Immunomodulatory effect of metabolites secreted by the probiotic strain of E. coli O83:K24:H31

doi:10.21203/rs.3.rs-5306705/v1

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Immunomodulatory effect of metabolites secreted by the probiotic strain of E. coli O83:K24:H31

https://doi.org/10.21203/rs.3.rs-5306705/v1

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Bacteria and their metabolic products profoundly affect the immune system. Research has shown that early postnatal supplementation with specific probiotic strains, such as Escherichia coli O83:K24:H31 (EcO83), can provide health benefits. EcO83 is a facultative anaerobe capable of generating energy through multiple metabolic pathways, an adaptation that allows it to thrive in the gastrointestinal tract where oxygen availability can vary. Despite these advantages, the precise mechanisms through which this probiotic, and in particular its metabolites, functions remain largely unknown.

In this study, we aimed to identify the metabolites that EcO83 produces when cultured under both aerobic and anaerobic conditions. We also aimed to investigate the immunomodulatory effects of these metabolites on human peripheral blood mononuclear cells (PBMCs), mouse splenocytes, and bone marrow-derived dendritic cells (BMDCs) in vitro.

Our results revealed the presence of short-chain fatty acids (SCFA), namely acetate and propionate, in the supernatants of both aerobic and anaerobic EcO83 cultures. Interestingly, the supernatants from the aerobic cultures induced greater production of Th1 cytokines, such as IFN-γ, in PBMCs, whereas anaerobic culture supernatants more prominently triggered the Th2 cytokine IL-13. Similarly, murine splenocytes exhibited increased IFN-γ production when stimulated with aerobic supernatants. Additionally, both aerobic and anaerobic EcO83 supernatants promoted the release of proinflammatory (TNF-α) and anti-inflammatory (IL-10) cytokines from the BMDCs, as well as their maturation, evidenced by the upregulation of surface marker CD80.

In conclusion, we are the first to report that EcO83 produces SCFA, including acetate and propionate, under varying oxygen conditions. Our findings suggest that probiotics can exert beneficial effects through their secreted metabolites, not solely through the presence of the bacterial cells themselves, making them a promising postbiotic solution for therapeutic administration.

EcO83

SCFA

propionate

acetate

cytokines

PBMC

splenocytes

BMDC

probiotic

immunoregulation

The human body hosts a wide variety of microorganisms, collectively referred to as microbiota. Our relationship with these microbes begins at birth, with initial colonization occurring as we pass through the birth canal, establishing our early microbiota, and continues throughout postnatal life. Newborns have an underdeveloped immune system dominated by Th2 responses [1, 2]. Interestingly, those born via caesarean section tend to exhibit altered microbiota, which may lead to delayed immune system maturation and increased susceptibility to immune-mediated disorders [3]. As the immune system matures during early childhood, exposure to microbes after birth aids in its development, helping to balance the Th1 and Th2 branches and establishing proper regulatory mechanisms to manage relatively harmless environmental antigens, such as food and microbiome compounds [4–6]. As our understanding of the role of microbiota in immune function grows, it has become clear that the metabolites produced by the microbiota play a crucial part [7–9].

Administering probiotics is considered an effective strategy to promote immune system maturation and support the development of healthy microbiota. Numerous studies have shown that probiotics can aid in immune system development [10–12] and help prevent the onset of certain diseases [13, 14]. However, the therapeutic use of probiotics in patients with gut inflammation has produced inconsistent results [15, 16]. Significantly, the immunomodulatory effects of probiotics are highly strain-specific, as indicated by several studies exploring the potential of different bacterial strains [17, 18]. This underscores the importance of understanding how probiotics influence the immune system to improve the efficacy of probiotic supplementation in preventing immune-mediated inflammatory diseases [19].

Probiotic bacteria exert their effects on host cells either directly or through secreted metabolites. Numerous studies reflect the fact that short-chain fatty acids (SCFA) have become a key focus of immunological research due to their robust immunomodulatory effects [20, 21]. SCFA are particularly crucial in inducing regulatory functions by enhancing regulatory T cells, thus mitigating unwanted proinflammatory responses, such as reducing the severity of colitis in experimental mouse models [22, 23]. Furthermore, SCFA strengthen barrier functions, which may reduce the severity of inflammatory disorders, as reviewed by Pérez-Reytor et al. and Venegas et al. [24, 25]. Importantly, the immunoregulatory effects of SCFA administered to mothers can be passed on to their offspring, showing their ability to induce and maintain long-term epigenetic changes that support an anti-inflammatory state [26].

