Abstract
Immune dysregulation is central in bacterial sepsis with lipopolysaccharide (LPS) of gram-negative bacteria acting as a key component in innate immune activation. Modulation of the host response by potential immunomodulatory agents may confer benefits. This ex vivo study compared cytokine responses induced by E. coli and P. aeruginosa LPS and evaluated the impact of cefiderocol on LPS-induced immune activation in human whole blood. Whole blood from ten healthy volunteers was stimulated ex vivo with E. coli or P. aeruginosa LPS in the presence or absence of cefiderocol and incubated for 4 h at 37 °C. Plasma concentrations of TNF-α, IL-1β, IL-6, IL-10, IFN-α, IFN-γ, and IL-8 were quantified at 0 h, 2 h, and 4 h using a multiplex immunoassay. Cytokine release was significantly higher after E. coli LPS stimulation than with P. aeruginosa LPS across all analytes. Mean TNF-α area under the curve (AUC₀–₄) was 2379 pg*h/mL with cefiderocol vs 1936 pg*h/mL without (percent difference 18.2% [95% CI, 5.9–31.4]) after E. coli LPS stimulation, and 278 pg/mL vs 242 pg*h/mL (12.9% [4.3–21.5]) after P. aeruginosa LPS stimulation. For IL-6, cefiderocol slightly increased AUC₀–₄ following E. coli LPS stimulation (4584 vs 3635 pg*h/mL; 20.7% [8.3–33.0]) but not with P. aeruginosa LPS (184 vs 201 pg*h/mL; − 9.3% [− 53.7–35.2]). IL-1β, IL-10, IFN-α, IFN-γ, and IL-8 levels were mostly unaffected by cefiderocol. Cefiderocol did not suppress cytokine release in human whole blood. In this study, E. coli LPS elicited a stronger immune response than P. aeruginosa LPS.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00430-026-00883-1.
Keywords: Sepsis, Immune system, Cytokine, Antibiotic
Introduction
Sepsis remains a major global health concern and a leading cause of morbidity and mortality worldwide [1]. Sepsis results from a dysregulated immune response to infection, leading to inflammation and organ dysfunction. An important mediator of this process is lipopolysaccharide (LPS), also known as endotoxin, a component found on the outer membrane of Gram-negative bacteria. LPS is a potent immunostimulatory component and can be recognized by the innate immune system by binding to Toll-like receptor 4 (TLR-4) through interaction with other molecules like MD-2, CD14 and LPS binding protein [2–4]. Consequently, downstream signaling pathways are activated resulting in the release of pro-inflammatory cytokines. Because of its immune-stimulating properties, LPS is used both in vitro and in vivo (i.e., the human endotoxin challenge model) to study inflammatory responses [5]. Escherichia coli-derived LPS is most commonly used in experimental study, although LPS from other Gram-negative pathogens is available and may elicit divergent immune responses. Timely administered and effective antimicrobial therapy is essential for improving outcomes in sepsis [6, 7]. Among the newer therapeutic options is cefiderocol, a siderophore cephalosporin, that has been developed to target multi-drug resistant Gram-negative bacteria with limited treatment options. Cefiderocol exhibits high affinity for iron and exploits bacterial iron transport systems to enter the cell. Once inside the periplasmic space, cefiderocol binds to penicillin-binding proteins, disrupting cell wall biosynthesis and ultimately leading to bacterial cell death [8]. Beyond their direct antimicrobial activity, it has been observed that some antimicrobial substances have additional immunomodulatory effects [9]. Such effects, including modulation of cytokine production, have been shown for several antibiotics, such as clarithromycin [10], tetracyclines [11] and fosfomycin [12], whereas data for beta-lactams remains limited [9]. Some evidence suggests that cefazolin may influence cytokine production [13]. Recent studies demonstrated that cefiderocol may attenuate cytokine release. [14, 15]. However, these studies used very high LPS concentrations and a murine macrophage model in one study [14] and purified peripheral blood mononuclear cells (PBMCs) in the other [15]. To extent these findings to a more clinically relevant context, we aimed to investigate both the stimulation potential of Escherichia coli (E. coli) versus Pseudomonas aeruginosa (P. aeruginosa) LPS and the effect of cefiderocol on LPS-induced cytokine responses in human whole blood ex vivo.
