
==== Front
Gut Microbes
Gut Microbes
Gut Microbes
1949-0976
1949-0984
Taylor & Francis

39284098
10.1080/19490976.2024.2399215
2399215
Version of Record
Research Article
Research Paper
Enterotoxigenic Escherichia coli heat labile enterotoxin affects neutrophil effector functions via cAMP/PKA/ERK signaling
J. MA ET AL.
GUT MICROBES
Ma Jinglin
Hermans Leen
Dierick Matthias
Van der Weken Hans
https://orcid.org/0000-0003-4281-2990
Cox Eric
https://orcid.org/0000-0002-3222-8769
Devriendt Bert
Laboratory Immunology, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University , Merelbeke, Belgium
CONTACT Bert Devriendt B.Devriendt@Ugent.be Laboratory Immunology, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke 9820, Belgium
16 9 2024
2024
16 9 2024
16 1 2399215Integra13 9 2024
Integra13 9 2024
18 4 2024
26 8 2024
28 8 2024
© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.
2024
The Author(s)
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in humans and animals, induced by enterotoxins produced by these pathogens. Despite the crucial role of neutrophils in combatting bacterial infections, our understanding of how enterotoxins impact neutrophil function is limited. To address this knowledge gap, we used heat-labile enterotoxin (LT) and heat-stable enterotoxin a (STa) to investigate their impact on the effector functions of neutrophils. Our study reveals that pSTa does not exert any discernible effect on the function of neutrophils. In contrast, LT altered the migration and phagocytosis of neutrophils and induced the production of inflammatory factors via activation of cAMP/PKA and ERK1/2 signaling. LT also attenuated the release of neutrophil extracellular traps by neutrophils via the PKA signaling pathway. Our findings provide novel insights into the impact of LT on neutrophil function, shedding light on the underlying mechanisms that govern its immunoregulatory effects. This might help ETEC in subverting the immune system and establishing infection.

KEYWORDS

Heat labile enterotoxin
ETEC
neutrophils
pig
cAMP/PKA/ERK signaling
China Scholarship Council 10.13039/501100004543 201906350196 Flemish fund for scientific research 3S036319 Special Research Fund of Ghent University BOF/STA/202009/021 J.M. is supported by a by a PhD grant from the China Scholarship Council (grant number 201906350196). M.D. is supported by the Flemish fund for scientific research (FWO-SB; 3S036319). This research is supported by grants from the Special Research Fund of Ghent University (BOF/STA/202009/021; BOF/BAS/2015/0029/01).
==== Body
pmcIntroduction

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children in and travelers to ETEC endemic regions as well as in livestock species, including swine.1–3 ETEC strains have two main classes of virulence factors: colonization factors or fimbriae, and enterotoxins.4,5 While the fimbriae are critically important to establish an infection by mediating the adhesion of ETEC to the gut epithelium, the enterotoxins trigger diarrhea, facilitating nutrient acquisition or bacterial transmission. Two types of secreted enterotoxins can be distinguished, the heat-labile enterotoxin (LT) and the heat-stable enterotoxins (STs).4,6 LT is an AB5 toxin that binds to the gut epithelium via its pentameric B subunit in a ganglioside M1 (GM1)-dependent manner. Upon internalization, the A subunit activates adenylate cyclase, leading to increased intracellular cyclic adenosine monophosphate (cAMP) levels. This in turn activates protein kinase A (PKA), leading to the opening of the cystic fibrosis transmembrane regulatory channel (CFTR) and the inhibition of the Na+/H+ ion exchanger isotype 3 (NHE3). Together, this causes the efflux of electrolytes and water into the gut lumen.7,8 Heat-stable enterotoxin a (STa, a short 18–19 aa peptide) on the other hand acts by activating guanylate cyclase on intestinal epithelial cells, leading to increased cyclic GMP (cGMP) levels. This in turn affects the function of both ion channels, resulting in a disruption of the electrolyte balance, which ultimately leads to diarrhea.9

The enterotoxicity of LT and STs is well known, along with the pathways they trigger in gut epithelial cells which ultimately lead to diarrhea. Intestinal epithelial cells respond to ETEC and its virulence factors by producing inflammatory mediators which attract and inform innate immune cells and help in coordinating immune responses to clear ETEC infections.10–12 In addition to this indirect effect on immune cells, LT is a potent immunogen and activates dendritic cells to promote mucosal immune responses.13,14 Intriguingly, LT also decreases phagocytosis by macrophages and reduces inflammation by driving the polarization of alternatively activated macrophages.15 The impact of STs on immune cells is less studied, although recent data indicate that STa does not directly affect macrophage function.6,15 Whether LT and STa can affect the function of other innate immune cells, like neutrophils, is unknown.

Neutrophils have potent antimicrobial activities and play a critical role in protecting the host against bacterial pathogens, also at barrier sites. Neutrophils migrate in abundant numbers to injured or infected sites, where they perform effector functions to contain and eliminate pathogens. These effector functions include the phagocytosis of bacteria, the release of host defense proteins, the generation of reactive oxygen species (ROS) and the formation of neutrophil extracellular traps (NETs).16 These web-like chromatin structures are released by the neutrophils and are composed of DNA and various antimicrobial components to trap and kill bacteria.17 However, some pathogens have evolved mechanisms to evade killing by NETs and even use these to their own benefit.17 In addition, neutrophils release cytokines and chemokines to help in orchestrating and regulating immune responses to infections.18 Intriguingly, neutrophils can migrate across the gut epithelium into the intestinal lumen both in steady state and during inflammation as well as ETEC infection.19–22 This offers an opportunity for direct contact between neutrophils and ETEC enterotoxins in the gut lumen.

Given the essential role of neutrophils in the defense against bacterial infections and in coordinating immune responses, we aimed to investigate whether the ETEC-derived enterotoxins LT and pSTa (STa secreted by porcine ETEC strains) can affect the function of primary porcine neutrophils.

Results

GM1 mediates the binding of LT to neutrophils, whereas neither LT nor pSta affects neutrophil viability

To understand whether LT and pSTa can affect the effector functions of neutrophils, LT was purified from the supernatant of a porcine wild type ETEC strain23 (Fig. S1), while pSTa was synthesized. The bioactivity of both LT and pSTa was evidenced by their ability to increase cAMP and cGMP levels in gut epithelial cells, respectively (Fig, S2), and by their capacity to induce swelling in gut organoids (Fig. S3, movie S1–3). A key step in the enterotoxicity of LT is its binding to GM14,24 however, whether LT binds to the membrane of neutrophils in a GM1-dependent manner is currently unknown. Thus, we set out to test this by incubating neutrophils with LT and evaluating its binding to the neutrophil membrane. As shown in Figure 1a, a dose-dependent increase in membrane bound LT was observed upon incubation of neutrophils with LT, consistent with confocal microscopy images (Fig. S4). Pre-treating LT with GM1 inhibited the binding of LT to the neutrophils, confirming that the binding of LT to neutrophils was indeed mediated by GM1 (Figure 1b). Figure 1. LT binds to neutrophils and triggers their migration.

(a) Neutrophils (2×105) were incubated with 0, 20 or 500 ng/mL LT for 1 h at 4°C. The binding of LT to the neutrophil membrane was analyzed by immunostaining and flow cytometry. (b) Pre-incubation of 0, 25 or 500 ng/mL LT with 1 μg/mL GM1 for 2 h inhibited the binding of LT to neutrophils. (c) Scheme of the transwell migration assay. Neutrophils (3×105) were added to the upper chamber of the transwells (pore size 3 μm) and 0, 20 and 500 ng/mL LT or 0, 100 and 500 ng/mL pSTa was added to the lower chamber. Upon incubation for 3 h at 37°C, the number of neutrophils in the lower chamber was assessed by flow cytometry. (d) Representative dot plots of neutrophil migration toward 500 ng/mL LT, 500 ng/mL pSTa and 100 ng/mL CXCL-8 for 3 h. Quantification analysis of migrated neutrophils toward 0, 20 and 500 ng/mL LT or 0, 100 and 500 ng/mL pSTa, added to the lower chamber. (e) Scheme of the transwell migration assay. Neutrophils (3×105) were pretreated with 0, 20 and 500 ng/mL LT or 0, 100 and 500 ng/mL pSTa for 2 or 8 h and then were added to the upper chamber, while 100 ng/mL CXCL-8 was added to the lower chamber. Upon incubation for 3 h at 37°C, the number of neutrophils in the lower chamber was assessed by flow cytometry. (f) Quantification analysis of migrated neutrophils toward 100 ng/mL CXCL-8 after treatment with 0, 20 and 500 ng/mL LT for 2 or 8 h or (g) 0, 100 and 500 ng/mL pSTa for 2 or 8 h. n = 4 individual blood donors. The error bars represent the mean ± SD. The data of the binding assay were analyzed with a non-parametric Friedman test. The migration assay data were analyzed with One-way ANOVA with a posthoc Tukey test. A paired Student's T test was used to compare two groups with or without GM1 or CXCL8. *, # p < 0.05; **, ## p < 0.01, ns: not significant.

To eliminate pathogens, neutrophils must however first migrate to the site of infection. Thus, we investigated whether LT and pSTa might affect the migration of neutrophils. To exclude a potential effect of the enterotoxins LT and pSTa on the viability of neutrophils, a cell viability assay was performed. Both LT and pSTa up to 500 ng/mL did not decrease the viability of neutrophils upon incubation for 2 or 8 h (Fig. S5). We then examined whether LT and pSTa could induce neutrophil migration in a transwell migration assay. To this end, neutrophils were added to the apical compartment of the transwells, while LT or pSTa were added to the basolateral compartment (Figure 1c). Upon incubation, neutrophils that migrated to the basolateral compartment were counted by flow cytometry. While pSTa did not influence neutrophil migration, LT clearly induced migration of neutrophils in a dose-dependent manner (Figure 1d). We also assessed whether LT and pSTa might influence the migration induced by CXCL-8, a potent chemotactic cue for neutrophils.25 Neutrophils were pretreated with LT or pSTa for 2 and 8 h and then added to the apical compartment of the transwells, while CXCL-8 was added to the basolateral compartment (Figure 1e). After incubation, neutrophils that migrated to the basolateral compartment were counted by flow cytometry. The results indicated that LT and pSTa did not affect the CXCL-8-dependent migration of neutrophils (Figure 1f,g).