The strain Escherichia coli A0 34/86, serotype O83:K24:H31 (EcO83) is a Gram-negative (G-), a facultative anaerobic probiotic bacterium (Colinfant New Born, Dyntec, Czech Republic) marketed for use in newborns and infants at risk of nosocomial infection and diarrhea [27, 28]. EcO83 can grow in both oxygen-rich and oxygen-poor environments [29]. Under aerobic conditions, EcO83 utilizes respiration to generate energy, but it can switch to anaerobic metabolism when oxygen is unavailable, such as in the gut.

Our previous research demonstrated that early postnatal supplementation with EcO83 reduces allergy development in neonates [30–32]. Furthermore, we have shown that EcO83’s immunomodulatory effects involve dendritic cell maturation and the enhancement of immunoregulatory functions in both in vitro and in vivo settings [30, 33]. Recent findings also suggest that certain E. coli strains may produce SCFA [34, 35]. In this study, we first explore whether EcO83 secretes SCFA when cultured under both aerobic and anaerobic conditions. We then assess the immunomodulatory effects of supernatants containing SCFA and other metabolites from both aerobic and anaerobic cultures on human peripheral blood mononuclear cells (PBMC). Finally, we compare the immune-modulating effects of the whole EcO83 bacterium with those of the EcO83 supernatant on murine splenocytes. Additionally, we examine the maturation, cytokine expression, and secretion of murine bone marrow-derived dendritic cells (BMDCs) after exposure to EcO83 supernatants.

2.1. Bacteria cultivation and supernatant preparation

The probiotic strain of EcO83 was cultured in Luria Bertani broth (LB) for six hours, reaching the plateau phase of its exponential growth curve. The bacterial suspension was centrifuged at 10,000 x g for 10 minutes at room temperature (RT), and the supernatant was collected for further analysis. To stimulate the cells, the supernatants were diluted as follows: 10% of the total culture volume (200 µl, marked as AE/AN 200), 2% (40 µl, marked as AE/AN 40), 0.4% (8 µl, marked as AE/AN 8), and 0.08% (1.6 µl, marked as AE/AN 1.6). Supernatants produced under aerobic conditions were labeled AE, and those produced under anaerobic conditions were labeled AN.

2.2. Detection of SCFAs in EcO83 supernatants by NMR spectroscopy

The presence of SCFAs in EcO83 supernatants was determined using nuclear magnetic resonance (NMR) spectroscopy, as previously described [36]. Briefly, the supernatants were prepared from the bacterial culture by centrifugation, and the samples were then measured on a 600 MHz Bruker Avance III spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with a 5-mm TCI cryogenic probe head. The samples were analyzed using the manufacturer’s software, Topspin 3.5. The concentration of SCFAs acetate, propionate, and butyrate was identified by comparison of proton and carbon chemical shifts with the HMDB database and expressed as PQN-normalized intensities of corresponding signals in one-dimensional projections of J-resolved spectra.

2.3. Cell culture preparation

Murine and human leukocyte populations were used to test the immunomodulatory effects of metabolites produced by EcO83 on immune cells. All cell cultivations were maintained in RPMI 1640 medium (Sigma) supplemented by 10% fetal bovine serum (FBS, Biosera), glutamine (200 nmol, Sigma), HEPES (Sigma), and gentamycin (40 µg/ml) unless stated otherwise.

2.3.1. Human PBMCs

Peripheral blood mononuclear cells (PBMCs) were obtained from a buffy coat using gradient centrifugation, as previously described [30]. In brief, blood was diluted with PBS (1:1) and centrifuged for 30 minutes at 20°C on polysaccharide Ficoll-Paque gradient (Cytiva). The fraction containing mononuclear cells was collected, washed three times, counted, and then seeded at a concentration of 10⁶ cells/1ml in a total volume of 2 ml per well. PBMCs were stimulated with EcO83 and four different concentrations of bacterial cell culture supernatants (as described in Section 2.1). PBMCs were harvested after 4 hours for total RNA extraction, and supernatants from the cell cultures were collected after 24 hours. All samples were stored at -80°C until further processing. Buffy coats were provided by the Institute for Haematology and Blood Transfusion, Prague, Czech Republic, following signed written informed consent. The Ethical Committee of the General University Hospital in Prague approved the study on 30/06/2020.

