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. Author manuscript; available in PMC: 2025 Aug 1.
Published in final edited form as: ChemMedChem. 2024 Jul 12;19(15):e202400011. doi: 10.1002/cmdc.202400011

Low-molecular weight branched polyethylenimine reduces cytokine secretion from human immune system monocytes stimulated with bacterial and fungal PAMPs

Neda Heydarian a, Maya Ferrell a, Ayesha S Nair b, Chase Roedl a, Zongkai Peng a, Tra D Nguyen a, William Best a, Karen L Wozniak b, Charles V Rice a,*
PMCID: PMC11463166  NIHMSID: NIHMS2015578  PMID: 38740551

Abstract

The innate immune system is an evolutionarily conserved pathogen recognition mechanism that serves as the first line of defense against tissue damage or pathogen invasion. Unlike the adaptive immunity that recruits T-cells and specific antibodies against antigens, innate immune cells express pathogen recognition receptors (PRRs) that can detect various pathogen-associated molecular patterns (PAMPs) released by invading pathogens. Microbial molecular patterns, such as lipopolysaccharide (LPS) from Gram-negative bacteria, trigger signaling cascades in the host that result in the production of pro-inflammatory cytokines. LPS stimulation produces a strong immune response and excessive LPS signaling leads to dysregulation of the immune response. However, dysregulated inflammatory response during wound healing often results in chronic non-healing wounds that are difficult to control. In this work, we present data demonstrating partial neutralization of anionic LPS molecules using cationic branched polyethylenimine (BPEI). The anionic sites on the LPS molecules from Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the lipid A moiety and BPEI binding create steric factors that hinder the binding of PRR signaling co-factors. This reduces the production of pro-inflammatory TNF-α cytokines. However, the anionic sites of Pseudomonas aeruginosa (P. aeruginosa) LPS are in the O-antigen region and subsequent BPEI binding slightly reduces TNF-α cytokine production. Fortunately, BPEI can reduce TNF-α cytokine expression in response to stimulation by intact P. aeruginosa bacterial cells and fungal zymosan PAMPs. Thus low-molecular weight (600 Da) BPEI may be able to counter dysregulated inflammation in chronic wounds and promote successful repair following tissue injury.

Keywords: lipopolysaccharide, branched polyethylenimine, cytokine, pathogen-associated molecular pattern molecules

Graphical Abstract

graphic file with name nihms-2015578-f0001.jpg

Non-healing wounds contain Gram-negative pathogens and fungi whose pro-inflammatory toxins interrupt wound healing. Clinicians rely on a wide array of products to treat slow-healing wounds, but these products often fail to prevent advanced stages of chronic wound development. As a result, the US Food and Drug Administration has identified new products for non-healing wounds as a priority area “due to high unmet need”. We demonstrate that branched polyethylenimine (BPEI) can neutralize these pro-inflammatory toxins.

INTRODUCTION:

Wound healing encompasses a complicated pathophysiological process that involves immune cells to provide protection against microbial pathogens. However, opportunistic microorganisms can become pathogenic and can cause persistent infections. Unlike acute wounds, which progress through the inflammatory phase and eventually heal normally, chronic wounds stagnate in the inflammatory phase (1). This is due in part to the presence of polymicrobial communities in chronic wounds, which include bacteria and fungi. These microbes contribute to the overproduction of cytokines, which in turn perpetuates inflammation (2–4). The persistent inflammation in chronic wounds can lead to delayed healing, deepening and expansion of the wound, and a significant decrease in the quality of life for those affected (5–7). Chronic wounds are a major global health problem which affect millions of people around the globe and cost healthcare systems billions of dollars each year (8). In the absence of FDA-approved drugs for healing chronic wounds, new therapeutic agents need to be developed (9). Ideally, these compounds should have multifunction properties that reduce cytokine production, kill pathogens, and disrupt biofilms (10).