Methods
Study design and participants
This ex vivo immunological study aimed to evaluate the immune-stimulatory potential of E. coli versus P. aeruginosa LPS and to compare the human immune response after LPS stimulation with and without cefiderocol addition. Ten healthy volunteers between the age of 18–45 years were included in the study. Main exclusion criteria were use of any medication within one week prior to study day, vaccination within four weeks, clinically relevant illness within four weeks, alcohol abuse, smoking of more than five cigarettes per day, and pregnancy. Baseline characteristics and vital parameters were recorded for all participants. A single blood sample was obtained from each participant by venipuncture in standardized manner. A total of 72 mL of venous blood was drawn into lithium heparinized tubes for the experiment and a routine laboratory assessment including inflammatory markers was performed. No additional procedures or follow-up assessments were conducted beyond this visit.
Written informed consent was obtained from all participants before enrolment in the study. The study protocol was approved by the Ethics Committee of the Medical University of Vienna (EK 1084/2024) and was conducted in accordance with the International Council for Harmonisation Good Clinical Practice guidelines.
Laboratory procedures
LPS and investigational product
E. coli LPS and P. aeruginosa LPS were added to whole blood at a final concentration of 50 pg/mL, a level comparable to concentrations reported in patients with sepsis [16]. For E.coli LPS, the National Institutes of Health (NIH), USA reference endotoxin derived from E.coli O113:H10 was used. It was produced in the United States using hot phenol extraction and subsequent purification steps including alcohol precipitation, enzymatic treatment, ultracentrifugation and lyophilization. The endotoxin underwent biological standardization and quality control testing [17]. For P.aeruginosa LPS, research-grade lipopolysaccharide from P.aeruginosa serotype 10 (Sigma Aldrich, USA) was used. It was purified by gel-filtration chromatography and supplied as a lyophilized powder preparation with a protein contamination of less than 3% [18]. Cefiderocol (Fetcroja, Shionogi B.V., Netherlands) was added at 100 µg/mL to one experimental group, corresponding to the maximum concentration (cmax) observed in humans after administration of 2 g cefiderocol [19]. An equivalent volume of 0.9% saline served as control.
Ex vivo LPS stimulation
The experiment is depicted in Figure S1. The experiment consisted of two parallel conditions: blood incubated in the presence of cefiderocol and blood incubated without antibiotic. Prior to LPS stimulation, samples in the cefiderocol arm were spiked with cefiderocol, whereas saline was added to the control arm. Following collection, baseline plasma samples were obtained immediately by centrifugation and stored at − 80 °C. For the LPS stimulation, aliquots were incubated for 4 h at 37 °C under continuous gentle agitation. LPS (either E. coli LPS or P. aeruginosa LPS) was added either at the start of incubation (4 h condition) or after 2 h of incubation (2 h condition). Moreover, negative controls without added LPS were performed. Consequently, the samples were incubated for 4 h, with LPS exposure lasting 4 h, 2 h, or none. At the end of the respective incubation period, samples were centrifuged (30 min, 500×g, 4 °C), and supernatant was stored at − 80 °C until analysis.
Cytokine quantification
The following cytokines were measured in duplicates using a Luminex 7-plex assay (Luminex Performance Assay, R&D Systems, USA) because they are known to be reliably released following LPS stimulation in humans in vivo and ex vivo [20, 21]: Tumour necrosis factor- α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interferon-α (IFN-α) and interferon-γ (IFN-γ).