2.3.2. Murine splenocytes

Splenocytes from BALB/c mice were isolated by mechanical disruption of the spleen tissue, followed by three washing steps. Cells were counted and seeded at a final concentration of 10⁶ cells/ml in 2 ml of supplemented RPMI 1640 medium. Various bacterial cell culture supernatant concentrations were tested (as described in Section 2.1). Cells were harvested after 4 hours for total RNA extraction. Supernatants from the cell cultures were collected after 24 hours and stored at -80°C until further processing.

2.3.3. Murine BMDCs

Bone marrow-derived dendritic cells (BMDCs) were prepared as previously described [17, 37]. In brief, bone marrow precursor cells were isolated from the femur and tibia, then cultured for seven days in RPMI medium (Lonza; supplemented as mentioned earlier) in the presence of recombinant murine growth factors rmIL-4 and rmGM-CSF (10µg/ml; Peprotech). On day 7, cells were harvested and seeded in 12-well plates (2 x 10⁶ cells/well in a total volume of 2 ml) for an additional three days, maintaining the growth factors. On day 10, BMDCs were stimulated with EcO83 in a ratio of 10 bacteria to one BMDC and varying concentrations of EcO83 supernatants (as detailed in section 2.1). After 4 hours, BMDCs were collected and centrifuged, and the total RNA was extracted for gene expression analysis of target genes using quantitative real-time PCR. Simultaneously, BMDCs were stimulated for 24 hours, and the influence of bacterial metabolites on BMDC maturation was examined via flow cytometry. Cytokine levels were measured from the supernatants of stimulated splenocytes using ELISA.

2.4. qRT-PCR

Total RNA was extracted from the cells using RNeasy Mini Kit (Qiagen) following the manufacturer’s recommendations. The extracted RNA was reverse transcribed using a High-Capacity RNA to cDNA kit (ThermoFisher). Gene expression of selected genes was quantified by quantitative real-time PCR, as previously described [2]. Specifically, TaqMan assays (ACTB Hs99999903_m1; IL2 Hs00174114_m1; IL10 Hs00174086_m1; IL13 Hs00174379_m1; IFNG Hs00174143_m1; Actb Mm00607939_s1; Il2 Mm00434256_m1; Il10 Mm00439616_m1; Il13 Mm99999190_m1; Ifng Mm00801778_m1; Tnfa Mm99999068_m1; all ThermoFisher) were used, and the results were normalized against the reference gene beta-actin. The ΔΔCt method was applied, and relative gene expression values were expressed as 2^{^−ΔΔCt}.

2.5. ELISA

Cytokine concentration in the cell culture supernatants was determined by ELISA, using reagents purchased from the R&D system. Cytokines were measured using DuoSet® ELISA kits (cat. no. DY402 for IL-2 detection and DY417 for IL-10 detection). Alternatively, a combination of capture antibodies, biotinylated secondary antibodies, and recombinant standard proteins was used (for IFN-γ: MAB785, 485-ML, BAF485 and for TNF-α: AF-410-NA, 410MT, BAF410). All samples were tested in duplicates, and concentrations were determined using calibration curves generated with KIM software (Schoeller).

2.4. Flow cytometry

The effect of metabolites produced by EcO83 on BMDC maturation was assessed by evaluating the expression of cell surface activation markers using flow cytometry. After 24-hour stimulation, BMDCs were stained with fluorochrome-labeled antibodies against CD11c PE-Cy7 (clone N417); CD80 FITC (clone 16-10A1); CD86 APC (clone GL1); MHCII PE (clone M5/11.15.1.) from eBioscience. The samples were then analyzed using BD FACS Canto II flow cytometer (Becton Dickinson), and the data was processed using FlowJo software (TreeStar).

2.6. Statistics

The data were graphically and statistically analyzed using GraphPad Prism software. Statistical significance was determined using non-parametric one-way ANOVA, followed by Dunnett's post-hoc multiple comparison test. A p-value of ≤ 0.05 was considered statistically significant.

3.1. EcO83 secretes SCFAs under both aerobic and anaerobic conditions

To evaluate the ability of EcO83 to produce SCFAs, bacterial cultures were grown under both aerobic and anaerobic conditions. After six hours of incubation, the EcO83 cultures were centrifuged, and the supernatants were collected. LB without EcO83 was used as a negative control. The presence of acetate and propionate was detected in both the aerobic and anaerobic cultures, while butyrate was not detected in any of the samples (Table 1). None of the analyzed SCFAs were detected in the media control (Table 1).