Prime targets for these new wound healing compounds are pathogen-associated molecular patterns (PAMPs) (11) such as bacterial lipopolysaccharide (LPS) and fungal glucan (zymosan) (12–14). LPS is an abundant glycolipid component present on the outer leaflet of Gram-negative bacteria. During infection, the immune system’s response to the highly conserved lipid A component of LPS is a severe generalized inflammation via toll-like receptor 4 (TLR4) that primarily recognizes, and is activated by LPS endotoxin, leading to signaling events that eventually culminate with the release of inflammatory cytokines (15–16). TLR4 activation requires interaction with several co-receptors and through a series of consecutive steps. LPS first combines with LPS-binding protein (LBP) and then LBP transfers LPS molecule to differentiation 14 (CD14) protein. CD14 in turn chaperones the formation of LPS and myeloid differentiation factor 2 (MD2) complex (15). MD2 directly recognizes and binds the lipophilic part (lipid A) of LPS to form a discrete aggregate. It non-covalently associates to TLR4 to form the final activated heterodimer of LPS/MD2/TLR4 that in its turn induces the downstream intracellular signal (Figure 1). Pro-inflammatory cytokines such as TNF-α are released systemically and, without regulation, initiate the storm of cytokines that cause tissue damage (12).

Figure 1.

Figure 1.

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LPS molecules activate TLR4 receptor and induce the downstream signaling cascade that ultimately produces inflammatory cytokines. TLR4 activation requires interaction with several co-receptors and through a series of consecutive steps. LPS first combines with LPS-binding protein (LBP) and then LBP transfers LPS molecule to cluster of differentiation 14 (CD14) protein. CD14 in turn chaperones the formation of LPS and myeloid differentiation factor 2 (MD2) complex.

Fungal pathogens, which include Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, can cause invasive fungal infections that are a major global health burden (13–14,17). The innate immune system identifies fungal cells as foreign invaders by detecting distinct fungal pathogens PAMPs (13,18). At the surface of fungal cells, various fungal PAMPs are present, such as glucans (β- and α-linked), chitin, chitosan, mannans, galactosaminogalactan, and fungal DNA (13–14). These critical fungal cell wall components are identified by pattern recognition receptors (PRRs) including TLRs (particularly TLR2 and TLR4), C-type lectin receptors (CLRs) such as Dectin-1 and Dectin-2, and galectin family proteins that are present on the surface of host cells (14,17,19). Zymosan, derived from the cell wall of Saccharomyces cerevisiae, is composed of protein and repeating glucose units linked by β-1,3-glycosidic bonds. Zymosan stimulates monocytes through the activation of TLR2. This activation leads to the induction of NF-kB signaling pathway and the subsequent production of various inflammatory cytokines such as TNF-α, IL-6, IL-8 (20–25). Zymosan is also recognized by Dectin-1 receptor which is expressed on the surface of monocytes, dendritic cells, and neutrophils (19,21,26).

In this work, we demonstrated the ability of 600 Da branched polyethylenimine (BPEI) to mitigate TNF-α cytokine production by THP-1 monocyte cells from exposure to LPS of Gram-negative bacteria, whole cell bacteria, and fungal zymosan. The significance of utilizing low molecular weight BPEI against bacterial and fungal PAMPs becomes more evident when considering its ability to disrupt biofilms and kill bacterial pathogens Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and carbapenem-resistant Enterobacteriaceae (27–39).

Methods

Cell culture

THP-1 human monocyte cell line was purchased from Sigma (St. Louis, MO, ECACC, 88081201) and maintained in RPMI 1640 medium containing L-glutamine and sodium bicarbonate (Sigma, St. Louis, MO, R8758), supplemented with 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT, SH30066.03), and 1% Pen Strep (10000 U/ml penicillin and 10000 μg/ml streptomycin, Gibco™ 15140122), at 37 °C and 5% CO2 (v/v) in a humidified incubator. Cells were grown in T-75-cm2 culture flasks (Corning, 431464) and sub-cultured every 5 or 6 days by three to five times dilution.