LT affects neutrophil phagocytosis without influencing ROS production, while pSta has no impact on neutrophil function

Neutrophils have potent antimicrobial effector functions, such as the production of ROS and phagocytosis. We next wondered whether LT and pSTa might also affect neutrophil effector functions. Neutrophils can generate reactive oxygen species (ROS) to destroy pathogens.16,26 However, to date it is still unclear whether LT and pSTa have an impact on neutrophil ROS production. Figure 2a clearly show that neither LT nor pSTa affected ROS production by neutrophils. Furthermore, treating neutrophils with LT or pSTa for 2 and 8 h did not influence ROS production by neutrophils induced by phorbol myristate acetate (PMA), a potent activator of neutrophils (Fig. S6). Figure 2. LT, but not pSta, decreases the phagocytosis ability of neutrophils.

(a) Neutrophils (2×105) were incubated with 0–500 ng/mL LT or 0–500 ng/mL pSTa for 2 h at 37°C and their ROS production was measured in a chemiluminescence assay. RLU: relative light units. (b) The gating strategy to assess uptake of pHrodoTM red E. coli by flow cytometry. (c) Representative histograms showing the phagocytosis of pHrodoTM red E. coli by neutrophils upon incubation with LT or (e) pSTa for 8 h at the indicated concentrations. (d) Phagocytosis of pHrodoTM red E. coli by neutrophils (2×105) after treatment with LT or (f) pSTa for 2 or 8 h at the indicated concentrations. n = 3 to 4 individual blood donors. The bars represent the mean ± SD. Data were analyzed with One-way ANOVA with a post hoc Tukey test to compare LT or pSTa treatment groups to the control group. **p < 0.01, ***p < 0.001. A paired Student's T test was used to compare two groups with or without E. coli. ###p < 0.001.

In addition to ROS production, phagocytosis by neutrophils is a crucial step to clear microbes, dead cells and damaged tissues.16,27 Here, pHrodo-labeled E. coli was used to evaluate the potential effects of LT and pSTa on the phagocytosis of bacteria by neutrophils. As shown in Figure 2b-d, LT significantly reduced the phagocytosis of E. coli by neutrophils, even at a concentration as low as 4 ng/mL. In contrast, pSTa did not change the ability of neutrophils to phagocytose E. coli (Figure 2e,f).

LT downregulates the cell surface expression of CD11b by neutrophils

In neutrophils, β2 integrins, composed of CD18 and CD11a or CD11b, regulate different cellular processes, such as cell migration, ROS production and phagocytosis.28,29 Given that LT induced neutrophil migration and decreased phagocytosis of bacteria by neutrophils, we investigated whether LT might induce changes in the cell surface expression levels of CD11b or CD11a of neutrophils. While CD11a expression levels were not altered in LT-stimulated neutrophils (Figure 3a), a 2 h stimulation of neutrophils with 500 ng/mL LT downregulated the expression of CD11b (Figure 3b). Furthermore, LT also downregulated CD11b surface expression in the presence of CXCL-8 (Figure 3c) and E. coli (Figure 3d). Figure 3. LT downregulated CD11b cell surface expression of neutrophils.

Neutrophils (2×105) were first pretreated with 0, 20 and 500 ng/mL LT for 2 or 8 h at 37°C. The expression of CD11a (a) and CD11b (b) was analyzed by flow cytometry. Neutrophils (2×105) were first pretreated with 0, 20 and 500 ng/mL LT for 2 h at 37°C and then incubated with 100 ng/mL CXCL-8 (c) or 6 × 106 pHrodoTM red E. coli (d) for another 2 h. The expression of CD11a was analyzed by flow cytometry. n = 3 to 4 individual blood donors. The bars represent the mean ± SD. Data were analyzed with One-way ANOVA with a posthoc Tukey test to compare LT treatment groups to the control group. *p < 0.05, **p < 0.01.

LT induces secretion of cytokines and chemokines by neutrophils

Upon recognition of pathogens, neutrophils secrete pro-inflammatory mediators to inform neighboring cells and attract other immune cells.18 To address whether LT affects the ability of neutrophils to produce these mediators, the mRNA expression of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α as well as the chemokine CXCL-8 by porcine neutrophils was assessed upon stimulation with LT. Upon a 2-hour exposure to LT, neutrophils upregulated the mRNA expression of IL-1β, TNF-α and CXCL-8 as compared to control cells (Figure 4a). As shown in Figure S7, LT also increased the mRNA expression of the chemokines CCL3 and CCL5 but decreased CCL2 transcript levels. Similar to the transcript levels, neutrophils secreted more IL-1β, TNF-α and CXCL-8 in response to LT, while IL-6 secretion levels were not influenced by LT (Figure 4b). Figure 4. Neutrophils respond to LT by increased production of pro-inflammatory cytokines and chemokines.

(a) Neutrophils (2×106) were treated with LT at the indicated concentrations for 2 h. IL-1β, IL-6, TNF-α and CXCL-8 transcript levels were evaluated by qPCR. (b) IL-1β, IL-6, CXCL-8 and TNF-α secretion levels were measured by ELISA in the culture supernatant of neutrophils (2×106) after treatment with LT at the indicated concentrations for 8 h. n = 3 to 4 individual blood donors. The bars represent the mean ± SD. Data were analyzed with One-way ANOVA with a posthoc Tukey test to compare LT treatment groups to the control group. *p < 0.05; **p < 0.01, ***p < 0.001.

LT promoted neutrophils to generate NETs in vitro.

A crucial effector function of neutrophils is the formation of neutrophil extracellular traps (NETs). These NETs are formed by expulsion of DNA and enzymes from the neutrophil.16 We thus examined NETs formation after LT treatment. The latter changed the morphology of the neutrophils from a round to an elongated shape in a time- and dose-dependent manner (Figure 5a). This change in morphology resembles NETs formation. To confirm this, we quantified the presence of extracellular DNA in LT-treated neutrophils. As shown in Figure 5b, LT treatment increased the presence of extracellular DNA on neutrophils in a dose-dependent manner (Figure 5b,c). Furthermore, staining for the NET marker myeloperoxidase (MPO) further confirmed the generation of NETs by LT treated neutrophils (Figure 5d,e). While NETs are known to kill extracellular bacteria, previous studies have demonstrated that pathogenic bacteria can evade entrapment and killing by NETs.30 To understand how LT-induced NETs affect the growth of bacteria, we cultured an E. coli lab strain (HB101) and an ETEC strain (GIS26) in the presence of neutrophils after NETosis induction. As shown in Figure 5f, both PMA- and LT-induced NETs inhibited the growth of the ETEC strain GIS26, while only PMA-induced NETs inhibited the growth of the E. coli strain HB101. Figure 5. LT promoted neutrophils to generate NETs in vitro.

(a) Neutrophils (2×105) were incubated with varying concentrations of LT (0, 4, 20, 100 and 500 ng/mL) at 37°C. The morphology of the neutrophils was observed under 200× magnification at 3, 6 and 9 h. Representative images of neutrophil morphology (n = 4). (b) Neutrophils (2×105) were incubated with 0, 20 and 500 ng/mL LT for 8 h. Extracellular DNA was stained with Sytox Green and intracellular DNA with Syto Red. Representative images are shown (n = 4). Scale bars, 100 μm. (c) Quantification of fluorescence intensity of Sytox green signal. (d) Neutrophils (2×106) were incubated with 0, 20 and 500 ng/mL LT for 8 h and stained for MPO. Representative confocal microscopy images of NETs (DNA: blue; MPO, green). Scale bars, 10 μm. (e) Quantification of the percentage of NETs in all observed neutrophils. (f) Neutrophils were pretreated with 500 ng/mL LT or 5 μg/mL PMA for 8 h, or pretreated with 10 µg/mL cytochalasin D (Cyto D) for 20 min. The E. coli strain HB101 and the ETEC strain GIS26 (2×106 CFU/ml) were grown for 2 h at 37°C in the presence of neutrophils. The OD600 was measured every 20 min. n = 3 to 6 individual blood donors. The bars represent the mean ± SD. Data of NETs formation were analyzed with one-way ANOVA with a posthoc Tukey test to compare LT treatment groups to the control group. *p < 0.05, **p < 0.01, ***p < 0.01. A paired Student's T test was used to compare two groups with or without PMA. ###, p < 0.001. Data of antimicrobial activity of NETs were analyzed with a paired Student’s T test to compare treatment groups to the control group at the 120 min timepoint. *p < 0.05; **p < 0.01.

LT augments intracellular cAMP levels of neutrophils and activates PKA signaling.

In intestinal epithelial cells, binding of LT to GM1 triggers uptake of LT, which in turn elicits increased cAMP levels and activation of PKA.4 Whether LT also activates the cAMP/PKA pathway in neutrophils was still unknown. Interestingly, we found that intracellular cAMP levels in neutrophils were elevated upon stimulation with LT for 90 min, while extracellular cAMP levels were not affected (Figure 6a). Subsequently, we examined additional PKA activation by quantifying the levels of both PKA and phosphorylated PKA. The results showed that LT did not change total PKA levels, nor increased the phosphorylation of PKA in porcine neutrophils (Figure 6b). This seems to indicate that the LT-induced increase in cAMP levels suffices to activate PKA in neutrophils. cAMP-dependent PKA belongs to the Arg-directed kinases or AGC kinase family.31 These serine/threonine protein kinases share a common recognition site characterized by the presence of an Arg at position −3 relative to the Ser/Thr. To investigate the potential activation of PKA upon LT stimulation of neutrophils, a phospho-PKA substrate antibody was used to evaluate the presence of phosphorylated PKA substrates. Upon 2 and 8 h stimulation of neutrophils with LT the levels of proteins phosphorylated at the AGC kinase recognition site were increased (Figure 6c). In an effort to confirm a role for PKA, neutrophils were pre-treated with the PKA inhibitor H89. The results indicated that PKA inhibition reduced the levels of phosphorylated proteins induced by stimulation of neutrophils with LT for 2 h. This reduction was not observed when neutrophils were stimulated with LT for 8 h (Figure 6d, S8). The AGC kinase family contains many members. Since inhibiting PKA did not completely prevent phosphorylation of target proteins, other kinases such as Akt or PKC might also be involved. To address this possibility, we pre-treated the neutrophils with the Akt inhibitor XI or the PKC inhibitor Go6983 and then evaluated the levels of phosphorylated substrates in the neutrophils upon LT stimulation. Inhibition of Akt and PKC did not prevent the phosphorylation of substrates triggered by LT (Figure 6e-f, S8). Notably, as PKC regulates migration and phagocytosis of neutrophils, we further assessed PKC activation. Western blot analysis showed that stimulation with LT did not affect the levels of phosphorylated PKC (Figure 6g). Figure 6. LT induced activation of the cAMP/PKA signaling pathway in porcine neutrophils.

