. coli resulted in a threefold increase in the level of H2S after 6 h infection (P < 0.05), This suggests that E. coli infection can cause an increase in H2S levels, which can be detected. Consequently, it is necessary to examine the key enzymes involved in H2S synthesis during dynamic infection process (Figure 1C). First, qPCR assay demonstrated CSE expression fold-change increased significantly at the transcriptional level in a time-dependent manner upon E. coli stimulation, comparing to other two enzymes (P < 0.05) (Figure 1D). This observation was further confirmed by Western blot analysis (P < 0.05) (Figure 1E). And we noticed that CBS levels increased significantly within 3–4 h of infection and both CBS and 3-MPST protein decreased within 5–6 h. Following a thorough analysis, we chose to conduct our subsequent experiment at 5 h of infection time. At this point, H2S generation is likely to be mainly driven by CSE.

Figure 1
figure 1

E. coli infection lead to an elevation of CSE in macrophages. A Positive staining for macrophage marker CD68 during E. coli infection. B Pathways of transsulfuration pathway in mammalian. C Detection of endogenous H2S in RAW264.7 cells during E. coli challenge. Relative mRNA expression D and protein expression levels (E) related to H2S synthesis in RAW264.7 cells during E. coli invasion. Data are presented as the means ± SEMs (n = 3). *(P < 0.05) = significantly different.

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CSE exacerbates the pro-inflammatory response of macrophages exposure to E. coli

To investigate the role of CSE in the pathogenesis of E. coli in the macrophage, a specific CSE inhibitor PAG was used to detect the effects. The effect of inhibitory effect was confirmed by performing Western blot analysis (Figure 2A), and take no effect on the growth of E. coli and the phagocytic capacity of macrophages (Figures 2B, C). As a rule, lactate dehydrogenase (LDH) release into the culture medium is an indicator for cell damage. And we found LDH release (Figure 2D) markedly decreased after employing PAG (P < 0.05). By detecting the mRNA levels of multiple cytokines in macrophages, we found that E. coli infection caused a sharp increase in the expression of Il-1β (Figure 2E), TNF-α (Figure 2F). Il-1β increased nearly 7000 times (P < 0.05). PAG pretreatment significantly decreased mRNA levels (P < 0.05), however, the mRNA levels of anti-inflammatory factors IL-10 (Figure 2G) show an opposite trend (P < 0.05). As shown in Figure 2I E. coli challenged significantly increased the pro-inflammatory factors of p65 phosphorylation levels and COX-2 protein (catalyzes the formation of prostaglandin) expression level, while PAG treatment significantly inhibited the above changes (P < 0.05). Meanwhile, the protein levels of iNOS and were significantly increased after infection and PAG reduced their expression levels (P < 0.05). Conversely, the protein level of Arg1 showed the opposite trend. These results were confirmed by the detection of HIF-1α both in transcript (Figure 2I) and protein level (P < 0.05). Conversely, these data demonstrated that up-regulated CSE could exacerbate inflammatory damage and affect the metabolism of macrophages induced by E. coli infection.

Figure 2
figure 2

CSE amplified the inflammatory response induced by E. coli. A Effect of CSE inhibition by PAG in RAW264.7 cells during E. coli infection. B Bacterial growth curves supplemented with PAG. C The impact of macrophage phagocytosis with PAG supplementation. Assessment of lactate dehydrogenase (LDH) activity (D) in the supernatants of RAW264.7 cells primed with PAG during E. coli infection. mRNA expression levels of pro-inflammatory IL-1β (E), IL-6 (F) and anti-inflammatory factors IL-10 (G) in RAW264.7 cells during E. coli invasion with or without PAG treatment. H Inflammation related protein expression during E. coli challenge with or without PAG treatment. Data are presented as the means ± SEMs (n = 3). *(P < 0.05) = significantly different.