Preparation of heat-killed P. aeruginosa bacteria

P. aeruginosa bacterial stocks were purchased from American Type Culture Collection (ATCC BAA-47, a PAO1 strain). A heat-killed preparation of bacteria (HKL) was prepared by growing bacterial cultures in cation-adjusted Mueller–Hinton broth (CAMHB) media overnight at 37°C on a rotator. Cultures in log-phase growth were harvested and bacteria concentration was enumerated by measuring their optical density at 600 nm. Then, the cells were centrifuged, and washed three times in PBS. The recovered bacteria were resuspended in PBS and incubated at 85°C for 5 minutes. Then, the bacterial cells were cooled down for 20 minutes at room temperature. After two additional washes in PBS, the absence of viable colonies was confirmed by lack of growth on nutrient agar plates. Optical density (OD) of the heat-killed P. aeruginosa (HKPA) was measured at 600 nm one more time to confirm their concentration. The HKPA were stored at −80 °C.

LPS, HKPA, and zymosan treatments of THP-1 cells

Before treatments, THP-1 cells were seeded onto 96-well plates (Greiner Bio-one, Stuttgart, Germany) at 2×106 cells/mL in RPMI 1640 complete medium (1% Pen Strep, and 10% FBS) and incubated overnight at 37 °C and 5% CO2. The day after, the THP-1 cells were stimulated with 100 ng/ml of E. coli O26:B6 LPS (eBioscience™ Lipopolysaccharide (LPS) Solution, Invitrogen, 00–4976-93) for 8 h, or 100 ng/ml of E. coli O111:B4 LPS (Lipopolysaccharides, from Escherichia coli O111:B4, Sigma, L2630) for 4 h, or 100 ng/ml of K. pneumoniae LPS (Lipopolysaccharides, from Klebsiella pneumoniae, Sigma, L4268) for 2 h, or 1 μg/ml of P. aeruginosa LPS (Lipopolysaccharides, from Pseudomonas aeruginosa 10, Sigma, L8643) for 6 h, or 25 μg/ml of zymosan (cell wall from Saccharomyces cerevisiae; TLR2 and Dectin-1 ligand, InvivoGen, tlrl-zyn) for 6 h. Whole-cell heat-killed P. aeruginosa bacteria BAA47 (HKPA) were also used for stimulation at 105 cells/mL final concentration for 2 h. All the solutions were prepared in endotoxin-free water. Concentrations of treatments were selected based on the optimum production of TNF-α cytokine. LPS, HKPA, and zymosan were reconstituted in LAL-grade water (InvivoGen). After stimulation, supernatants were collected at certain timepoints. To protect TNF-α cytokine protein from degradation by endogenous proteases released during protein extraction, halt protease inhibitor cocktail (Thermo Scientific, 87786) was used. Immediately after supernatant collection, 10 μL of protease inhibitor was added per one milliliter of supernatant to produce a 1X final concentration. Cell medium was frozen at −80 °C until analysis. Untreated cells were used as controls.

Time-point assays of LPS, HKPA, and zymosan

To explore at what time point the Gram-negative and fungal PAMPs have the most inflammatory effect on TNF-α cytokine secretion, we did a time-point assay for each PAMP. THP-1 cells were plated in 96-well plates at 2×106 cells/mL in RPMI complete medium. After 24 h incubation, the THP-1 cells were treated with 100 ng/ml of E. coli O26:B6 LPS, or 100 ng/ml of E. coli O111:B4 LPS, or 100 ng/ml of K. pneumoniae LPS, or 1 μg/ml of P. aeruginosa LPS, or 25 μg/ml of zymosan, or 105 cells/mL of HKPA. The solutions were prepared in endotoxin-free water. The plates were then incubated for 2, 4, 6, 8, and 24 h, and supernatants were collected at the end of each time-point. To protect TNF-α cytokine protein from degradation by endogenous proteases released during protein extraction, halt protease inhibitor cocktail was used at the final 1X concentration. Time durations of treatments were selected based on the optimum production of TNF-α cytokine. Cell medium was frozen at − 20 °C until analysis. Untreated cells were used as controls.