(a) The cAMP level in lysed neutrophils and neutrophil supernatant after treatment with 0, 20 and 500 ng/mL LT for 90 min. (b) The total PKA and phosphorylated PKA levels in neutrophils after treatment with LT for 2 or 8 h. (c) Representative immunoblots of substrates phosphorylated by PKA (phospho-PKA) in neutrophils after treatment with LT for 2 or 8 h. (d) Representative immunoblots of phospho-PKA substrates in neutrophils pre-treated with 10 μM H89 for 2 h, and then treated with LT for 2 or 8 h. (e) Representative immunoblot of phospho-PKA substrates in neutrophils pre-treated with the Akt inhibitor XI (10 μM) or (f) the PKC inhibitor Go6983 (10 μM) for 2 h, and then treated with LT for another 2 h. (g) The expression of β-actin and phosphorylated PKC in neutrophils after treatment with LT for 2 h or 8 h. n = 3 to 4 individual blood donors. The bars represent the mean ± SD. Data were analyzed with One-way ANOVA with a posthoc Tukey test to compare LT treatment groups to the control group. *p < 0.05, ***p < 0.01.

ERK1/2 is activated upon LT induced activation of PKA signaling.

In addition to the cAMP/PKA pathway, the activation of the ERK1/2 pathway in intestinal epithelial cells and dendritic cells has also been reported in studies investigating LT.32,33 Guided by these previous studies, we examined the activation status of ERK1/2 in neutrophils. Interestingly, western blot analysis indicated that LT induced ERK1/2 phosphorylation (Figure 7a), which was inhibited upon pretreatment of the neutrophils with the ERK1/2 inhibitor U0126 (Figure 7b). Previous research showed a crosstalk between the cAMP/PKA and ERK1/2 signaling pathways.34,35 Our results showed that the PKA inhibitor H89 inhibited the phosphorylation of ERK1/2 (Figure 7c). In addition, inhibition of ERK1/2 activity resulted in decreased levels of phosphorylated PKA substrates in neutrophils induced by LT (Figure 7d). Figure 7. LT induced ERK1/2 phosphorylation in neutrophils.

(a) Total ERK1/2 and phosphorylated ERK1/2 levels in neutrophils after treatment with LT for 2 or 8 h. (b) Total ERK1/2 and phosphorylated ERK1/2 levels in neutrophils pre-treated with 10 μM U0126 for 2 h, and then treated with LT for 2 h. (c) Protein levels of total ERK1/2 and phosphorylated ERK1/2 in neutrophils pre-treated with 10 μM H89 for 2 h, and then treated with LT for 2 h. (d) Representative immunoblot of phospho-PKA substrates in neutrophils pre-treated with 10 μM U0126 for 2 h, and then treated with LT for another 2 h. n = 3 to 4 individual blood donors. The bars represent the mean ± SD. Data were analyzed with One-way ANOVA with a posthoc Tukey test to compare LT treatment groups to the control group. *p < 0.05, **p < 0.01. A paired Student's T test was used to compare two groups with or without inhibitors. ###, < 0.01.

PKA and ERK1/2 signaling regulate neutrophil effector functions triggered by LT

Our results demonstrated that LT reduced phagocytosis of E. coli by neutrophils, triggered neutrophil migration and induced the production of inflammatory mediators by neutrophils. Concurrently, we showed that LT activated the cAMP/PKA pathway and ERK1/2 pathway in neutrophils. To determine whether the observed impact of LT on these effector functions correlates with the activation of these two pathways, neutrophils were pretreated with the PKA inhibitor H89 or the ERK1/2 inhibitor U0126. Upon LT stimulation, phagocytosis, migration, cytokine secretion and NET formation were assessed. Our findings demonstrated that H89 reduced the phagocytic activity of neutrophils in the absence of LT, while U0126 did not affect neutrophil phagocytosis (Figure 8a). Inhibiting PKA (H89) or ERK1/2 (U0126) signaling did not prevent the LT-induced reduction of bacterial phagocytosis by neutrophils. In contrast, pretreatment of neutrophils with H89 and U0126 decreased migration of neutrophils induced by LT, indicating that both PKA and ERK1/2 activation play an important role in LT-induced neutrophil migration (Figure 8b). We also investigated whether inhibiting PKA and ERK1/2 signaling might affect the LT-induced secretion of IL-1β, CXCL-8, and TNF-α. As shown in Figure 8c, pretreatment of neutrophils with H89 and U0126 significantly inhibited the LT-induced secretion of IL-1β and TNF-α by neutrophils. Of note, inhibiting PKA and ERK1/2 had opposing effects on the LT-mediated CXCL-8 secretion by neutrophils. While pretreatment with the PKA inhibitor H89 led to a significant increase in CXCL-8 secretion, pretreatment with the ERK1/2 inhibitor U0126 significantly decreased this. Furthermore, pretreatment of neutrophils with both H89 and U0126 resulted in nearly complete inhibition of the CXCL-8 secretion induced by LT (Figure 8d). Finally, we assessed whether PKA and ERK1/2 are involved in NET formation upon LT treatment of neutrophils. Our results showed that pretreatment with H89 or U0126 decreased NETs generation induced by LT (Figure 8e). Altogether, these data point to an interaction between LT-induced neutrophil effector functions and the activation of the cAMP/PKA/ERK pathway. Figure 8. Inhibition of PKA and ERK1/2 affected migration and inflammatory factor production induced by LT.

(a) Neutrophils (2×106) were pre-incubated with DMSO, 10 μM H89 or 10 μM U0126 for 2 h, and then treated with LT for another 2 h. Phagocytosis of pHrodoTM red E. coli by neutrophils was assessed by flow cytometry. (b) Neutrophils (2×106) were incubated with DMSO, 10 μM H89 or 10 μM U0126 for 2 h and neutrophil migration induced by LT was evaluated in a transwell assay as shown in Figure 1c. (c) Neutrophils (2×106) were pre-incubated with DMSO, 10 μM H89 or 10 μM U0126 for 2 h, and then treated with LT for another 8 h. IL-1β, TNF-α and CXCL-8 secretion levels were determined in neutrophil supernatant by ELISA. (d) Neutrophils (2×106) were pre-incubated with DMSO or 10 μM H89 and 10 μM U0126 for 2 h, and then treated with LT for another 8 h. CXCL-8 secretion levels were determined in neutrophil supernatant by ELISA. (e) Neutrophils (2×105) were pre-incubated with DMSO, 10 μM H89 or 10 μM U0126 for 2 h, and then treated with LT for another 2 or 8 h. NETs formation was determined by measuring the release of extracellular DNA. n = 3 to 4 individual blood donors. The bars represent the mean ± SD. Data were analyzed with One-way ANOVA with a posthoc Tukey test to compare LT treatment groups to the control group. *p < 0.05, **p < 0.01, ***p < 0.001. A paired Student's T test was used to compare two groups with or without inhibitors. #p < 0.05, ##p < 0.01, ###p < 0.001.

Discussion

ETEC causes diarrhea in humans and livestock species.4 While the pathways that finally result in diarrhea, triggered by the ETEC enterotoxins LT and ST in enterocytes, are well known, knowledge on the impact of these enterotoxins on neutrophils was completely lacking.6 As innate immune cells, neutrophils play a critical role in clearing bacterial infections and contribute to the initiation and regulation of adaptive immunity.16 Here, we addressed this knowledge gap and investigated whether LT and pSTa influence the effector functions of porcine neutrophils. Our findings revealed that pSTa did not affect neutrophil effector functions. In contrast, we showed that LT reduced the phagocytic capacity of neutrophils, increased neutrophil migration and NET formation and augmented the secretion of IL-1β, CXCL-8, and TNF-α, while ROS production was unaffected by this enterotoxin. Moreover, we demonstrate that the LT-induced migration and cytokine secretion by neutrophils can be attributed to the activation of a cAMP/PKA/ERK signaling pathway in these cells (Figure 9).Figure 9. Graphical summary of the results. The full lines indicate direct effects, while the dashed lines indicate indirect effects of LT on neutrophils.

In intestinal epithelial cells, LT binds to GM-1, resulting in its internalization. This uptake leads to a proteolytic cleavage of the A subunit into the A1 and A2 domains. The A1 domain contains the catalytic activity and irreversibly activates adenylate cyclase, which converts ATP in cAMP. When cAMP binds to the regulatory subunit of PKA, it triggers the dissociation of the catalytic subunits, leading to the phosphorylation of downstream target proteins and transcription factors.31,36 Our results indicate that a similar mechanism occurs in neutrophils. GM1 is present in the membrane of human neutrophils37 and adding GM-1 to LT blocked its binding to porcine neutrophils, suggesting that GM1 may serve as a receptor for LT on neutrophils. Notably, we observed an increase in intracellular cAMP levels in neutrophils after LT treatment, leading to PKA activation and subsequent phosphorylation of PKA substrates. In addition to the cAMP/PKA pathway, activation of ERK1/2 by LT has also been reported in epithelial cells as well as in dendritic cells.32,33,38,39 Similar to these studies, our results also show that LT activates the ERK1/2 signaling pathway in neutrophils. Like other studies showing a multifaceted interplay between these two signaling pathways,34,35,40 our data also point toward a crosstalk between the PKA and ERK1/2 signaling pathways, since the ERK1/2 inhibitor attenuated LT-induced activation of the cAMP/PKA pathway, and the PKA inhibitor reciprocally curtailed LT-induced activation of ERK1/2. Further research is needed to comprehensively elucidate the role of this interplay in the impact of LT on neutrophil effector functions.