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E. coli infected macrophages reactive oxygen species production and polarization were related to CSE

To test the hypothesis that the release of reactive oxygen species (ROS) resulting from the aforementioned inflammatory condition, we examined the role of CSE in cellular ROS production in E. coli-infected macrophages, and found PAG intervention can effectively reduced the ROS level result from E. coli infection (Figure 3A). Furthermore, overexpression of CSE by plasmid transfection in RAW 264.7 cells was investigated, after gene transfection, we observed a significant alteration in cell morphology, with cells no longer clustered and producing antennae (Figure 3B). As iNOS expression is an important marker for M1 macrophage differentiation, the protein level of iNOS was increased after CSE overexpression (P < 0.05) (Figure 3C). Taken together, our data indicated that CSE engaged in the generation of ROS and polarizing macrophages.

Figure 3
figure 3

CSE promoted macrophages reactive oxygen species generation and polarization induced by E. coli. A Reactive oxygen species were evaluated using DCFH-DA probes in RAW264.7 cells during E. coli invasion with or without PAG treatment. B RAW264.7 cells were transfected CSE overexpression plasmids and control cells were transfected with empty vector. C The overexpression of CSE was validated by Western blot analysis and the polarization marker iNOS detection.

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Upregulated CSE may involved in lipid metabolism upon E. coli infection

Mounting studies have revealed that lipid droplets play a significant role in regulating lipid homeostasis and participating in inflammatory responses [17]. Additionally, CSE has been closely associated with lipid metabolism. Herein, we found that PAG pretreatment can dramatically lower the content of triglycerides (TGs) (Figure 4A) and total cholesterol (TC) (Figure 4B), which were significantly increased when encounter E. coli infection (P < 0.05). The aforementioned results were further confirmed by BODIPY staining analysis (Figure 4C). To validate these findings, we examined the mRNA levels of lipid metabolism related genes by qRT-PCR, E. coli stimulation increased expressions of Srebf1, Srebf2, while PAG suppressed the increased (P < 0.05), the variation tendency of Acox1 was opposite to that (P < 0.05) and there were no significant changes in Scd1 and Fasn genes (Figure 4D). Then we analyzed LKB1/AMPK/ACC signaling, which is a classical pathway involved in lipid metabolism regulation. Then results showed that E. coli infection could increase phosphorylation of LKB1 and AMPK, inhibition of CSE by PAG lead to the level of phosphorylation decrease (P < 0.05). Interestingly, phosphorylation of ACC continue to increase, which was different from our expectations (Figure 4E). Moreover, it can be seen that the use of PAG alone can also result in an increase in the phosphorylation of ACC, indicating PAG could potentially have a direct regulatory effect on ACC. Therefore, these findings indicate that CSE plays a role in regulating the LKB1/AMPK pathway related to lipid metabolism in response to E. coli infection, while the impact of PAG on ACC phosphorylation could be more significant than the influence of increased AMPK phosphorylation.

Figure 4
figure 4

Inhibiting CSE can reduce macrophage lipid droplet accumulation against E. coli infection. A, B TG and TC contents in RAW264.7 cells during E. coli invasion. C Relative mRNA expression levels related to lipid synthesis in RAW264.7 cells during E. coli challenge. Heatmap (on the left) and statistical chart (on the right). D BODIPY 493/503 staining for LD visualization (green) increased in E. coli infection. F Expression of lipid metabolism related proteins in E. coli infection. Data are presented as the means ± SEMs (n = 3). *(P < 0.05) = significantly different.