Table 1

Levels of SCFA in the EcO83 cultures under aerobic and anaerobic conditions
	Acetate (mM)	Propionate (mM)	Butyrate (nM)
Media control	not detectable	not detectable	not detectable
Aerobic culture	1.8	3.1	not detectable
Anaerobic culture	1.6	3.0	not detectable

3.2. Aerobic and anaerobic EcO83 supernatants differentially affect cytokine gene expression in human PBMCs

To evaluate how metabolites produced by EcO83 affect immune cells, we examined the expression of various Th1, Th2, and Treg cytokine genes in PBMCs following 4 hours of stimulation with different concentrations of aerobic and anaerobic EcO83 supernatants. PBMCs exposed to EcO83 cells served as a positive control, while those stimulated with LB broth (Medium) acted as a negative control. The highest concentration of supernatants from aerobic EcO83 cultures (10% = AE 200 and 2% = AE 40), but not 0.4% (AE 8) and 0.08% (AE 1.6), led to an increase in IL2 expression, similar to the EcO83 cells. Anaerobic EcO83 supernatants did not elevate the expression of this cytokine (Fig. 1A). Expression of IL10 and IL13 was increased only by AN 200 and AN 40 and by the stimulation with the EcO83 cells (Fig. 1B and C). Finally, EcO83 significantly upregulated IFNG expression, with aerobic supernatants showing a concentration-dependent increase and being more effective at enhancing IFNG expression than anaerobic supernatants, which had no effect (Fig. 1D).

3.3. PBMCs stimulated by both aerobic and anaerobic EcO83 supernatants show significant IL-10 production

To confirm the gene expression results and assess the effect of EcO83 supernatants on cytokine production, we measured cytokine levels in PBMC cultures after 24 hours of in vitro stimulation using ELISA. IL-1β, IL-2, IL-4, and IL-13 were undetectable in PBMCs treated with either aerobic or anaerobic EcO83 supernatants (data not shown). The ELISA results demonstrated that the positive control, whole EcO83 bacteria, triggered both IL-10 and IFN-γ production in the PBMCs (Fig. 2). Aerobic supernatants (AE 40, 8 and 1.6) triggered an IL-10 response, but not at the highest concentration (AE 200). All concentration of anaerobic supernatants, including the lowest (AN 1.6) increased IL-10 production (Fig. 2A). Anaerobic EcO83 supernatants had no effect on IFN-γ production, which remained low across all tested concentrations, whereas aerobic supernatants induced significant IFN-γ levels, but only at the highest concentration (AE 200) (Fig. 2B). These findings suggest that while both aerobic and anaerobic EcO83 supernatants promote IL-10 production, aerobic supernatants also stimulate IFN-γ, unlike anaerobic supernatants.

3.4. Aerobic and anaerobic EcO83 supernatants differentially affect cytokine gene expression in mouse splenocytes

To evaluate the effect of EcO83 metabolites in a different experimental model, mouse splenocytes were stimulated with EcO83 cells as well as aerobic and anaerobic EcO83 supernatants. Cytokine gene expression was assessed by qRT-PCR after 4 hours of stimulation (Fig. 3). None of the stimuli significantly increased the expression of Il2 (Fig. 3A) and Il10 expression was significantly increased only in splenocytes treated with the whole bacterium and high concentrations of the aerobic supernatants (AE 200 and AE 40) (Fig. 3B). Similarly, the expression of Il13 increased in response to EcO83 and aerobic supernatants AE 200 and AE 40 (Fig. 3C). The highest concentration of anaerobic supernatants, AN 200, also induced expression of Il13 (Fig. 3C). Lastly, Ifng expression was significantly increased only in response to the bacterial cells and high concentrations of aerobic supernatants (AE 200 and AE 40) (Fig. 3D).

3.5. Differential effect of aerobic and anaerobic EcO83 supernatants on cytokine production in mouse splenocytes

To further characterize the effects of EcO83 metabolites on cytokine production, cytokine levels were measured in splenocyte cell cultures after 24 hours of stimulation. The mRNA data were corroborated by cytokine release in the splenocyte cultures. IL-10 production was significantly increased in response to the highest concentrations of both aerobic (AE 200) and anaerobic (AN 200) EcO83 supernatants (Fig. 4A). IFN-γ production was significantly elevated only by AE 40 at the protein level (Fig. 4B). Neither aerobic nor anaerobic EcO83 supernatants induced the production of IL-4 or IL-13 (data not shown).