Migration of neutrophils to inflamed or infected sites is regulated by chemokine gradients secreted by cells within or surrounding the infection site.25 Interestingly, LT can induce the secretion of CXCL-8 and IL33 by epithelial cells,12,33,41,42 suggesting that LT might affect the migration of neutrophils. For instance, intradermal administration of LT to mice recruits neutrophils to the skin.43 In addition to responding to chemokine gradients, a recent study showed that neutrophils can directly recognize and respond to bacterial toxins from S. aureus to migrate to the infection site.44 In our study, LT directly induced neutrophil migration, suggesting that neutrophil migration upon recognition of secreted bacterial products might be conserved across tissues and species. This migration of neutrophils toward LT involved PKA and ERK signaling. While ERK1/2 signaling has a known role in neutrophil migration,45,46 the impact of intracellular cAMP levels and PKA activation on neutrophil chemotaxis remains a topic of debate. Some studies suggested that the cAMP/PKA pathway plays a crucial role in neutrophil migration,47,48 while others proposed that its activation may hinder chemotaxis.45,49 The reason for this discrepancy remains unknown and further work will be necessary to fully define the role of these signaling pathways in neutrophil migration.

At the site of infection, neutrophils execute several antimicrobial activities to clear the threat, including phagocytosis and the production of superoxides.50 Here, we showed that neither LT nor pSTa induced ROS production by neutrophils, nor did they inhibit ROS production induced by a potent neutrophil activator (PMA). In contrast, LT diminished the ability of neutrophils to phagocytose E. coli, independent from PKA and ERK1/2 signaling. The reduced phagocytosis triggered by LT might be due to the reduced CD11b expression levels in LT treated neutrophils, as CD11b/CD18 plays an important role in phagocytosis by neutrophils.51,52 The LT-induced reduction of neutrophil phagocytosis is in line with a recent report showing that LT reduced the ability of macrophages to phagocytose ETEC, resulting in a heightened bacterial burden.15 These findings collectively suggest that ETEC uses LT to subvert phagocytosis by innate immune cells (neutrophils and macrophages) and as such may enhance the persistence of ETEC within the host. Notably, our findings indicated that the Akt inhibitor XI almost completely blocked neutrophil phagocytosis (Figure S9), indicating that Akt signaling plays a crucial role in regulating phagocytosis by neutrophils. Further research is needed to understand whether LT can inhibit Akt signaling to control neutrophil phagocytosis.

Neutrophils also combat pathogens by releasing NETs, a process called NETosis.16 NETs are web-like chromatin structures contain DNA, histones and various antimicrobial components such as antimicrobial peptides, myeloperoxidase, and cathepsin G17. While NETosis often leads to the death of neutrophils, sometimes neutrophils respond to pathogens with vital NETosis, in which the neutrophils stay alive, allowing them to continue performing their functions after releasing NETs.53 Here, we showed that LT the triggers the formation of NETs. Since neutrophil viability remained unaffected by LT treatment, this seems to indicate that LT induces NET formation via the vital NETosis pathway.54 Further experiments are warranted to confirm this. The mechanisms of NETs formation are incompletely understood. While some research indicates that the ROS pathway plays a critical role in NETosis, other studies have shown that vital NETosis can occur independently of ROS.17 In this study, LT treatment did not induce ROS production, suggesting that ROS might not be involved in the LT-induced NETs formation. While some studies pointed to a role for ERK1/2 in NETs formation,55,56 our results indicated that inhibition of ERK signaling did not prevent NETs formation induced by LT. This implies a role for alternate pathways in LT-induced NETs generation. Interestingly, activation of PKA can inhibit NETs formation.57,58 Here, we found that inhibiting PKA enhanced NETs formation in the absence of LT. This seems to indicate that PKA prevents NETosis in steady conditions and that LT activates PKA to dampen NET formation, at least temporarily. Although NETs are a defense mechanism against pathogens, some pathogenic bacteria can escape entrapment and killing by NETs and even use these NETs as a nutrient source.59,60 Our results showed that LT-induced NETs inhibited the growth of ETEC similarly to PMA, indicating that ETEC did not develop strategies to counteract the antimicrobial activity of NETs.

In addition to these antimicrobial activities, neutrophils can also release a spectrum of inflammatory mediators, including cytokines, chemokines, leukotrienes, and prostaglandins, which can subsequently regulate the activity of other immune cells and stromal cells.61 In this study, LT triggered an increased secretion of IL-1β, TNF-α and CXCL8 in neutrophils. Other innate immune cells like dendritic cells and monocytes also secrete pro-inflammatory cytokines upon contact with LT.32,62 When investigating the involved signaling pathways, our results illustrated that PKA and ERK1/2 signaling are involved in the LT-induced upregulation of IL-1β and TNF-α secretion by neutrophils. Although it is known that ERK activation can induce production of IL-1β and TNF-α,63 the influence of PKA activation on their production by neutrophils remains incompletely understood. While some studies showed that PKA activation induces production of IL-1β and TNF-α,64,65 others demonstrated that PKA activation inhibits IL-1β and TNF-α production.66,67 A possible explanation for the conflicting results may result from the different stimulus and treatment time used in the different cellular contexts. In the context of CXCL-8 secretion, our findings suggest that PKA and ERK1/2 signaling have opposing functions in the response of neutrophils to LT. More specifically, the PKA signaling cascade activated by LT controls CXCL-8 secretion by neutrophils and also activates ERK1/2, which then attenuates the magnitude of the CXCL-8 response.

Our experiments show that the enterotoxin LT affects neutrophil effector functions in part through the cAMP/PKA/ERK signaling pathway using validated inhibitors. Nevertheless, a main limitation to this study is the lack of knockout and overexpression data to elucidate the role of key proteins in these LT-induced changes in neutrophil effector functions. Neutrophils are terminally differentiated cells and do not survive long both in circulation and in culture. This short lifespan of primary neutrophils limits the feasibility of generating gene deletion and overexpression mutants in these cells with current technologies. Future research should focus on developing innovative methods and tools, like conditional neutrophil-specific transgenic pigs, to enable this research in large animal models.

In conclusion, while the heat stable enterotoxin pSTa did not affect neutrophil effector functions, ETEC-derived LT induced neutrophil migration and production of inflammatory mediators by activating the cAMP/PKA and ERK1/2 signaling pathways. This might aid in clearance of ETEC infections by neutrophils and other immune cells. However, LT shuts down neutrophil phagocytosis and dampens NET formation which may be advantageous for ETEC to establish and maintain infection in the gut.

Materials and methods

Enterotoxins

pSTa was synthesized by Bachem company (Bachem Holding). LT was purified from the supernatant of ETEC strain IMM07 (O147:K88, F4ac, LT+STa−STb+) as previously described68 with some modifications. The bacterial strain was grown in CAYE medium for 24 h at 37°C with vigorous stirring. Bacteria were pelleted by centrifuging the culture medium at 5000 g for 20 min at 4°C. Upon collection of the supernatant, ammonium sulfate (Sigma) was added to the supernatant until a 55% saturation level. Upon stirring overnight incubation at 4°C, the precipitated proteins were harvested by two rounds of centrifugation at 5000 g for 30 min at 4°C. The pellet was dissolved in 200 mL TEAN buffer (50 mM Tris-HCl (pH 7.5), l mM EDTA and 200 mM NaCl) and dialyzed against TEAN buffer overnight at 4°C. The resulting solution was loaded on a galactose agarose (Thermo Fisher Scientific) column at a flow rate of 0.5 mL/min. Unbound proteins were removed with 10 column volumes of TEAN buffer. The bound proteins were eluted with TEAN buffer containing 0.12 M galactose (Sigma) and then dialyzed against distilled water overnight at 4°C. Protein precipitates were removed by centrifugation at 12,000 g for 10 min, 4°C, and then pooled using a centrifugal filter (Millipore). The presence of LT in the elution fractions was determined with western blotting, while the purity of LT was determined by SDS-PAGE and a silver staining kit (Thermo Fisher Scientific). The concentration of LT was determined by GM1-ELISA using rabbit anti-heat labile toxin antibody (Abcam) as described (Wang et al., 2020). The purified LT was stored at 4°C until use.

Isolation of neutrophils

The neutrophils were isolated from blood as described previously.69 In brief, peripheral blood was collected from 10 to 24-week-old pigs via the jugular vein on heparin. After mixing with the same volume of RPMI 1640 medium (Gibco), neutrophils were isolated by density gradient centrifugation on a discontinuous Percoll gradient (68% and 75%, GE Healthcare). Neutrophils were resuspended at a density of 2 × 106 cells/mL in phenol red-free RPMI 1640 medium (Gibco) containing 10% fetal calf serum (FCS, Greiner) and 1% penicillin/streptomycin (Gibco). All animal experiments were approved by the animal care and ethics committee of the Faculty of Veterinary Medicine, Ghent University (EC2017/121 and EC2023/22).

cAMP and cGMP ELISA

The bioactivity of purified LT was determined by measuring the cAMP production by IPEC-J2 cells, which was originally isolated from jejunal epithelium of neonatal piglet. IPEC-J2 cells were maintained in DMEM/F12 medium supplemented with 5% FCS, 1% penicillin-streptomycin, 1% insulin-transferrin-selenium (ITS, Sigma), 2% L-glutamine and 5 ng/mL epidermal growth factor (EGF, Gibco) and incubated at 37°C, 5% CO2 and 95% humidity. The bioactivity of the synthesized pSTa (Bachem) was determined by measuring the cGMP production by T84 cells which was derived from a colon carcinoma in a 72-year-old man. T84 cells were cultured in DMEM/F12 medium supplemented with 5% FCS and 1% P/S and incubated at 37°C, 5% CO2 and 95% humidity. IPEC-J2 cells (1 × 105 cells/well) and T84 cells (2 × 105 cells/well) were seeded into 24-well plates and cultured for two days to reach at least 90% confluence. LT (0, 20, 100, 500 ng/mL) was added to IPEC-J2 cells, while pSTa (0, 100 and 500 ng/mL) was added to the T84 cells. The cells were subsequently incubated for 1 h at 37°C, 5% CO2 and 95% humidity. After removal of the culture medium, the cells were lysed with 0.1 M HCl to stop endogenous phosphodiesterase activity. Upon centrifugation at 660 g for 10 min at room temperature to remove cellular debris, the IPEC-J2 lysate was assayed for cAMP using a Direct cAMP ELISA Kit (Enzo Life Sciences) and the T84 lysate was assayed for cGMP using a Direct cGMP ELISA Kit (Enzo Life Sciences), following the manufacturer’s guidelines.