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CSE is responsible for inducing autophagy during an E. coli challenge

Given that AMPK is the main sensor and regulator of cell metabolism, we try to explore how LKB1-AMPK function. We focused on its downstream molecule ULK1, the mammalian homolog of yeast Atg1, for autophagosome formation. In the absence of E. coli infection, CSE overexpression resulted in P62 protein increased, accompanying LC3 protein decreased (P < 0.05) (Figure 5A), which suggested a possible relevance of CSE with the autophagic pathway. For the purpose of further corroborating the above conclusion, we utilized PAG to reverse the observed impact of overexpressed CSE. Upon doing so, we observed that PAG was able to effectively reverse the influence of LC3 and p62 proteins resulting from CSE overexpression (Additional file 3). When cells were exposed to E. coli, we found the autophagy proteins ATG5, P62 and ULK1 phosphorylation increased markedly, however PAG can reverse such changes (P < 0.05). Instead, LC3 protein raised significantly, and a significant decreasing trend was observed after PAG pretreatment (P < 0.05) (Figure 5B). Overall, results above confirmed that E. coli infection activated AMPK-ULK1 and consequently promotes autophagy, while this phenomenon could be reversed by inhibiting CSE.

Figure 5
figure 5

CSE induced autophagy during E. coli challenge. A RAW264.7 cells were transfected with CSE overexpression plasmids, while control cells were transfected with an empty vector. Immunoblots were performed to detect changes in autophagy-related proteins. B The signalling pathways connected to autophagy. during E. coli challenge with or without PAG treatment. Data are presented as the means ± SEMs (n = 3). *(P < 0.05) = significantly different.

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Inhibiting excessive autophagy attenuated inflammation upon E. coli infection

Since we observed that CSE affected autophagy in E. coli-infected macrophages, we asked whether autophagy could regulate inflammation. Hence, we used the AMPK inhibitor compound C and autophagy inhibitor chloroquine to treat E. coli-infected macrophages, and we found significant reduction of proinflammatory factors in mRNA expression levels after both compound C and chloroquine pretreatment, such as IL-1β (Figure 6A), IL-6 (Figure 6B), and TNF-α (P < 0.05) (Figure 6C). We then investigated COX-2 protein levels and LC3 protein levels using Western blotting. Like in the previous results, E. coli infection caused LC3 and COX-2 protein level increased. After employing two inhibitors, both LC3 and COX-2 protein level decreased (P < 0.05) (Figure 6D, E). Collectively, we have elucidated that inhibiting CSE attenuated excessive inflammation in an autophagic machinery-dependent manner.

Figure 6
figure 6

Inhibiting excessive autophagy resulted in the attenuated inflammation upon E. coli infection. Relative mRNA expression levels of pro-inflammatory IL-1β (A), IL-6 (B) and TNF-α (C) in RAW264.7 cells during E. coli invasion with or without compound C and chloroquine. D Immunoblots were performed to detect changes in autophagy-related protein (LC3B) and inflammatory-related proteins (COX-2), after pretreat compound C (D) and chloroquine (E). Data are presented as the means ± SEMs (n = 3). *(P < 0.05) = significantly different.

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H2S donor GYY4137 displayed stronger protection against E. coli infection

Next, we investigated the role of exogenous H2S play in this process. Firstly, it was observed that GYY4137 was not cytotoxic to RAW 264.7 cells at concentrations under 200 μM (P < 0.05) (Figure 7A). We also examined the expression of iNOS and Arg1 protein using Western blot analysis and discovered that pretreatment with GYY4137 was able to effectively decrease the levels of iNOS protein whilst increasing Arg1 protein, thus partially reversing the changes caused by E. coli infection (P < 0.05) (Figure 7B). Overall, the role of exogenous H2S did not align with that of endogenous H2S in macrophages during E. coli infection.

Figure 7
figure 7

GYY4137 effectively protected against E. coli infection. A CCK8 assay for cellular viability. B Inflammation-related protein expression during E. coli challenge with or without GYY4137 treatment. Data are presented as the means ± SEMs (n = 3). *(P < 0.05) = significantly different.