Neutralizing immune response induced by LPS, HKPA, and zymosan using BPEI

Low-molecular weight (600 Da) branched polyethylenimine (BPEI) was purchased from Polysciences, Inc. The effect of BPEI against the Gram-negative and fungal PAMPs-induced TNF-α cytokine production was determined. Briefly, THP-1 cells were plated in 96-well plates at 2×106 cells/mL in RPMI complete medium and incubated overnight. The day after, different concentrations of BPEI were prepared. For each neutralizing experiment, the THP-1 cells were treated with either PAMP alone, combinations of an equivalent amount of the PAMP with each of the BPEI concentrations, and BPEI concentrations alone. All the solutions were prepared in endotoxin-free water. The latter was used as negative control and the cells treated with PAMP alone represented the positive control. The combo conditions were incubated for 30 minutes before being added to THP-1 cells. Untreated cells were also prepared as control. Then, supernatants were collected after certain hours of incubation. In HKPA neutralizing experiment, combo conditions were added to the cells immediately without prior incubation.

Enzyme-linked immunosorbent assay (ELISA) measurements

Concentrations of TNF-α cytokine were determined using DuoSet ELISA kits (R&D Systems, DY210) in THP-1 cell supernatants treated by each of the Gram-negative and fungal PAMPs along with BPEI. A 96-well plate was coated with 100 μl per well of TNF-α specific capture antibody diluted in endotoxin-free PBS (Endotoxin-free Dulbecco’s PBS 1X, Milipore Sigma, TMS-012-A) and incubated overnight at room temperature. The day after, each well was washed three times with 300 μl of washing buffer. Next, the plate was blocked with 300 μl of reagent diluent (R&D Systems, DY995) and incubated at room temperature for a minimum of 1 h and then washed three times with washing buffer. Subsequently, 100 μl of standards or collected supernatants was added to the plate and incubated at room temperature for 2 h, followed by washing. Then, 100 μl of TNF-α specific detection antibody was added and the plate was incubated for 2 h at room temperature and then washed. After that, 100 μl of streptavidin-HRP was added, and the plate was incubated for a minimum of 30 min at room temperature in dark, followed by washing. Then, 100 μl of substrate solution (equal volume of hydrogen peroxide and tetramethylbenzidine (R&D Systems, DY999)) was added and the plate was once again incubated in dark at room temperature till the color developed. Reaction was stopped by adding 50 μl of 2 N sulfuric acid as stop solution. The absorbance of each well was immediately determined using a microplate reader set at 450 nm. For wavelength correction, the reading was set to 540 nm as well. Optical imperfections in the plate were corrected by subtracting readings at 540 nm from readings at 450 nm.

Statistical Analysis

All experiments were performed in triplicate (n=3) and the presented data are representative results of the means ± standard error of the mean (SEM). Differences in cytokine production were analyzed using one-way ANOVA with Tukey’s post-test. A 95% confidence value with a p-value consisting of p < 0.05 was considered statistically significant. Data were analyzed using GraphPad Prism 6.01 software (GraphPad Software Inc., USA) and Adobe Illustrator.

Results

Optimization of TNF-α protein production induced by Gram-negative and fungal PAMPs

The major component of outer leaflet of Gram-negative bacteria is called lipopolysaccharide (LPS), which has a crucial role in the interaction between the host and pathogen within the innate immune system. LPS acts as a potent immunostimulant endotoxin that can stimulate the immune system strongly, even when present in nanogram amounts (40–41). In order to optimize the secretion of TNF-α cytokine induced by the Gram-negative endotoxin, LPS molecules from different Gram-negative strains were selected to stimulate THP-1 cells. The monocyte cells were treated with 100 ng/ml of either E. coli O26:B6 LPS, E. coli O111:B4 LPS, or K. pneumoniae LPS. In the case of P. aeruginosa LPS, 1 μg/ml of the molecules were utilized to treat THP-1 cells. Moreover, heat-killed P. aeruginosa bacteria BAA47™ (HKPA) were prepared and used intact at the concentration of 105 cells/mL. To generate an immune response stimulated by a fungal PAMP, THP-1 cells were treated with 25 μg/ml of zymosan. The plates were then incubated for 2, 4, 6, 8, and 24 h, and supernatants were collected at the end of each time-point. Untreated cells were incubated for each of the time points as control. Levels of TNF-α cytokine protein were measured in cell supernatants using ELISA. As shown in Panel A of Figures 2–6, each of the Gram-negative and fungal PAMPs showed a time-dependent manner in inducing TNF-α cytokine secretion, reaching a maximum after 4–8 hours for E. coli and P. aeruginosa LPS. However, TNF-α cytokine expression was highest after 2 hours for K. pneumoniae LPS (Figure 4A) and HKPA (Figure 6A). As expected, levels of TNF-α proteins were the lowest at 24 h. None of the untreated cells in each time-point assay showed any signal for TNF-α production in the ELISA data.