Neutrophils were seeded at 2 × 106 cells/well in 24-well plates. After 1 h incubation, LT (0, 100 and 500 ng/mL) was added to the culture medium and incubated at 37°C for 10 or 90 min. Next, the neutrophils were collected and centrifuged at 400 g for 5 min, 4°C. The supernatant was transferred into a new tube and the cells were lysed with 300 μL 0.1 M HCl. The cAMP and cGMP levels in the supernatant and cell lysates were then quantified using a Direct cAMP or cGMP ELISA Kit according to the manufacturer’s instructions.

Swelling assay in gut organoids

Small intestinal crypts were isolated from 8-week-old piglets and maintained until enteroids could be passaged. These enteroids were then used to perform a swelling assay as previously described.70 Briefly, enteroid fragments were cultured in 6 μL Matrigel (growth factor reduced, Corning) containing 1 μL organoids growth medium (OGM, 1:1 mix of OGM human basal medium and organoid supplement, Stem Cell Technologies) and 10 μM Y-inhibitor (Sigma) as well as 50 μL OGM medium for two days until small enteroids developed. Then, LT (100 ng/mL) or pSTa (1 μg/mL) were added to the culture medium. The enteroids were monitored for three hours using a live-cell microscope (Olympus IX81) with controlled temperature (37 °C) and CO2 (5%). Five to ten enteroids were selected at random and every 10 min the enteroids were imaged. The resulting time-lapse was analyzed with ImageJ. At the 80 min and 180 min timepoint, the surface area of the enteroids was measured manually. The relative increase in surface area at the indicate timepoint was then calculated by dividing the area measured at that timepoint by the area of the initial state (T = 0).

LT binding assay

The binding of LT to the membrane of neutrophils was measured by flow cytometry. Neutrophils were seeded at 2 × 105 cells/well in 96-well plates and incubated at 37°C, 5% CO2 for 1 h. Then, neutrophils were incubated with 0, 20 or 500 ng/mL LT for another 30 min on ice. After incubation, neutrophils were washed 3 times with ice-cold PBS and were incubated for 3 h on ice with rabbit anti- E. coli LT polyclonal antibody (1:300, Abcam) in ice-cold PBS with 1% FCS. Upon three washes with ice-cold PBS, neutrophils were stained for 1 h on ice with PE-labeled goat anti-rabbit lgG polyclonal antibody (1:300, Invitrogen) in ice-cold PBS. Upon washing 3 times, neutrophils were stained with the viability dye SytoxTM Blue (1 μM, Thermo Fisher Scientific) in PBS and analyzed by flow cytometry (Cytoflex, Beckman Coulter). The data were analyzed using CytExpert software (Beckman Coulter). For the GM1 inhibition assay, 0, 25 or 500 ng LT was preincubated with 1 μg GM1 (Sigma) for 2 h at 37°C before adding to the neutrophils.

Cell viability assay

The viability of the neutrophils upon enterotoxin stimulation was measured by flow cytometry (Cytoflex, Beckman Coulter). Neutrophils were seeded at 2 × 105 cells/well in 96-well plates and incubated at 37°C and 5% CO2 for 1 h. Then, neutrophils were treated with various concentrations of LT (0, 4, 20, 100 and 500 ng/mL) or pSTa (0, 100, 200, 300, 400 and 500 ng/mL) for another 4 or 10 h. After toxin treatment, neutrophils were harvested, transferred to 96-well V-bottom plates and stained with 1 μg/mL propidium iodide (PI, Sigma) on ice for 5 min. Upon doublet discrimination, neutrophils were selected based on their FSC-A/SSC-A properties. Live/dead discrimination was performed based on PI staining using a minimal event count of 10,000 neutrophils. The data were analyzed using CytExpert software (Beckman Coulter).

Analysis of reactive oxygen species production

A luminol chemiluminescence assay was performed to evaluate the ROS production by enterotoxin-stimulated neutrophils as described previously.69 Neutrophils (2×105 cells/well) were seeded in a 96-well white microplate and incubated for 1 h at 37°C, 5% CO2. The culture medium was then replaced by luminol buffer (100 μg/mL) and the background levels were measured. Then, LT (0, 32, 160, 800, 4000 ng/mL) or pSTa (0, 800 and 4000 ng/mL) was added and the chemiluminescence was continuously measured every 5 min for a period of 2 h at 37°C using a microplate reader (MTX lab system). Neutrophils stimulated with 50 μg/mL PMA (Sigma) were used as a positive control. To determine the effect of LT and pSTa on the ROS production induced by PMA, the neutrophils were pretreated with LT (0, 4, 20, 100, 500 ng/mL) or pSTa (0, 4, 20, 100, 500 ng/mL) for 2 or 8 h and then stimulated with PMA. Chemiluminescence was monitored as described above.

Chemotaxis assay

Neutrophil chemotaxis was assessed using polycarbonate transwell inserts (6.5 mm diameter, 3 μm pore size, Falcon). To detect neutrophil migration induced by LT or pSTa, neutrophils (3×105 cells) were added to the upper chamber and culture medium containing LT (20 and 500 ng/mL) or pSTa (100 and 500 ng/mL) was added to the bottom chamber. Culture medium without enterotoxin was used as a negative control, while recombinant porcine CXCL-8 (100 ng/mL; R&D systems) was used as a positive control. Upon 3 h, neutrophils in the lower chamber were collected and stained with propidium iodide (1 μg/mL in PBS). The number of cells was determined by flow cytometry (Beckman Coulter), and the data were analyzed using CytExpert software (Beckman Coulter). To evaluate whether LT and pSTa influence the migration of neutrophils induced by CXCL-8, neutrophils (3×105) were pretreated with various concentrations of LT or pSTa at 37°C, 5% CO2 for 2 or 8 h. The pretreated neutrophils were then transferred to the upper chamber, and culture medium containing 100 ng/mL CXCL-8 was added to the bottom chamber. Culture medium without CXCL-8 was used as a control. After 3 h of incubation, the number of neutrophils in the lower chamber was determined as described above. To evaluate the role of PKA or ERK1/2 signaling in LT-induced neutrophil chemotaxis, neutrophils were preincubated with diluent control (DMSO), the PKA inhibitor H89 (10 μM, Sigma) or the ERK1/2 inhibitor U0126 (10 μM, Cell signaling Technology) for 2 h at 37 before conducting the chemotaxis assay. The used concentrations of H89 and U0126 did not affect neutrophil viability.

Phagocytosis assay

Neutrophils (2 × 105 cells/well) were pretreated with LT (0, 4, 20, 100, 500 ng/mL) or pSTa (0, 100 and 500 ng/mL) for 2 or 8 h. After incubation, pHrodoTM red E. coli (6 × 106 particles, Thermo Fisher Scientific) were added to the cells and incubated at 37°C for 2 h. Subsequently, neutrophils were collected and washed 3 times with cold PBS to remove unbound particles. Finally, neutrophils were stained with SytoxTM Blue (1 μM, Thermo Fisher Scientific) and analyzed by flow cytometry (Cytoflex, Beckman Coulter). The data were analyzed using CytExpert software (Beckman Coulter). For the inhibition assay, the neutrophils were preincubated with 10 μM H89 or U0126 for 2 h at 37°C before LT was added to neutrophils.

Analysis of CD11a and CD11b expression

To check the cell surface expression levels of CD11a and CD11b by neutrophils, neutrophils (2×105 cells/well) were treated with LT (0, 20 and 500 ng/mL) for 2 or 8 h. After this treatment, the neutrophils were stained with FITC-conjugated anti-porcine CD11a antibody (1:10 dilution, Bio-Rad) or FITC-conjugated anti-porcine CD11b (1:10 dilution, Abcam) antibody in PBS + 1%FCS for 40 min on ice. Upon washing to remove any unbound antibodies, the neutrophils were stained with 1 μM SytoxTM Blue and measured using flow cytometry (Cytoflex, Beckman Coulter). The data were analyzed using CytExpert software (Beckman Coulter).