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Discussion

E. coli is a significant contributor to infections globally, particularly diarrheal diseases, which result in considerable economic losses. The intestine houses the highest concentration of immune cells in the body, of which macrophages are predominant in the lamina propria [18]. To investigate the pathogenesis of enteric infection, we first developed infection models in mice through the oral administration of E. coli, then created an in vitro model of E. coli infection using murine monocytic RAW264.7 cells. In this study, a high number of macrophages were recruited to the colon of infected mice, as expected in an inflammatory process. Further research was conducted based on the crucial role of H2S in numerous pathophysiological conditions. In fact, bacterial infection can cause changes in H2S synthases, but infection was found to be a highly dynamic process. Hence, various enzymes may catalyze distinct stages of an infection, resulting in the production of H2S. In this study, gene and protein expression results indicated that CSE expression fluctuation was stronger compared to CBS and MPST in infected cells, suggesting that CSE is a major H2S producer in macrophages during E. coli infection, particularly in 5 h infection. Previous studies have demonstrated significant increases in both CBS and CSE expression with the same Mycobacterium Tuberculosis (Mtb) [19, 20], while Staphylococcus aureus [21], Helicobacter pylori [22], and Pseudomonas aeruginosa [23] can result in an increase in CSE expression.

In previous studies, the role of CSE in inflammation concerning H2S production has been explored. However, there is no consensus on the matter and findings largely depend on exogenous versus endogenous enzymatic H2S derivation. For example, Rahman et al. demonstrated that CSE promotes an excessive innate immune response, suppresses the adaptive immune response, and reduces circulating IL-1β, IL-6, TNF-α, and IFN-γ levels in response to Mtb infection [19]. However, host CSE derived H2S protects against Pseudomonas aeruginosa sepsis [23]. The contrasting outcomes highlight the intricate and adaptable nature of the cellular immune reaction. In our research, we observed that CSE distinctly augmented inflammatory responses in macrophages, potentially linked to elevated ROS production and macrophage polarization.

Given that mammalian lipid droplets possess a protein-mediated antimicrobial capability, which is heightened during poly-microbial sepsis and in response to LPS [24, 25], and it is noteworthy that H2S has the capacity to increase both the size and quantity of lipid droplets while simultaneously reducing lipolysis [26]. HIF-1α, as a transcription factor, plays an important role between metabolic activities and immune response [27]. Firstly, we observed a significant decrease in HIF-1α expression after inhibiting CSE during E. coli infection. We then focused on lipid metabolism and found that PAG reduced the accumulation of lipid droplets in RAW264.7 macrophages. Additionally, we examined the gene expressions of several other factors related to lipid metabolism and observed that PAG decreased the expression of lipogenesis genes (Srebf1 and Srebp2) while increasing the expression of steatolysis gene Acox1. It is known that AMPK can be activated through both LKB1 and LKB1-independent pathways [28], and there are multiple mechanisms through which H2S may impact LKB1 [29, 30]. As ACC acts downstream of AMPK in fatty acid metabolism, AMPK can inhibit ACC activity through phosphorylation, thereby reducing fatty acid synthesis. Despite this, we found that the phosphorylation of ACC increased instead of decreased upon adding PAG before and after E. coli infection in present study, which suggests that PAG may have a direct impact on ACC. However, the exact mechanism requires further investigation.

We propose that AMPK may activate ULK1 through phosphorylation, thereby regulating cellular autophagy. Recent studies indicate that endogenously produced and/or exogenously administered H2S may exhibit two opposing effects on autophagy in various disease models [31, 32]. These effects could be attributed to factors such as concentration, time frame, and reaction time of H2S, as well as differences between disease stages or models. It is generally believed that autophagy is positively correlated with LC3 and negatively correlated with p62. The former was markedly decreased and the latter was increased following CSE overexpression alone, which indicates that autophagic degradation is inactive. However different manifestations were found after E. coli infection, which led to the upregulated expression of autophagy related proteins. It is well-known that autophagy is a double-edged sword, and the exact threshold at which protective autophagy becomes cytotoxic autophagy remains unknown. For example, Kong et al. discovered that excessive autophagy disorders promote inflammatory responses in the pancreas [33]. Our data demonstrate that inhibition of autophagy by CQ and AMPK inhibitor compound C also significantly reduces the inflammatory response. This effect is consistent with previous findings that treatment with CSE inhibitors produces similar results. However, it is important to note that autophagy not only eliminates unnecessary or dysfunctional parts of the cell but also releases amino acids and molecules that support the cell's metabolic functions. Cysteine, for example, can be used as a substrate for the enzymatic creation of H2S via CSE. Inhibiting autophagy can directly hinder the production of CSE/H2S, which can lead to the alleviation of inflammation. Therefore, future studies should be performed to validate these conclusions.