Statistical analysis
The primary endpoint was the difference in cytokine concentrations after LPS stimulation in the absence or presence of cefiderocol. An additional endpoint was the comparison of cytokine responses between E. coli and P. aeruginosa LPS. Baseline data is presented as median with interquartile range (IQR) for continuous variables and number (percentage) for categorical variables. Cytokine data were summarized descriptively as mean ± standard deviation or standard error. Cytokine responses were further quantified as the area under the curve (AUC₀–₄) from baseline to 4 h, calculated using the trapezoidal rule. Paired comparisons (cefiderocol-spiked vs. cefiderocol-free samples, E. coli vs. P. aeruginosa LPS) were analysed using the paired t-test. We calculated percent differences with 95% confidence intervals to express the relative difference in group comparisons. A two-sided p-value < 0.05 was considered statistically significant. Values below the Lower Limit of Quantification were substituted for 0. Statistical analyses and graphical presentations were performed using GraphPad Prism (GraphPad Software, Boston, MA, USA).
Role of the funding source
This study was funded by Shionogi. The sponsor had no impact on data analysis or interpretation.
Results
Participant characteristics
A total of 10 participants were included in the analysis, with baseline characteristics summarized in Table 1. The median age (interquartile range [IQR]) was 27 years (IQR 25–34) and 8 of the 10 participants were female. The median body temperature was within the normal range at 36.4 °C (IQR 36.0–36.5). Routine laboratory assessment indicated no evidence of systemic inflammation with a median leukocyte count of 5.5 G/L (IQR 5.0–6.8) and a low mean CRP concentration of 0.05 mg/dL (IQR 0.03–0.08).
Table 1.
Participant characteristics
| Variable | Reported as | Value | |
|---|---|---|---|
| n | 10 | ||
| Age | median (IQR) | 27 (25–34) | |
| Sex | n (%) | Male | 2 (20.0) |
| Female | 8 (80.0) | ||
| Body temperature (°C) | median (IQR) | 36.35 (36.00–36.50) | |
| Total leukocyte count (G/L) | median (IQR) | 5.45 (5.04–6.79) | |
| Total neutrophile count (G/L) | median (IQR) | 2.80 (2.50–3.83) | |
| CRP (mg/dL) | median (IQR) | 0.05 (0.03–0.08) |
Cytokine levels
Cytokine levels were significantly higher after stimulation with E. coli LPS compared to P. aeruginosa LPS across all tested cytokines. Time-concentration profiles are shown in Fig. 1, and mean concentrations at baseline and 4 h in Fig. 2. Table 2 depicts AUC values with and without cefiderocol and corresponding absolute and percent differences. For TNF-α, the mean (SE) AUC0-4 was significantly higher in the presence of cefiderocol than without, both for E.coli (2379 ± 613 pg*h/mL vs. 1936 ± 717 pg*h/mL, percent difference 18.2% [95% CI, 5.9–31.4]) and P. aeruginosa (278 ± 106 pg*h/mL vs. 242 ± 86 pg*h/mL; percent difference 12.9% [95% CI, 4.3–21.5]). Similarly, for IL-6, cefiderocol increased cytokine release following E. coli LPS stimulation (4584 ± 1575 pg*h/mL vs. 3635 ± 1213 pg*h/mL; percent difference 20.7% [95% CI, 8.4–33.0]), whereas no significant difference was observed with P. aeruginosa LPS (184 ± 55 pg*h/mL vs. 201 ± 98 pg*h/mL; percent difference -9.3% [95% CI, -53.7–35.2]). For IL-1β, AUCs were comparable with and without cefiderocol for both E.coli (219 ± 58 pg*h/mL vs. 209 ± 60 pg*h/mL; percent difference 4.3% [95% CI, 23.1–31.7]) and P. aeruginosa (46 ± 13 pg*h/mL vs. 47 ± 15 pg*h/mL; percent difference -2.8% [95% CI, -32.6–27.0]).
Fig. 1.
Time-concentration curves of TNF-α, IL-6, IL-1β, IL-8, IL-10, IFN-α and IFN-γ. Data is shown as mean ± standard deviation
Fig. 2.
Mean concentration of TNF-α, IL-6, IL-1β, IL-8, IL-10, IFN-α and IFN-γ at baseline and at 4h. Data is shown as mean (horizontal number) ± standard deviation
Table 2.