To check the cell surface expression levels of CD11b in absence of CXCL-8 and E. coli, neutrophils (2×105 cells/well) were pretreated with LT (0, 20 and 500 ng/mL) for 2 h. After pretreatment, neutrophils were incubated with CXCL-8 (1 μg/mL) or pHrodoTM red E. coli (6×106 particles) at 37°C for another 2 h. Then, neutrophils were washed 3 times with cold PBS to remove excess CXCL-8 or E. coli and stained with FITC-conjugated anti-porcine CD11a antibody (1:10 dilution, Bio-Rad) or FITC-conjugated anti-porcine CD11b (1:10 dilution, Abcam) antibody in PBS + 1%FCS for 40 min on ice. Upon washing to remove any unbound antibodies, the neutrophils were stained with 1 μM SytoxTM Blue and measured using flow cytometry (Cytoflex, Beckman Coulter). The data were analyzed using CytExpert software (Beckman Coulter).

qPCR

Neutrophils were treated with different concentrations of LT for 2 or 8 h. Next, neutrophils were collected and total RNA was isolated using the Qia Shredder and RNeasy Mini Kit (Qiagen) according to the manufacturer’s guidelines. The RNA concentration and purity were determined by microvolume UV-Vis spectrophotometry (DeNovix, Wilmington, DE, USA) and the RNA integrity was evaluated by agarose gel electrophoresis. RNA (500 ng) was treated with RQ1 RNase-Free DNase (Promega) and subsequently reverse transcribed into cDNA using SuperScript III Reverse Transcriptase kit (Invitrogen) in the presence of a recombinant ribonuclease inhibitor (RNase OUT; Invitrogen) according to the manufacturer’s instructions. The resulting cDNA served as a template for the qPCR assay. Primers (table S1) were designed with Primer-BLAST (NIH, USA) or taken from literature and synthesized by Integrated DNA Technologies (IDT, Coralville, IA). Quantitative PCR was performed using 25 ng cDNA template at 60°C annealing temperature using a StepOnePlus real-time PCR system (Applied Biosystems) with SYBR green master mix (Applied Biosystems), following the protocol provided by the manufacturer. The final volume of qPCR mix is 20 μL. The cycle conditions were 1 cycle of 10 min at 95°C and 40 cycles of 15 s at 95°C, 30 s at the annealing temperature (60°C), and 30 s at 72°C. After the cycles, the melt curve analysis was performed. The results were analyzed by the double delta threshold cycle method and normalized to the expression level of the reference genes (β-actin and GAPDH) and to the control condition. Reference genes were selected based on geNorm analysis using qBase+ software.

Cytokine and chemokine secretion

Neutrophils (2 × 106 cells) were treated with different concentrations of LT for 8 h, upon which the supernatant was collected and centrifuged at 400 g for 5 min. The IL-6, CXCL-8 and TNF-α concentration in the cell-free supernatant were measured using commercial DuoSet ELISA kits (R&D systems) according to the manufacturer’s instructions. For the role of PKA and ERK1/2 signaling in LT-induced cytokine secretion, neutrophils were preincubated with diluent control (DMSO), 10 μM H89 or U0126 for 2 h at 37°C before LT was added to the neutrophils.

Quantifying NETs formation

Neutrophils were seeded in 96-well plates (2×105 cells/well), incubated at 37°C with 5% CO2 for 1 h and then stimulated with various concentrations of LT (0, 4, 20, 100, and 500 ng/mL). Cells were imaged using a Thunder microscope upon 3, 6, and 9 h of incubation.

NETs formation was quantified by detecting DNA release from the neutrophils. Neutrophils (2×105 cells/well) were incubated at 37°C with 5% CO2 for 1 h and then treated with 0, 20, and 500 ng/mL LT for an additional 8 h. PMA (5 μg/mL) was included as a positive control. For the inhibition assay, neutrophils were preincubated with 10 μM H89 or U0126 for 2 h at 37°C before LT was added to neutrophils. The neutrophils were then stained with Sytox Green (1 μM, Invitrogen) for 15 min at 37°C, followed by a Syto Red stain (1 μM, Invitrogen) for another 15 min at 37°C. Images were acquired using a Thunder microscope (Leica) and analyzed using Image J software to quantify NETs formation.

Confocal microscopy to examine NETs formation

Neutrophils were seeded (2×106 cells/well) on poly-L-lysine-coated coverslips and allowed to rest for 1 h at 37°C. Subsequently, the neutrophils were treated with varying concentrations of LT (0, 20, and 500 ng/mL) or PMA (5 μg/mL) for an additional 8 h. Following treatment, the neutrophils were fixed with 4% paraformaldehyde in PBS for 10 min at 37°C and then permeabilized with 0.1% Triton X-100 in PBS for 20 min. Upon three washes with PBS and blocking with 1% BSA containing 5% goat serum in PBS for 2 h at 37°C, the cells were stained overnight at 4°C with rabbit anti-MPO antibodies (diluted 1:200, R&D systems). Upon washing to remove unbound antibodies, the neutrophils were stained with FITC-conjugated goat anti-rabbit IgG (diluted 1:200, Sigma) antibodies for 2 h at 37°C. Upon washing, nuclei were counterstained with Hoechst 33342 for 5 min at room temperature. Finally, the neutrophils were washed and mounted to observe MPO using confocal microscopy (Leica). The acquired images were analyzed using Image J software.

Antimicrobial activity of NETs

The antimicrobial activity of NETs was measured following the method described in a previous study with some modifications.71 Neutrophils were seeded at 2 × 105 cells/well in 96-well plates and incubated at 37°C and 5% CO2 for 1 h. Then, neutrophils were treated with 0 or 500 ng/mL LT for another 8 h. PMA (5 μg/mL) was used as a positive control. As a negative control, neutrophils were preincubated with the phagocytosis inhibitor cytochalasin D (10 µg/ml, Sigma Aldrich, Taufkirchen) for 20 min. The neutrophil supernatant was then removed upon centrifugation (400 g, 5 min) and 100 μL RPMI 1640 containing the porcine ETEC strain GIS26 (F4+, LT+STa+STb+)23 or the E. coli lab strain HB101 at 2 × 106 CFU/mL was added to each well and incubated at 37°C for 120 min. Growth of the bacteria was monitored by measuring the OD600 at 20 min intervals (Tecan Spark).

Western blotting

To analyze phosphorylation of PKA, neutrophils (2×106 cells) were treated with LT (0, 20 or 500 ng/mL) for 2 or 8 h. Then, the cells were lysed using 60 μL RIPA buffer containing a protease and phosphatase inhibitor cocktail (1:100; Thermo Fisher Scientific) and EDTA (0.05 M; Thermo Fisher Scientific). Upon determining the protein concentration using a BCA kit (Thermo Fisher Scientific), proteins were denatured by adding 10 μL 6× loading buffer, 3 μL β-mercaptoethanol and heating for 5 min at 95°C. Then, equal amounts of proteins were separated on a 10% SDS – PAGE and subsequently transferred onto PVDF membranes (Amersham). After blocking in PBS + 0.05% Tween 20 + 5% BSA for 1 h at room temperature, membranes were probed overnight at 4°C with rabbit polyclonal anti-PKA C-α (1:1000, Cell signaling Technology), rabbit polyclonal anti-phospho- PKA C (Thr197, 1:500, Cell signaling Technology), rabbit monoclonal anti-phospho-PKA substrate (1:500, Cell signaling Technology), rabbit monoclonal anti-phospho-PKC (gamma Thr514, 1:500, Cell signaling Technology), rabbit polyclonal anti-p44/42 MAPK (1:1000, Cell signaling Technology), rabbit polyclonal anti-phospho-p44/42 MAPK (Thr202/Tyr204, 1:300, Cell signaling Technology) or rabbit monoclonal anti-β-actin (1:1000; Cell Signaling Technology) antibodies. Then, membranes were washed 3 times and incubated with HRP-conjugated porcine anti-rabbit IgG antibody (1:1000, Dako) for 1 h at room temperature. After washing 3 times, protein bands were visualized using a SuperSignalTM ultimate sensitivity chemiluminescent substrate kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The relative band intensities were determined using Image Lab software (Bio-Rad). To evaluate the role of certain signaling pathways, the neutrophils were preincubated with diluent control 10 μM H89, Akt inhibitor XI (Santa Cruz Biotechnology), Gö 6983 (MedChemExpress) or U0126 for 2 h at 37°C before LT was added.

Statistical analysis

Statistical analysis was performed with IBM SPSS Statistic 26 (USA). Homogeneity of variances was assessed with Levene’s test. Student’s T test, Friedman test or one-way ANOVA with posthoc Tukey test were used for statistical analysis as indicated in the figure legends. Data are presented as the mean ± standard deviation (SD). A p-value <0.05 was considered significant.

Supplementary Material

Supplemental Material

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

J.M. designed and carried out the experiments, performed data analysis and drafted the manuscript. L.H. assisted in collecting blood, flow cytometry and RT-qPCR. M.D. assisted in purifying LT and western blotting. H.V.d.W. assisted in ELISA, Western blotting and confocal microscopy. E.C. supervised the project and provided critical feedback. B.D. conceived the presented idea, designed the experiments, supervised the project, provided critical feedback and drafted the manuscript. All authors reviewed the manuscript.

Data availablity

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Ethics declarations

The authors declare no competing interests.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2024.2399215
==== Refs
References