Figure 2.

Figure 2.

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600 Da BPEI neutralization of LPS from E. coli O26:B6. Panel A shows the timepoint assay of TNF-α production from THP-1 cells were treated with 0.1 μg/ml of E. coli O26:B6 LPS. The cells were then incubated for 2, 4, 6, 8, and 24 h, and supernatants were collected at the end of each time-point. Levels of TNF-α cytokines were quantified using ELISA. Untreated cells were used as controls. Timepoints that resulted in the highest secretion of TNF-α protein were selected as the optimum timepoints for stimulating THP-1 cells with E. coli O26:B6 LPS. Panel B shows reduced TNF-α cytokine levels when the E. coli O26:B6 LPS was premixed with 0.01, 0.1, and 1 μg/mL of 600 Da BPEI. All experiments were performed in triplicate and the presented data are representative results of the means ± standard error of the mean (SEM). A 95% confidence value with a p-value consisting of p < 0.05 was considered statistically significant.

Figure 6.

Figure 6.

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600 Da BPEI neutralization of whole-cell P. aeruginosa bacteria. Panel A shows the timepoint assay of TNF-α production from THP-1 cells were exposed to 105 cells/ml of whole-cell P. aeruginosa bacteria. The cells were then incubated for 2, 4, 6, 8, and 24 h, and supernatants were collected at the end of each time-point. Levels of TNF-α cytokines were quantified using ELISA. Untreated cells were used as controls. Timepoints that resulted in the highest secretion of TNF-α protein were selected as the optimum timepoints for stimulating THP-1 cells. Panel B shows reduced TNF-α cytokine levels when whole-cell P. aeruginosa bacteria were mixed with 0.1, 1.0, and 10 μg/mL of 600 Da BPEI. Panel C shows reduced TNF-α cytokine levels when whole-cell P. aeruginosa bacteria were mixed with 50, 100, and 150 μg/mL of 600 Da BPEI. All experiments were performed in triplicate and the presented data are representative results of the means ± standard error of the mean (SEM). A 95% confidence value with a p-value consisting of p < 0.05 was considered statistically significant.

Figure 4.

Figure 4.

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600 Da BPEI neutralization of LPS from K. pneumoniae. Panel A shows the timepoint assay of TNF-α production from THP-1 cells were treated with 0.1 μg/ml of K. pneumoniae LPS. The cells were then incubated for 2, 4, 6, 8, and 24 h, and supernatants were collected at the end of each time-point. Levels of TNF-α cytokines were quantified using ELISA. Untreated cells were used as controls. Timepoints that resulted in the highest secretion of TNF-α protein were selected as the optimum timepoints for stimulating THP-1 cells with K. pneumoniae LPS. Panel B shows reduced TNF-α cytokine levels when the K. pneumoniae LPS was premixed with 0.1 and 1 μg/mL of 600 Da BPEI whereas Panel C shows data for premixing LPS with 25, 50, 75, and 100 μg/mL. All experiments were performed in triplicate and the presented data are representative results of the means ± standard error of the mean (SEM). A 95% confidence value with a p-value consisting of p < 0.05 was considered statistically significant.

Neutralizing the immunostimulatory effects of Gram-negative and fungal PAMPs with BPEI

THP-1 cells were treated with each PAMP molecule (E. coli O26:B6 LPS, E. coli O111:B4 LPS, K. pneumoniae LPS, P. aeruginosa LPS, HKPA, and zymosan). Parallel experiments were performed with each PAMP mixed with different concentrations of 600 Da BPEI. The amount of secreted TNF-α cytokines was measured using ELISA. The data shown in Figures 2B and 3B demonstrate the modulation effect of BPEI on the production of TNF-α proteins stimulated with LPS molecules from two strains of E. coli bacteria. Against 0.1 μg/mL LPS molecules purified from E. coli O26:B6 bacteria, three concentrations of BPEI were used (0.01, 0.1, and 1 μg/mL). These concentrations corresponded to LPS to BPEI mass ratios of 0.1:1, 1:1, and 1:10, respectively. Although we did notice a decrease in the quantity of TNF-α cytokine resulting from all BPEI concentrations, the mitigating effect was modest (Figure 2B). This suggests that BPEI possesses some antagonistic effects on the immune response, yet BPEI is unable to completely neutralize the LPS stimulation of THP-1 cells. Likewise, BPEI exhibited a modest reduction in the levels of secreted TNF-α in response to E. coli O111:B4 LPS (Figure 3B).