Cytokine AUC0-4 values (pg*h/mL) with vs without cefiderocol and corresponding differences
| Cytokine | AUC with CDFC | AUC w/o CDFC | Absolute Difference | Percent difference | p value | ||||
|---|---|---|---|---|---|---|---|---|---|
| mean (SE) | mean (SE) | mean | 95% CI | mean | 95% CI | ||||
| E. coli | |||||||||
| TNF-α | 2379.40 (613.2) | 1935.80 (716.90) | 433.50 | 140.50 | 746.60 | 18.22 | 5.90 | 31.38 | 0.01 |
| IL-6 | 4584.00 (1575.00) | 3635.00 (1213.00) | 948.60 | 382.70 | 1515.00 | 20.69 | 8.35 | 33.05 | 0.00 |
| IL-1β | 218.60 (57.96) | 209.20 (60.44) | 9.45 | -50.39 | 69.29 | 4.32 | -23.05 | 31.70 | 0.73 |
| IL-8 | 5205.00 (1478.00) | 4677.00 (1368.00) | 528.30 | 152.30 | 904.30 | 10.15 | 2.93 | 17.37 | 0.01 |
| IL-10 | 1012.00 (182.80) | 939.20 (150.30) | 72.43 | -36.82 | 181.70 | 7.16 | -3.64 | 17.95 | 0.17 |
| IFN-α | 44.61 (10.59) | 39.88 (12.06) | 4.73 | -0.18 | 9.64 | 10.60 | -0.40 | 21.61 | 0.06 |
| IFN-γ | 29.65 (14.59) | 26.76 (11.61) | 2.89 | -3.45 | 9.24 | 9.76 | -11.65 | 31.16 | 0.33 |
| P. aeruginosa | |||||||||
| TNF-α | 278.20 (105.90) | 242.2 (85.61) | 35.91 | 11.90 | 59.93 | 12.91 | 4.28 | 21.54 | 0.01 |
| IL-6 | 184.00 (54.79) | 201.1 (98.18) | -17.03 | -98.87 | 64.82 | -9.26 | -53.73 | 35.23 | 0.65 |
| IL-1β | 45.54 (12.69) | 46.81 (15.46) | -1.27 | -14.84 | 12.30 | -2.78 | -32.59 | 27.01 | 0.84 |
| IL-8 | 1914.00 (812.10) | 2048.00 (1211.00) | -133.40 | -1021.00 | 754.60 | -6.97 | -53.34 | 39.43 | 0.74 |
| IL-10 | 521.60 (136.30) | 521.70 (155.70) | 0.04 | -59.85 | 59.93 | 0.01 | -11.47 | 11.49 | 1.00 |
| IFN-α | 19.62 (10.50) | 20.54 (9.88) | -0.92 | -4.10 | 2.25 | -4.71 | -20.91 | 11.48 | 0.53 |
| IFN-γ | 26.38 (12.86) | 24.96 (11.27) | 1.42 | -3.15 | 6.00 | 5.40 | -11.94 | 22.74 | 0.50 |
AUC Area-under-the-curve; CDFC cefiderocol; w/o without; p < 0.05 was considered statistically significant.
Among IL-10, IFN-α, IFN-γ and IL-8, there were no significant differences in cytokine levels between groups except for IL-8 where a significant increase in AUC following E. coli LPS stimulation in the presence of cefiderocol was observed (percent difference 10.1% [95% CI, 2.9–17.4]).
Discussion
This ex vivo immunological study investigated two aspects: (i) differences in cytokine production following stimulation with E. coli LPS versus P. aeruginosa LPS, and (ii) the effect of cefiderocol on LPS-induced cytokine levels in human whole blood. Stimulation with E. coli LPS elicited a markedly stronger immune response compared to P. aeruginosa LPS, resulting in significantly higher cytokine levels across analytes. In contrast, P. aeruginosa LPS did not have a substantial stimulation potential when compared to the 4 h negative control indicating limited immunostimulatory capacity. Cefiderocol did not exhibit anti-inflammatory effects in human whole blood. Cytokine levels were generally higher in cefiderocol-treated samples than in native samples, however, these differences were small and unlikely to be clinically relevant.