1. Anderson JD, Bagamian KH, Muhib F, Amaya MP, Laytner LA, Wierzba T, Rheingans R. Burden of enterotoxigenic and shigella non-fatal diarrhoeal infections in 79 low-income and lower middle-income countries: a modelling analysis. Lancet Glob Health. 2019;7 (3 ):E321–23. doi:10.1016/S2214-109x(18)30483-2.30784633
2. Luppi A, Gibellini M, Gin T, Vangroenweghe F, Vandenbroucke V, Bauerfeind R, Bonilauri P, Labarque G, Hidalgo Á. Prevalence of virulence factors in enterotoxigenic Escherichia coli isolated from pigs with post-weaning diarrhoea in Europe. Porcine Health Manag. 2016;2 (1 ):20. doi:10.1186/s40813-016-0039-9.28405446
3. Taylor DN, Hamer DH, Shlim DR. Medications for the prevention and treatment of travellers’ diarrhea. J Travel Med. 2017;24 :S17–S22. doi:10.1093/jtm/taw097.28520998
4. Dubreuil JD, Isaacson RE, Schifferli DM, Donnenberg MS. Animal enterotoxigenic Escherichia coli. EcoSal Plus. 2016;7 (1 ). doi:10.1128/ecosalplus.ESP-0006-2016.
5. von Mentzer A, Svennerholm AM. Colonization factors of human and animal-specific enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 2023;32 (5 ):448–464. doi:10.1016/j.tim.2023.11.001.38052687
6. Wang HX, Zhong ZF, Luo Y, Cox E, Devriendt B. Heat-stable Enterotoxins of Enterotoxigenic Escherichia coli and their impact on Host immunity. Toxins. 2019;11 (1 ):24. doi:10.3390/toxins11010024.30626031
7. O’Neal CJ, Jobling MG, Holmes RK, Hol WGJ. Structural basis for the activation of cholera toxin by human ARF6-GTP. Science. 2005;309 (5737 ):1093–1096. doi:10.1126/science.1113398.16099990
8. Viswanathan VK, Hodges K, Hecht G. Enteric infection meets intestinal function: how bacterial pathogens cause diarrhoea. Nat Rev Microbiol. 2009;7 (2 ):110–119. doi:10.1038/nrmicro2053.19116615
9. Weiglmeier PR, Rosch P, Berkner H. Cure and curse: E-coli heat-stable enterotoxin and its receptor guanylyl cyclase C. Toxins. 2010;2 (9 ):2213–2229. doi:10.3390/toxins2092213.22069681
10. Devriendt B, Stuyven E, Verdonck F, Goddeeris BM, Cox E. Enterotoxigenic (K88) induce proinflammatory responses in porcine intestinal epithelial cells. Dev And Comp Immunol. 2010;34 (11 ):1175–1182. doi:10.1016/j.dci.2010.06.009.20600278
11. Luo Y, Xu J, Zhang C, Jiang C, Ma Y, He H, Wu Y, Devriendt B, Cox E, Zhang H, et al. Toll-like receptor 5-mediated IL-17C expression in intestinal epithelial cells enhances epithelial host defense against F4 ETEC infection. Vet Res. 2019;50 (1 ). doi:10.1186/s13567-019-0665-8.
12. Motyka NI, Stewart SR, Hollifield IE, Kyllo TR, Mansfield JA, Norton EB, Clements JD, Bitoun JP. Elevated extracellular cGMP produced after exposure to Enterotoxigenic heat-stable toxin induces epithelial IL-33 release and alters intestinal immunity. Infect Immun. 2021;89 (4 ). doi:10.1128/IAI.00707-20.
13. Bauer DL, Bachnak L, Limbert VM, Horowitz RM, Baudier RL, D’Souza SJ, Immethun VE, Kurtz JR, Grant SB, McLachlan JB, et al. The adjuvant combination of dmLT and monophosphoryl lipid a activates the canonical, Nonpyroptotic NLRP3 Inflammasome in dendritic cells and significantly interacts to expand antigen-specific CD4 T cells. J Immunol. 2023;210 (10 ):1519–1530. doi:10.4049/jimmunol.2200221.37023458
14. Clements JD, Norton EB, Papasian CJ. The mucosal vaccine adjuvant LT(R192G/L211A) or dmLT. Msphere. 2018;3 (4 ):e00215–00218. doi:10.1128/mSphere.00215-18.30045966
15. Hollifield IE, Motyka NI, Fernando KA, Bitoun JP. Heat-labile enterotoxin decreases macrophage phagocytosis of Enterotoxigenic Escherichia coli. Microorganisms. 2023;11 (8 ):2121. doi:10.3390/microorganisms11082121.37630681
16. Burn GL, Foti A, Marsman G, Patel DF, Zychlinsky A. The neutrophil. Immunity. 2021;54 (7 ):1377–1391. doi:10.1016/j.immuni.2021.06.006.34260886
17. Papayannopoulos V. Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol. 2018;18 (2 ):134–147. doi:10.1038/nri.2017.105.28990587
18. Cassatella MA, Ostberg NK, Tamassia N, Soehnlein O. Biological roles of neutrophil-derived granule proteins and cytokines. Trends Immunol. 2019;40 (7 ):648–664. doi:10.1016/j.it.2019.05.003.31155315
19. Chin AC, Lee WY, Nusrat A, Vergnolle N, Parkos CA. Neutrophil-mediated activation of epithelial protease-activated receptors-1 and-2 regulates barrier function and transepithelial migration. J Immunol. 2008;181 (8 ):5702–5710. doi:10.4049/jimmunol.181.8.5702.18832729
20. Gul E, Enz U, Maurer L, Abi Younes A, Fattinger SA, Nguyen BD, Hausmann A, Furter M, Barthel M, Sellin ME, et al. Intraluminal neutrophils limit epithelium damage by reducing pathogen assault on intestinal epithelial cells during Salmonella gut infection. PloS Pathog. 2023;19 (6 ):e1011235. doi:10.1371/journal.ppat.1011235.37384776
21. Sumagin R, Robin AZ, Nusrat A, Parkos CA. Transmigrated neutrophils in the intestinal lumen engage ICAM-1 to regulate the epithelial barrier and neutrophil recruitment. Mucosal Immunol. 2014;7 (4 ):905–915. doi:10.1038/mi.2013.106.24345805
22. Rose R, Moon HW. Elicitation of enteroluminal neutrophils by enterotoxigenic and nonenterotoxigenic strains of Escherichia coli in swine. Infect Immun. 1985;48 (3 ):818–823. doi:10.1128/iai.48.3.818-823.1985.3888845
23. Wang HX, Garcia RS, Cox E, Devriendt B, Dudley EG. Porcine enterotoxigenic Escherichia coli strains differ in their capacity to secrete enterotoxins through varying YghG levels. Appl Environ Microb. 2020;86 (24 ):e00523–00520. doi:10.1128/AEM.00523-20.
24. Chatterjee A, Chowdhury R. Bile and unsaturated fatty acids inhibit the binding of cholera toxin and Escherichia coli heat-labile enterotoxin to GM1 receptor. Antimicrob Agents Ch. 2008;52 (1 ):220–224. doi:10.1128/Aac.01009-07.
25. de Oliveira S, Rosowski EE, Huttenlocher A. Neutrophil migration in infection and wound repair: going forward in reverse. Nat Rev Immunol. 2016;16 (6 ):378–391. doi:10.1038/nri.2016.49.27231052
26. El-Benna J, Hurtado‐Nedelec M, Marzaioli V, Marie J-C, Gougerot‐Pocidalo M-A, Dang PMC. Priming of the neutrophil respiratory burst: role in host defense and inflammation. Immunol Rev. 2016;273 (1 ):180–193. doi:10.1111/imr.12447.27558335
27. Mayadas TN, Cullere X, Lowell CA. The multifaceted functions of neutrophils. Annu Rev Pathol-Mech. 2014;9 (1 ):181–218. doi:10.1146/annurev-pathol-020712-164023.
28. Bouti P, Webbers SDS, Fagerholm SC, Alon R, Moser M, Matlung HL, Kuijpers TW. β2 integrin signaling cascade in neutrophils: more than a single function. Front Immunol. 2021;11 :619925. doi:10.3389/fimmu.2020.619925.33679708
29. Sekheri M, Othman A, Filep JG. β2 Integrin regulation of neutrophil functional plasticity and fate in the resolution of inflammation. Front Immunol. 2021;12 :660760. doi:10.3389/fimmu.2021.660760.33859651
30. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A. Neutrophil extracellular traps kill bacteria. Science. 2004;303 (5663 ):1532–1535. doi:10.1126/science.1092385.15001782
31. Ramms DJ, Raimondi F, Arang N, Herberg FW, Taylor SS, Gutkind JS. G α s–protein kinase a (PKA) pathway signalopathies: the emerging genetic landscape and therapeutic potential of human diseases driven by aberrant G α s-pka signaling. Pharmacol Rev. 2021;73 (4 ):155–197. doi:10.1124/pharmrev.120.000269.34663687
32. Brereton CF, Sutton CE, Ross PJ, Iwakura Y, Pizza M, Rappuoli R, Lavelle EC, Mills KHG. Escherichia coli heat-labile enterotoxin promotes protective Th17 responses against infection by driving innate IL-1 and IL-23 production. J Immunol. 2011;186 (10 ):5896–5906. doi:10.4049/jimmunol.1003789.21490151
33. Wang XG, Gao XF, Hardwidge PR. Heat-labile enterotoxin-induced activation of nf-κB and MAPK pathways in intestinal epithelial cells impacts enterotoxigenic Escherichia coli (ETEC) adherence. Cell Microbiol. 2012;14 (8 ):1231–1241. doi:10.1111/j.1462-5822.2012.01793.x.22452361
34. Stork PJS, Schmitt JM. Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol. 2002;12 (6 ):258–266. doi:10.1016/S0962-8924(02)02294-8.12074885
35. Werry TD, Sexton PM, Christopoulos A. ‘Ins and outs’ of seven-transmembrane receptor signalling to ERK. Trends Endocrin Met. 2005;16 (1 ):26–33. doi:10.1016/j.tem.2004.11.008.
36. Musheshe N, Schmidt M, Zaccolo MC. From long-range second Messenger to Nanodomain Signalling. Trends Pharmacol Sci. 2018;39 (2 ):209–222. doi:10.1016/j.tips.2017.11.006.29289379
37. Zhang T, de Waard AA, Wuhrer M, Spaapen RM. The role of glycosphingolipids in immune cell functions. Front Immunol. 2019;10 :90. doi:10.3389/fimmu.2019.00090.30761148
38. Bone H, Eckholdt S, Williams, Williams NA. Modulation of B lymphocyte signalling by the B subunit of Escherichia coli heat-labile enterotoxin. Int Immunol. N.A;14 (6 ):647–658. doi:10.1093/intimm/dxf029.12039916