Figure 3.

Figure 3.

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600 Da BPEI neutralization of LPS from E. coli O111:B4. Panel A shows the timepoint assay of TNF-α production from THP-1 cells were treated with 0.1 μg/ml of E. coli O111:B4 LPS. The cells were then incubated for 2, 4, 6, 8, and 24 h, and supernatants were collected at the end of each time-point. Levels of TNF-α cytokines were quantified using ELISA. Untreated cells were used as controls. Timepoints that resulted in the highest secretion of TNF-α protein were selected as the optimum timepoints for stimulating THP-1 cells with E. coli O111:B4 LPS. Panel B shows reduced TNF-α cytokine levels when the E. coli O111:B4 LPS was premixed with 0.1 and 1 μg/mL of 600 Da BPEI. All experiments were performed in triplicate and the presented data are representative results of the means ± standard error of the mean (SEM). A 95% confidence value with a p-value consisting of p < 0.05 was considered statistically significant.

When K. pneumoniae LPS was used to treat THP-1 cells for two hours, it provoked a significant secretion of TNF-α as shown in Figure 4A. This observation suggests that K. pneumoniae LPS may induce a more robust immune response compared to E. coli and P. aeruginosa LPS. Combinations of either 0.01 μg/mL or 0.1 μg/mL of BPEI molecules with 0.1 μg/mL of K. pneumoniae LPS resulted in a slight neutralization in the immune response (Figure 4B). The neutralization effect increased when BPEI concentrations were increased, (Figure 4C). This finding indicates a dose-dependent relationship between BPEI and the disruption of TNF-α production induced by K. pneumoniae LPS.

The binding of 600 Da BPEI to P. aeruginosa LPS has been reported (34,39). Thus, it was expected that BPEI would effectively reduce the TNF-α secretion induced by P. aeruginosa LPS. However, contrary to these expectations, 0.01, 0.1, 1.0, or 10 μg/mL of BPEI mixed with 0.1 μg/mL P. aeruginosa LPS did not exhibit any reduction in TNF-α secretion (Figure 5B). Greater success was achieved when 25, 50, 75, or 100 μg/mL BPEI was used for neutralization. Future studies will investigate the reason(s) why BPEI neutralization is dependent on the bacterial strain used to obtain the LPS PAMPs. It is likely that structural and/or compositional differences between the LPS from E. coli and P. aeruginosa may explain the observed differences in the ability of cationic BPEI (Figure 8) to neutralize the LPS from these pathogens. For instance, the LPS from P. aeruginosa contains numerous anionic carboxylate groups in the O-antigen region (Figure 8B) (42–45) that provide binding sites for interactions with BPEI. However, these binding sites are located away from the alkyl tail region that is the binding site for LBP and subsequent co-receptors that lead to TLR2 activation. In this scenario, BPEI binding with isolated and soluble P. aeruginosa LPS may not hinder cytokine release. In contrast to this hypothesis, the LPS from E. coli does not contain anionic charges in the O-antigen region (Figure 8C) and thus BPEI will bind with the anionic sites of the inner core and lipid A regions (42–45). Thus, BPEI binding introduces steric barriers to interfere with LBP binding and TLR2 activation. These effects are not observed with low amounts of BPEI mixed with P. aeruginosa LPS because BPEI can bind with the distal O-antigen region. Hence, lower amounts of secreted cytokine are not observed unless higher BPEI concentrations are used to occupy both the O-antigen and lipid A regions.

Figure 5.

Figure 5.