Finally, researches have demonstrated that H2S produced by the intestinal microbiota can have the same effect as endogenously-produced H2S. This is noteworthy because intestinal cells are also exposed to H2S from the luminal side in addition to the H2S produced internally [34]. The results of monitoring the level of macrophage polarization are interesting because they show that the use of slow-releasing H2S donor GYY4137 reduces polarization in macrophages. This finding is in line with a previous study which demonstrated that GYY4137 is effective in inhibiting NLRP3 inflammasome activity in macrophages [35]. Furthermore, GYY4137 reduces the secretion of inflammatory factors and limits the production of ROS in cardiomyocytes [36].

In summary, our study highlights the significance of CSE in controlling the macrophage-mediated response to E. coli (Figure 8). Prolonged overexpression of CSE in macrophages results in inflammation upon exposure to E. coli. Alternatively, curbing CSE activity can curtail it, which could be connected to surplus autophagy. The use of PAG and GYY4137 could have crucial therapeutic applications for combating infections caused by E. coli and other related pathogens. Future studies could investigate the potential relationship between CSE and Desulfovibrio, which are known to be the major producers of exogenous H2S in the gastrointestinal tract. Additionally, it would be interesting to develop drugs that could regulate the generation of exogenous H2S.

Figure 8
figure 8

Proposed mechanism for CSE/H2S exacerbating inflammation in macrophages. Red arrows represent increase; blue arrows represent decrease.

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Data Availability

All data included in this study are available upon request by contact with the corresponding author.

References

  1. Wang X, Teng D, Guan Q, Mao R, Hao Y, Wang X, Yao J, Wang J (2017) Escherichia coli outer membrane protein F (OmpF): an immunogenic protein induces cross-reactive antibodies against Escherichia coli and Shigella. AMB Express 7:155

    Article  PubMed  PubMed Central  Google Scholar 

  2. Fang D, Xu T, Sun J, Shi J, Li F, Yin Y, Wang Z, Liu Y (2023) Nicotinamide mononucleotide ameliorates sleep deprivation-induced gut microbiota dysbiosis and restores colonization resistance against intestinal infections. Adv Sci 10:e2207170

    Article  Google Scholar 

  3. Dutta S, Guin S, Ghosh S, Pazhani GP, Rajendran K, Bhattacharya MK, Takeda Y, Nair GB, Ramamurthy T, Gordon SV (2013) Trends in the prevalence of diarrheagenic Escherichia coli among hospitalized diarrheal patients in Kolkata, India. PLoS One 8:e56068

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Jia Y, Yang B, Shi J, Fang D, Wang Z, Liu Y (2022) Melatonin prevents conjugative transfer of plasmid-mediated antibiotic resistance genes by disrupting proton motive force. Pharmacol Res 175:105978

    Article  PubMed  CAS  Google Scholar 

  5. Yang L, Shen Y, Jiang J, Wang X, Shao D, Lam MMC, Holt KE, Shao B, Wu C, Shen J, Walsh TR, Schwarz S, Wang Y, Shen Z (2022) Distinct increase in antimicrobial resistance genes among Escherichia coli during 50 years of antimicrobial use in livestock production in China. Nat Food 3:197–205

    Article  PubMed  CAS  Google Scholar 

  6. Liu Q, Liu S, Cao H, Shen Z (2021) Ramulus Mori (Sangzhi) Alkaloids (SZ-A) Ameliorate glucose metabolism accompanied by the modulation of gut microbiota and ileal inflammatory damage in type 2 diabetic KKAy mice. Front Pharmacol 12:642400

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Ge X, Xue G, Ding Y, Li R, Hu K, Xu T, Sun M, Liao W, Zhao B, Wen C, Du J. The loss of YTHDC1 in gut macrophages exacerbates inflammatory bowel disease. Adv Sci. 2023;e2205620.