3.6. Differential changes in cytokine gene expression in BMDCs upon stimulation with EcO83 supernatants

To characterize the impact of EcO83 supernatants on cytokine expression in dendritic cells (DCs), which play a crucial role in initiating adaptive immune responses, bone marrow-derived DCs (BMDCs) were stimulated with various concentrations of EcO83 supernatants. Consistent with our previous study [33], Tnfa expression was significantly elevated by EcO83 cells (Fig. 5A). The highest concentration of both aerobic and anaerobic EcO83 supernatants (AE 200 and AN 200) significantly increased Tnfa expression (Fig. 5A). Lower concentrations of anaerobic EcO83 supernatants also stimulated Tnfa expression, albeit to a lesser but still statistically significant extent (Fig. 5A). Il10 expression was increased only with the highest concentrations of EcO83 supernatants (AE 200 and AN 200) in both aerobic and anaerobic groups (Fig. 5B).

3.7. Distinct cytokine responses to aerobic and anaerobic EcO83 supernatants

To validate the mRNA data, cytokine levels in BMDCs cultures stimulated with whole EcO83 bacteria and various concentrations of EcO83 supernatants were measured by ELISA (Fig. 6). TNF-α levels increased following stimulation with EcO83 bacteria, as well as with both aerobic and anaerobic EcO83 supernatants (Fig. 6A). IL-10 was significantly elevated by EcO83 cells, and also by the highest concentration of aerobic and anaerobic supernatants (AE 200, AE 40, and AN 200) (Fig. 6B). Although both bacteria and AE 200 induced comparable levels of TNF-α, AE 200 stimulated higher levels of IL-10, suggesting the anti-inflammatory properties of this postbiotic preparation.

3.8. Impact of aerobic and anaerobic EcO83 supernatants on dendritic cell maturation and activation markers

The effect of aerobic and anaerobic EcO83 supernatants on the maturation status of dendritic cells was evaluated. BMDCs were stimulated with EcO83 cells and supernatants, and changes in surface maturation and activation markers (CD80, CD86, MHCII) were evaluated by flow cytometry (Fig. 7). CD80 expression was significantly elevated by AE 200, AE 40, and AN 200, AN 40, AN 8 compared to the negative control, indicating a strong activation of dendritic cells by these treatments (Fig. 7A). Notably, significant CD86 expression was observed only following stimulation with EcO83 bacteria (Fig. 7B). MHC II expression remained consistent across all groups, suggesting that MHC II is relatively stable and less influenced by these treatments (Fig. 7C).

E. coli O83 is a facultative anaerobic probiotic with known beneficial effects on the host immune system. In previous studies, both we and others have demonstrated the anti-allergic properties of the live bacteria or its extracellular vesicles derived from aerobic cultures [29, 30, 32, 38–40]. This study aimed to compare the effects of metabolites produced under aerobic and strictly anaerobic conditions across three in vitro models: human PBMC, mouse splenocytes, and BMDC. Our findings offer new insights into how postbiotics, in the form of secreted metabolites, can induce immunomodulation. Furthermore, we observed that EcO83 produced the SCFA acetate and propionate in both aerobic and anaerobic cultures.

Some of the most significant bacterial products with well-documented beneficial effects on the mammalian immune system are SCFAs, particularly acetate, pyruvate, and butyrate. SCFA, commonly produced through the bacterial fermentation of indigestible oligosaccharides, are associated with maintaining gut health, shaping the epigenetic landscape of the mucosal immune system [41], serving as an essential substrate for colonocytes [42], and promoting tolerogenic responses, such as the induction of regulatory T cells [43]. SCFA are produced by certain gut-resident bacteria including Faecalibacterium prausnitzii, Eubacterium rectale, and Roseburia spp. [44], as well as some probiotic strains like Lactobacillus rhamnosus GG, Lactobacillus acidophilus CRL 1014, and Bifidobacterium spp. [45]. Probiotic administration is a promising strategy for immune modulation in various clinical contexts, ranging from allergies [46, 47] to autoimmune diseases [48, 49]. In SCFA-producing probiotic strains, these metabolites account for a substantial part of their probiotic effects, as demonstrated in several autoimmune diseases [48–50].