39. Ye XL, Li PC, Yu QH, Yang Q. Bacillus subtilis inhibition of enterotoxic Escherichia coli-induced activation of MAPK signaling pathways in caco-2 cells. Ann Microbiol. 2013;63 (2 ):577–581. doi:10.1007/s13213-012-0506-8.
40. Godoy V, Banales JM, Medina JF, Pastor-Anglada M. Functional crosstalk between the adenosine transporter CNT3 and purinergic receptors in the biliary epithelia. J Hepatol. 2014;61 (6 ):1337–1343. doi:10.1016/j.jhep.2014.06.036.25034758
41. Read LT, Hahn RW, Thompson CC, Bauer DL, Norton EB, Clements JD. Simultaneous exposure to Escherichia coli heat-labile and heat-stable Enterotoxins increases fluid secretion and alters cyclic nucleotide and cytokine production by intestinal epithelial cells. Infect Immun. 2014;82 (12 ):5308–5316. doi:10.1128/Iai.02496-14.25287923
42. Motyka NI, Stewart SR, Porretta CP, Hollifield IE, Bauer DL, Bitoun JP. Enterotoxigenic Escherichia coli Enterotoxins regulate epithelial to immune Relay of IL-33 and IL-1Ra cytokines. Infect Immun. 2022;90 (3 ):e0063721. doi:10.1128/iai.00637-21.35191758
43. Greene CJ, Hu JC, Vance DJ, Rong Y, Mandell L, King-Lyons N, Masso-Welch P, Mantis NJ, Connell TD. Enhancement of humoral immunity by the type II heat-labile enterotoxin LT-IIb is dependent upon IL-6 and neutrophils. J Leukocyte Biol. 2016;100 (2 ):361–369. doi:10.1189/jlb.3A0415-153RR.27059843
44. Nguyen TH, Cheung GYC, Rigby KM, Kamenyeva O, Kabat J, Sturdevant DE, Villaruz AE, Liu R, Piewngam P, Porter AR, et al. Rapid pathogen-specific recruitment of immune effector cells in the skin by secreted toxins. Nat Microbiol. 2022;7 (1 ):62–72. doi:10.1038/s41564-021-01012-9.34873293
45. Mizuno R, Kamioka Y, Kabashima K, Imajo M, Sumiyama K, Nakasho E, Ito T, Hamazaki Y, Okuchi Y, Sakai Y, et al. In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines. J Exp Med. 2014;211 (6 ):1123–1136. doi:10.1084/jem.20132112.24842369
46. Sun CX, Wu MH, Guo M, Day ML, Lee ES, Yuan SY. ADAM15 regulates endothelial permeability and neutrophil migration via Src/ERK1/2 signalling. Cardiovasc Res. 2010;87 (2 ):348–355. doi:10.1093/cvr/cvq060.20189953
47. Cali B, Deygas M, Munari F, Marcuzzi E, Cassará A, Toffali L, Vetralla M, Bernard M, Piel M, Gagliano O, et al. Atypical CXCL12 signaling enhances neutrophil migration by modulating nuclear deformability. Sci Signal. 2022;15 (761 ):1552. doi:10.1126/scisignal.abk2552.
48. Watson RL, Buck J, Levin LR, Winger RC, Wang J, Arase H, Muller WA. Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration. J Exp Med. 2015;212 (7 ):1021–1041. doi:10.1084/jem.20150354.26101266
49. Insuela DBR, Ferrero MR, Gonçalves-de-Albuquerque CF, Chaves ADS, da Silva AYO, Castro-Faria-Neto HC, Simões RL, Barja-Fidalgo TC, Silva PMRE, Martins MA, et al. Glucagon reduces neutrophil migration and increases susceptibility to sepsis in diabetic mice. Front Immunol. 2021;12 :633540. doi:10.3389/fimmu.2021.633540.34295325
50. Naish E, Wood AJ, Stewart AP, Routledge M, Morris AC, Chilvers ER, Lodge KM. The formation and function of the neutrophil phagosome. Immunol Rev. 2023;314 (1 ):158–180. doi:10.1111/imr.13173.36440666
51. Hussen J, Shawaf T, Al-Mubarak AIA, Humam N, Almathen F, Schubert HJ. Leukocytes immunophenotype and phagocytosis activity in pregnant and nonpregnant dromedary she camels. Pak Vet J. 2020;40 (2 ):239–243. doi:10.29261/pakvetj/2019.117.
52. Le Cabec V, Carreno S, Moisand A, Bordier C, Maridonneau-Parini I. Complement receptor 3 (CD11b/CD18) mediates type I and type II phagocytosis during nonopsonic and opsonic phagocytosis, respectively. J Immunol. 2002;169 (4 ):2003–2009. doi:10.4049/jimmunol.169.4.2003.12165526
53. Jorch SK, Kubes P. An emerging role for neutrophil extracellular traps in noninfectious disease. Nat Med. 2017;23 (3 ):279–287. doi:10.1038/nm.4294.28267716
54. Yipp BG, Petri B, Salina D, Jenne CN, Scott BNV, Zbytnuik LD, Pittman K, Asaduzzaman M, Wu K, Meijndert HC, et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking. Nat Med. 2012;18 (9 ):1386–1393. doi:10.1038/nm.2847.22922410
55. Nie M, Yang L, Bi X, Wang Y, Sun P, Yang H, Liu P, Li Z, Xia Y, Jiang W, et al. Neutrophil extracellular traps induced by IL8 promote diffuse large B-cell lymphoma progression via the TLR9 signaling. Clin Cancer Res. 2019;25 (6 ):1867–1879. doi:10.1158/1078-0432.Ccr-18-1226.30446590
56. Douda DN, Khan MA, Grasemann H, Palaniyar N. SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx. P Natl Acad Sci USA. 2015;112 (9 ):2817–2822. doi:10.1073/pnas.1414055112.
57. Domingo-Gonzalez R, Martínez-Colón GJ, Smith AJ, Smith CK, Ballinger MN, Xia M, Murray S, Kaplan MJ, Yanik GA, Moore BB, et al. Inhibition of neutrophil extracellular trap formation after stem cell transplant by prostaglandin E 2. Am J Resp Crit Care. 2016;193 (2 ):186–197. doi:10.1164/rccm.201501-0161OC.
58. Zhao J, Liu Y, Shi X, Dang J, Liu Y, Li S, Cai W, Hou Y, Zeng D, Chen Y, et al. Infusion of GMSCs relieves autoimmune arthritis by suppressing the externalization of neutrophil extracellular traps via PGE2-PKA-ERK axis. J Adv Res. 2024;58 :79–91. doi:10.1016/j.jare.2023.05.001.37169220
59. de Buhr N, Bonilla MC, Pfeiffer J, Akhdar S, Schwennen C, Kahl BC, Waldmann K-H, Valentin-Weigand P, Hennig-Pauka I, von Köckritz-Blickwede M, et al. Degraded neutrophil extracellular traps promote the growth of actinobacillus pleuropneumoniae. Cell Death Dis. 2019;10 (9 ). doi:10.1038/s41419-019-1895-4.
60. Walker MJ, Hollands A, Sanderson-Smith ML, Cole JN, Kirk JK, Henningham A, McArthur JD, Dinkla K, Aziz RK, Kansal RG, et al. DNase Sda1 provides selection pressure for a switch to invasive group a streptococcal infection. Nat Med. 2007;13 (8 ):981–985. doi:10.1038/nm1612.17632528
61. Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011;11 (8 ):519–531. doi:10.1038/nri3024.21785456
62. Hajishengallis G, Tapping RI, Martin MH, Nawar H, Lyle EA, Russell MW, Connell TD. Toll-like receptor 2 mediates cellular activation by the B subunits of type II heat-labile enterotoxins. Infect Immun. 2005;73 (3 ):1343–1349. doi:10.1128/Iai.73.3.1343-1349.2005.15731031
63. Geest CR, Buitenhuis M, Groot Koerkamp MJA, Holstege FCP, Vellenga E, Coffer PJ. Tight control of MEK-ERK activation is essential in regulating proliferation, survival, and cytokine production of CD34-derived neutrophil progenitors. Blood. 2009;114 (16 ):3402–3412. doi:10.1182/blood-2008-08-175141.19667405
64. Liu XL, Huang D, Guo P, Wu Q, Dai M, Cheng G, Hao H, Xie S, Yuan Z, Wang X, et al. PKA/CREB and NF-κB pathway regulates AKNA transcription: a novel insight into T-2 toxin-induced inflammation and GH deficiency in GH3 cells. Toxicology. 2017;392 :81–95. doi:10.1016/j.tox.2017.10.013.29079362
65. Prince LR, Prosseda SD, Higgins K, Carlring J, Prestwich EC, Ogryzko NV, Rahman A, Basran A, Falciani F, Taylor P, et al. NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival. Blood. 2017;130 (8 ):1014–1025. doi:10.1182/blood-2017-03-770164.28637666
66. Bae GH, Kim YS, Park JY, Lee M, Lee SK, Kim JC, Kim JG, Shin YJ, Lee H, Kim S-Y, et al. Unique characteristics of lung-resident neutrophils are maintained by PGE/PKA/Tgm2-mediated signaling. Blood. 2022;140 (8 ):889–899. doi:10.1182/blood.2021014283.35679477
67. Hu X, Yan J, Huang L, Araujo C, Peng J, Gao L, Liu S, Tang J, Zuo G, Zhang JH, et al. INT-777 attenuates NLRP3-ASC inflammasome-mediated neuroinflammation via TGR5/cAMP/PKA signaling pathway after subarachnoid hemorrhage in rats. Brain Behav Immun. 2021;91 :587–600. doi:10.1016/j.bbi.2020.09.016.32961266
68. Grange PA, Parrish LA, Erickson AK. Expression of putative Escherichia coli heat-labile enterotoxin (LT) receptors on intestinal brush borders from pigs of different ages. Vet Res Commun. 2006;30 (1 ):57–71. doi:10.1007/s11259-005-3225-9.16362611
69. Baert K, Sonck E, Goddeeris BM, Devriendt B, Cox E. Cell type-specific differences in β-glucan recognition and signalling in porcine innate immune cells. Dev Comp Immunol. 2015;48 (1 ):192–203. doi:10.1016/j.dci.2014.10.005.25453580
70. Vermeire B, Gonzalez LM, Jansens RJJ, Cox E, Devriendt B. Correction to: porcine small intestinal organoids as a model to explore etec–host interactions in the gut. Vet Res. 2021;52 (1 ):107. doi:10.1186/s13567-021-00977-z.34344443
71. Bruns S, Kniemeyer O, Hasenberg M, Aimanianda V, Nietzsche S, Thywißen A, Jeron A, Latgé J-P, Brakhage AA, Gunzer M, et al. Production of extracellular traps against aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by Hydrophobin RodA. PloS Pathog. 2010;6 (4 ):e1000873. doi:10.1371/journal.ppat.1000873.20442864