  8. Liu F, Smith AD, Solano-Aguilar G, Wang TTY, Li RW (2020) Mechanistic insights into the attenuation of intestinal inflammation and modulation of the gut microbiome by krill oil using in vitro and in vivo models. Microbiome 8:83

    Article  PubMed  PubMed Central  Google Scholar 

  9. Sun H, Wu Z, Nie X, Wang X, Bian J (2021) An updated insight into molecular mechanism of hydrogen sulfide in cardiomyopathy and myocardial ischemia/reperfusion injury under diabetes. Front Pharmacol 12:651884

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Faris P, Negri S, Faris D, Scolari F, Montagna D, Moccia F (2023) Hydrogen sulfide (H2S): as a potent modulator and therapeutic prodrug in cancer. Curr Med Chem 30:4506–4532

    Article  PubMed  CAS  Google Scholar 

  11. Wallace JL, Wang R (2015) Hydrogen sulfide-based therapeutics: exploiting a unique but ubiquitous gasotransmitter. Nat Rev Drug Discov 14:329–345

    Article  PubMed  CAS  Google Scholar 

  12. Zhang H, Du J, Huang Y, Tang C, Jin H (2023) Hydrogen sulfide regulates macrophage function in cardiovascular diseases. Antioxid Redox Signal 38:45–56

    Article  PubMed  CAS  Google Scholar 

  13. Panagaki T, Randi EB, Augsburger F, Szabo C (2019) Overproduction of HS, generated by CBS, inhibits mitochondrial Complex IV and suppresses oxidative phosphorylation in Down syndrome. Proc Natl Acad Sci USA 116:18769–18771

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Gotor C, Aroca A, Romero LC (2022) Persulfidation is the mechanism underlying sulfide-signaling of autophagy. Autophagy 18:695–697

    Article  PubMed  CAS  Google Scholar 

  15. Wu D, Wang H, Teng T, Duan S, Ji A, Li Y (2018) Hydrogen sulfide and autophagy: a double edged sword. Pharmacol Res 131:120–127

    Article  PubMed  CAS  Google Scholar 

  16. Liu CM, Chiu KL, Chen TS, Chang SM, Yang SY, Chen LH, Ni YL, Sher YP, Yu SL, Ma WL (2015) Potential therapeutic benefit of combining gefitinib and tamoxifen for treating advanced lung adenocarcinoma. Biomed Res Int 2015:642041

    PubMed  PubMed Central  Google Scholar 

  17. Wan Z, Fu S, Wang Z, Xu Y, Zhou Y, Lin X, Lan R, Han X, Luo Z, Miao J (2022) FABP4-mediated lipid droplet formation in Streptococcus uberis-infected macrophages supports host defence. Vet Res 53:90

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Altemus J, Dadgar N, Li Y, Lightner AL (2022) Adipose tissue-derived mesenchymal stem cells’ acellular product extracellular vesicles as a potential therapy for Crohn’s disease. J Cell Physiol 237:3001–3011

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Rahman MA, Cumming BM, Addicott KW, Pacl HT, Russell SL, Nargan K, Naidoo T, Ramdial PK, Adamson JH, Wang R, Steyn AJC (2020) Hydrogen sulfide dysregulates the immune response by suppressing central carbon metabolism to promote tuberculosis. Proc Natl Acad Sci USA 117:6663–6674

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Saini V, Chinta KC, Reddy VP, Glasgow JN, Stein A, Lamprecht DA, Rahman MA, Mackenzie JS, Truebody BE, Adamson JH, Kunota TTR, Bailey SM, Moellering DR, Lancaster JR Jr, Steyn AJC (2020) Hydrogen sulfide stimulates Mycobacterium tuberculosis respiration, growth and pathogenesis. Nat Commun 11:557