Using NMRS, we confirmed that both the aerobic and the anaerobic supernatants of EcO83 contained comparable levels of acetate (1.8 and 1.6 mM in aerobic and anaerobic samples, respectively) and propionate (3.1 and 3.0 mM in aerobic and anaerobic samples, respectively), while no butyrate was detected, regardless of the conditions. Despite E. coli being one of the most well-characterized bacteria, only a few studies have convincingly demonstrated its SCFA production. For instance, SCFAs were detected in E. coli Nissle 1917, another probiotic strain [51, 52]. As E. coli is also a prominent model organism in bioengineering research, several inactive or cryptic metabolic pathways with potential for SCFA production have been identified. These include the “sleeping beauty mutase,” an inactive operon capable of propionate production when activated [53, 54]. Given the genetic and metabolic flexibility of bacteria, it is conceivable that these latent genes could confer unusual metabolic functions under specific conditions or in particular strains, such as SCFA production. In 2020, Nakkarach et al. isolated several potentially probiotic E. coli strains capable of SCFA production from the stool of three healthy donors [35], later demonstrating one of these strains’ anti-inflammatory and anti-cancer effects [55]. To our knowledge, this study is the first to report acetate and propionate production in EcO83, and these SCFAs likely play a key role in the probiotic activity of this strain.

In facultative anaerobes such as E. coli, three distinct metabolic modes are generally utilized under aerobic, anaerobic, and microaerophilic conditions [56]. This metabolic flexibility allows the bacteria to adapt to both healthy (predominantly anaerobic) and pathological conditions in the gut, where increased oxygenation often accompanies gut pathologies such as idiopathic bowel diseases [57] and drives dysbiosis in gut infection, among other conditions [58]. Probiotic strains capable of producing SCFA under these varying conditions may help mitigate dysbiosis, potentially contributing to EcO83’s ability to prevent nosocomial infections in children [31].

Although SCFAs are the most extensively studied metabolites, especially in probiotic research, many other bacterial products, which are less well-researched, undoubtedly contribute to the observed effects [59, 60]. The production of these compounds may vary depending on whether the bacteria are in aerobic, anaerobic, or microaerophilic conditions. Metabolomic studies exploring the broader range of metabolites produced by probiotic bacteria under these diverse conditions are therefore warranted. Novel models for more effective in vitro simulation of gut mucosa-microbe interactions, as reviewed by Martels et al. [61], will be highly valuable in advancing this area of research.

The impact of EcO83 supernatants on human PBMCs revealed significant differences in cytokine production depending on the metabolic environment. IFN-γ production was significantly elevated only in response to aerobic supernatants, indicating that metabolites generated under high oxygen conditions may stimulate inflammatory pathways through distinct mechanisms. Additionally, IL-10 production was more pronounced under anaerobic conditions, implying a greater need for regulatory responses in this environment. The distinct cytokine profiles observed indicate that the metabolic state of the probiotic culture significantly affects the nature and magnitude of the immune response in PBMCs, with potential implications for modulating systemic inflammation.

A different pattern emerged in splenocytes, particularly in regulating IL-10, an anti-inflammatory cytokine crucial for maintaining immune tolerance. Aerobic supernatants strongly induced IL-10 production, highlighting their potential role in promoting a more tolerogenic or anti-inflammatory environment within the spleen. This response could be leveraged to control excessive inflammatory responses in autoimmune or inflammatory diseases. The anaerobic supernatants, however, did not significantly alter IL-10 levels, suggesting that the anaerobic metabolites might support a more balanced or even pro-inflammatory response in splenocytes. This differential effect underscores the complexity of immune modulation in splenocytes, where the balance between pro- and anti-inflammatory signals is critical for maintaining immune homeostasis.

Dendritic cells (DCs) are pivotal in initiating and steering T-cell responses, contributing to the balance between different branches of the immune system and driving either inflammatory or tolerogenic outcomes. As such, DCs are potentially key mediators of probiotic effect, with various strains [62, 63], including EcO83, having been shown to exert a complex influence on these cells, as highlighted in our previous work [33]. TNF-α promotes immune activation, essential for effective pathogen elimination, while IL-10 is critical in maintaining immune tolerance and preventing excessive immune activation, which could otherwise result in tissue damage or autoimmune diseases.