Other studies have also demonstrated significantly lower cytokine levels following ex vivo stimulation of cells with P. aeruginosa LPS compared to E.coli LPS [22, 23]. The observed differences in cytokine release between these species might be attributable to structural variations in the LPS molecule itself. The Lipid A domain, which mediates activation of the innate immune system [4], differs among bacterial species in aspects such as phosphorylation pattern, number of acyl chains or composition of fatty acids [24]. In addition, Lipid A structure may also vary within the same species due to differences in growth conditions, environmental exposure or isolation sources [25]. Such alterations can influence immune recognition and downstream signaling through TLR-4 [2]. The low immunostimulatory activity of P. aeruginosa LPS in experimental studies has been attributed to a lower degree of Lipid A acylation [23]. Conversely, LPS derived from P. aeruginosa isolates in cystic fibrosis patients exhibits distinct Lipid A modifications, which, however, were associated with an enhanced inflammatory response compared to non cystic fibrosis isolates [26].
Cytokine production following LPS stimulation is complex and influenced by multiple interacting factors including bacterial species, serotype, isolate source, environmental factors and experimental set-up. The NIH E. coli reference endotoxin used in this study represents a highly standardized and reproducible LPS preparation that has been widely used for human challenge studies and bioanalytical assays for several decades [27]. The P. aeruginosa LPS used in this study represents a purified preparation from a single serotype prepared for research purposes. Therefore, neither preparation can fully reflect the structural diversity of LPS encountered in clinical infections. Furthermore, differences in extraction, purification, and standardization procedures between the two LPS preparations may have influenced LPS structures and thereby contributed to differences in stimulatory activity.
In contrast to our findings previous studies have reported that cefiderocol might attenuate the immune response ex vivo. Hildebrand et al. [15] observed that cefiderocol lead to decreased cytokine production (IL-6, IL-1β and TNF-α) in a dose-dependent manner. However, their experiments used extremely high LPS concentrations (100 ng/ml), which exceeds concentrations typically observed in septic patients approximately 1000-fold [16], and were conducted in isolated peripheral blood mononuclear cells (PBMCs) rather than whole blood. Similarly, Ju et al. [14] reported reduced levels of IL-6, TNF-α, and IL-1β and increased IL-10 levels; however, cultured mouse macrophage cells were used along with excessive LPS levels (100 ng/mL). By contrast, we used freshly drawn human whole blood, which better preserves the complexity of the immune system and used “physiologic” LPS levels observed in human sepsis which may therefore more accurately represent the immune response in vivo.
Our study has some limitations. First, the sample size was small and consisted predominantly of white, young, healthy female participants which may limit generalizability of our findings to other populations. Second, various LPS variants within a species exist and structural heterogeneity between variants may influence Toll-like receptor 4 activation and downstream cytokine release. Third, as this was an ex vivo study, results cannot directly be translated to patients and may not fully reflect the complex host–pathogen interactions occurring in septic patients in vivo. LPS structure may undergo in vivo modifications depending on bacterial strain and host factors, potentially altering immunostimulatory activity. Therefore, extrapolation to clinical settings must be made with caution.
In conclusion, in our study, LPS derived from E.coli elicited a robust cytokine response compared to P. aeruginosa LPS. Cefiderocol did not demonstrate an immunomodulatory effect in ex vivo in human whole blood.
Supplementary Information
Below is the link to the electronic supplementary material.
Author contributions
All listed authors meet all criteria for authorship in the ICMJE Recommendations. LP and MZ conceived the study idea. LP, MW and SE performed the research. LP, AJ and MZ performed the statistical analysis and had direct access to the data. LP and AJ prepared the figures and tables. LP and AJ analysed the data and drafted the manuscript. All authors critically revised the manuscript and approved the final version of the manuscript.
Funding
Open access funding provided by Medical University of Vienna. This study was funded by Shionogi.
Data availability
Data will be shared upon reasonable request.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Data Availability Statement
Data will be shared upon reasonable request.