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Gobert AP, Latour YL, Asim M, Finley JL, Verriere TG, Barry DP, Milne GL, Luis PB, Schneider C, Rivera ES, Lindsey-Rose K, Schey KL, Delgado AG, Sierra JC, Piazuelo MB, Wilson KT (2019) Bacterial pathogens hijack the innate immune response by activation of the reverse transsulfuration pathway. MBio 10:e02174-e2219

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Latour YL, Sierra JC, Finley JL, Asim M, Barry DP, Allaman MM, Smith TM, McNamara KM, Luis PB, Schneider C, Jacobse J, Goettel JA, Calcutt MW, Rose KL, Schey KL, Milne GL, Delgado AG, Piazuelo MB, Paul BD, Snyder SH, Gobert AP, Wilson KT (2022) Cystathionine γ-lyase exacerbates Helicobacter pylori immunopathogenesis by promoting macrophage metabolic remodeling and activation. JCI insight 7:e155338

    Article  PubMed  PubMed Central  Google Scholar 

  23. Renieris G, Droggiti DE, Katrini K, Koufargyris P, Gkavogianni T, Karakike E, Antonakos N, Damoraki G, Karageorgos A, Sabracos L, Katsouda A, Jentho E, Weis S, Wang R, Bauer M, Szabo C, Platoni K, Kouloulias V, Papapetropoulos A, Giamarellos-Bourboulis EJ (2021) Host cystathionine-γ lyase derived hydrogen sulfide protects against Pseudomonas aeruginosa sepsis. PLoS Pathog 17:e1009473

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Bosch M, Sánchez-Álvarez M, Fajardo A, Kapetanovic R, Steiner B, Dutra F, Moreira L, López JA, Campo R, Marí M, Morales-Paytuví F, Tort O, Gubern A, Templin RM, Curson JEB, Martel N, Català C, Lozano F, Tebar F, Enrich C, Vázquez J, Del Pozo MA, Sweet MJ, Bozza PT, Gross SP, Parton RG, Pol A (2020) Mammalian lipid droplets are innate immune hubs integrating cell metabolism and host defense. Science 370:eaay8085

    Article  PubMed  CAS  Google Scholar 

  25. Knight M, Braverman J, Asfaha K, Gronert K, Stanley S (2018) Lipid droplet formation in Mycobacterium tuberculosis infected macrophages requires IFN-γ/HIF-1α signaling and supports host defense. PLoS Pathog 14:e1006874

    Article  PubMed  PubMed Central  Google Scholar 

  26. Tsai CY, Peh MT, Feng W, Dymock BW, Moore PK (2015) Hydrogen sulfide promotes adipogenesis in 3T3L1 cells. PLoS One 10:e0119511

    Article  PubMed  PubMed Central  Google Scholar 

  27. Gonzalez FJ, Xie C, Jiang C (2018) The role of hypoxia-inducible factors in metabolic diseases. Nat Rev Endocrinol 15:21–32

    Article  PubMed  PubMed Central  Google Scholar 

  28. Sundararaman A, Amirtham U, Rangarajan A (2016) Calcium-oxidant signaling network regulates AMP-activated Protein Kinase (AMPK) activation upon matrix deprivation. J Biol Chem 291:14410–14429

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Cui C, Fan J, Zeng Q, Cai J, Chen Y, Chen Z, Wang W, Li SY, Cui Q, Yang J, Tang C, Xu G, Cai J, Geng B (2020) CD4+ T-cell endogenous cystathionine γ lyase-hydrogen sulfide attenuates hypertension by sulfhydrating liver kinase B1 to promote T regulatory cell differentiation and proliferation. Circulation 142:1752–1769

    Article  PubMed  CAS  Google Scholar 

  30. Kundu S, Pushpakumar S, Khundmiri SJ, Sen U (2014) Hydrogen sulfide mitigates hyperglycemic remodeling via liver kinase B1-adenosine monophosphate-activated protein kinase signaling. Biochim Biophys Acta 1843:2816–2826