We also investigated the impact of EcO83 supernatants on the maturation of bone marrow-derived dendritic cells (BMDCs), focusing on the expression of key activation markers such as CD80, CD86, and MHC II. Both aerobic and anaerobic supernatants, particularly at higher concentrations, were notably effective in increasing CD80 expression, which is essential for optimal T cell priming and activation. This suggests that EcO83 enhances dendritic cell maturation and subsequent adaptive immune responses under both aerobic and anaerobic conditions. However, MHC II expression remained consistently high across all treatments, indicating that the fundamental antigen-presenting capability of BMDCs was preserved, irrespective of the metabolic environment. These results indicate that the metabolic conditions in which probiotics are cultured can greatly influence their ability to modulate dendritic cell function, which is critical in shaping the immune response's direction and intensity. EcO83 metabolites, produced under both aerobic and anaerobic conditions, triggered a complex response in dendritic cells, including the upregulation of inflammatory, Th1, Th2, and regulatory cytokines, along with cell maturation, which aligns with our previous findings [30, 33].

Here, we demonstrated that the probiotic EcO83 produced SCFAs, namely acetate and propionate, under both aerobic and anaerobic conditions. EcO83 culture supernatants elicited complex immunomodulatory effects on both human and murine cells, with aerobic samples favoring a Th1 response and anaerobic samples more strongly promoting a Th2 response, particularly in the human cells. These observations are consistent with the previously described mechanisms of EcO83 culture in aerobic conditions, including its effects on dendritic cell maturation and its support of IFN-γ and IL-10 production in murine and human in vitro models [30, 33].

In conclusion, our study highlights the metabolic flexibility of EcO83 in producing SCFAs and modulating immune responses under both aerobic and anaerobic conditions, reinforcing its potential as a versatile probiotic. Future research should focus on detailed metabolomic profiling to further elucidate the full spectrum of bioactive compounds involved, which may open new avenues for therapeutic applications in immune-related disorders.

Conflict of Interest

The authors declare no competing interests.

Funding

This project was supported by the Charles University Cooperation IMMU 207032 research program and the Grant Agency of Charles University GAUK 478119, Ministry of Education, Youth and Sports 8J23AT019, OEAD CZ 07/2023, CZ 15/2023, OEAD CZ 15/2023, Austrian Science Fund P 34867. The work was also supported by the Programme Johannes Amos Comenius project MSCA fellowships CZ-UK2 (reg.n. CZ.02.01.01/00/22_010/0008115).

Author Contribution

Conceptualization, JH, AIK, and IS; methodology, VČ, PP, ON, MK, LS, EK, JH; writing—original draft preparation, JH, VČ; writing—review and editing, JH, VČ, EK, AIK, IS; supervision JH; funding acquisition, JH, LS, IS and AIK.

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No competing interests reported.

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Immunomodulatory effect of metabolites secreted by the probiotic strain of E. coli O83:K24:H31

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Abstract

Figures

1. Introduction

2. Material and methods

2.1. Bacteria cultivation and supernatant preparation

2.2. Detection of SCFAs in EcO83 supernatants by NMR spectroscopy

2.3. Cell culture preparation

2.3.1. Human PBMCs

2.3.2. Murine splenocytes

2.3.3. Murine BMDCs

2.4. qRT-PCR

2.5. ELISA

2.4. Flow cytometry

2.6. Statistics

3. Results

3.1. EcO83 secretes SCFAs under both aerobic and anaerobic conditions

3.2. Aerobic and anaerobic EcO83 supernatants differentially affect cytokine gene expression in human PBMCs

3.3. PBMCs stimulated by both aerobic and anaerobic EcO83 supernatants show significant IL-10 production

3.4. Aerobic and anaerobic EcO83 supernatants differentially affect cytokine gene expression in mouse splenocytes

3.5. Differential effect of aerobic and anaerobic EcO83 supernatants on cytokine production in mouse splenocytes

3.6. Differential changes in cytokine gene expression in BMDCs upon stimulation with EcO83 supernatants

3.7. Distinct cytokine responses to aerobic and anaerobic EcO83 supernatants

3.8. Impact of aerobic and anaerobic EcO83 supernatants on dendritic cell maturation and activation markers

4. Discussion

Declarations

Conflict of Interest

Funding

Author Contribution

References

Additional Declarations

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