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Wu J, Tian Z, Sun Y, Lu C, Liu N, Gao Z, Zhang L, Dong S, Yang F, Zhong X, Xu C, Lu F, Zhang W (2017) Exogenous H2S facilitating ubiquitin aggregates clearance via autophagy attenuates type 2 diabetes-induced cardiomyopathy. Cell Death Dis 8:e2992

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Xiao J, Zhu X, Kang B, Xu J, Wu L, Hong J, Zhang Y, Ni X, Wang Z (2015) Hydrogen sulfide attenuates myocardial hypoxia-reoxygenation injury by inhibiting autophagy via mTOR activation. Cell Physiol Biochem 37:2444–2453

    Article  PubMed  CAS  Google Scholar 

  33. Kong L, Deng J, Zhou X, Cai B, Zhang B, Chen X, Chen Z, Wang W (2021) Sitagliptin activates the p62-Keap1-Nrf2 signalling pathway to alleviate oxidative stress and excessive autophagy in severe acute pancreatitis-related acute lung injury. Cell Death Dis 12:928

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Blachier F, Beaumont M, Kim E (2019) Cysteine-derived hydrogen sulfide and gut health: a matter of endogenous or bacterial origin. Curr Opin Clin Nutr Metab Care 22:68–75

    Article  PubMed  CAS  Google Scholar 

  35. Castelblanco M, Lugrin JM, Ehirchiou D, Nasi S, Ishii I, So A, Martinon F, Busso N (2017) Hydrogen sulfide inhibits NLRP3 inflammasome activation and reduces cytokine production both in vitro and in a mouse model of inflammation. J Biol Chem 293:2546–2557

    Article  PubMed  PubMed Central  Google Scholar 

  36. Zhou T, Qian H, Zheng N, Lu Q, Han Y (2022) GYY4137 ameliorates sepsis-induced cardiomyopathy via NLRP3 pathway. Biochim Biophys Acta Mol Basis Dis 1868:166497

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Dr Howard Gelberg (Oregon State University) for editing this manuscript.

Funding

This project was supported by grants from the National Natural Science Foundation of China (No. 32072867 and 32273013), the Jiangsu Province Key Research and Development Program (No. BE2022384) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Authors and Affiliations

  1. MOE Joint International Research Laboratory of Animal Health and Food Safety, Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China

    Shaodong Fu, Zhenglei Wang, Yuanyuan Xu & Jinfeng Miao

  2. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China

    Xiangan Han

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  1. Shaodong Fu
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  2. Zhenglei Wang
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  3. Xiangan Han
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  4. Yuanyuan Xu
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Contributions

SF: conceptualization, visualization, data curation, formal analysis, writing-review and editing, project administration, software. ZW: data curation, software, project administration. YX: data curation, software, writing-review and editing. XH: conceptualization, visualization, writing-original draft, writing-review and editing. JM: conceptualization, visualization, funding acquisition, writing-original draft, writing-review and editing. All authors read and approved the final manuscript.

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Correspondence to Jinfeng Miao.

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Handling editor: Tina Dalgaard.

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Supplementary Information

Additional file 1

: Key resources table.

Additional file 2

: Oligonucleotide sequences used for qPCR.

Additional file 3

: PAG reversed the observed effect on autophagy of CSE overexpression. RAW264.7 cells were transfected with CSE overexpression plasmids, with or without PAG, while control cells were transfected with an empty vector. Immunoblots were performed to detect changes in autophagy-related proteins. Data are presented as the means ± SEMs (n = 3). *(P < 0.05) = significantly different.

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Fu, S., Wang, Z., Han, X. et al. The therapeutic potential for targeting CSE/H2S signaling in macrophages against Escherichia coli infection. Vet Res 54, 71 (2023). https://doi.org/10.1186/s13567-023-01203-8

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Keywords

Veterinary Research

ISSN: 1297-9716