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600 Da BPEI neutralization of LPS from P. aeruginosa. Panel A shows the timepoint assay of TNF-α production from THP-1 cells were treated with 0.1 μg/ml of P. aeruginosa LPS. The cells were then incubated for 2, 4, 6, 8, and 24 h, and supernatants were collected at the end of each time-point. Levels of TNF-α cytokines were quantified using ELISA. Untreated cells were used as controls. Timepoints that resulted in the highest secretion of TNF-α protein were selected as the optimum timepoints for stimulating THP-1 cells with P. aeruginosa LPS. Panel B shows reduced TNF-α cytokine levels when the P. aeruginosa LPS was premixed with 0.01, 0.1, 1, and 10 μg/mL of 600 Da BPEI whereas Panel C shows data for premixing LPS with 25, 50, 75, and 100 μg/mL. All experiments were performed in triplicate and the presented data are representative results of the means ± standard error of the mean (SEM). A 95% confidence value with a p-value consisting of p < 0.05 was considered statistically significant.

Figure 8.

Figure 8.

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Low-molecular weight (600 Da) BPEI is contains numerous cationic primary amines (Panel A). The lipopolysaccharide (LPS) structures from P. aeruginosa and E. coli bacteria are shown in Panels B and C, respectively. Both LPS molecules contain three main components: lipid A, a core oligosaccharide, and O-antigen. The O-antigen region in E. coli LPS is composed of D-Galactose. The O-antigen region in P. aeruginosa LPS contains several carboxyl groups which add to the negativity of the overall charge of the molecule. However, in the E. coli LPS structure, O-antigen is constituted of a polysaccharide chain attached to the core oligosaccharide and is free of charge. The O-antigen region is comprised of N-acetyl-α-D-fucosamine (Fuc2NAc, labeled as 1); 2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid (ManNAc(3NAc)A, labeled as 2); and 2-acetamido-3-acetamidino-2,3-dideoxy-β-D-mannuronic acid (ManNAc(3NAm)A, labeled as 3). This O-antigen composition is known as B-band, whereas A-band O-antigen is comprised solely of D-rhamnose groups. The carboxylate groups in the B-band O-antigen provide anionic sites to attract cationic 600 Da BPEI and cationic PEG350-BPEI to the LPS molecules in the bacterial outer membrane. Assembly of the B-band involves enzymes in the Wbp and Wzy pathways. GlcN = D-Glucosamine; KDO = Ketodeoxyoctonic acid; Hep = Heptose; Glc = D-Glucose; Gal = D-Galactose; NHAc = N-acetyl group; NHAm = N-amidine.

Regarding intact whole bacterial cells, the LPS molecules are not isolated but instead reside in a dense monolayer to form the outer leaflet of the bacterial outer membrane. This arrangement exposes the O-antigen region to the extracellular environment where it can interact with the host cells. While whole cell bacteria are known to stimulate TLRs, the mechanism and identification of the signaling initiation is not well understood. It is likely that LPS is involved, but it is also possible that other bacterial cell surface molecules participate and provide redundancy in the recognition of the immune system. Therefore, when BPEI binds to whole cells it can interfere with the signaling cascade by limiting recognition of the LPS O-antigen or other nearby molecules on the cell surface. We measured the ability of BPEI to neutralize the production of TNF-α induced by heat-killed P. aeruginosa bacteria (HKPA). As shown in Figure 6B and 6C, BPEI resulted in a small reduction in the immune response although increasing BPEI concentrations (50–150 μg/mL) resulted in up to ~ 30% reduction in TNF-α protein levels.

Finally, because fungal PAMPs also activate the innate immune response, we examined the interaction between fungal zymosan and BPEI and the effect on TNF-α secretion. Zymosan, a cell wall component derived from the yeast S. cerevisiae, can activate TLR receptors and induce the production of TNF-α (21–22). For this purpose, THP-1 cells were stimulated with different combinations of zymosan (25 μg/mL) and BPEI (ranging from 50 to 125 μg/mL). Interestingly, the results depicted in Figure 7 show that 600 Da BPEI effectively neutralized zymosan, leading to a significant reduction in cytokine production. Furthermore, BPEI exhibited a dose-dependent pattern in mitigating the production of TNF-α protein, with a 50% reduction observed at a concentration of 125 μg/mL. The binding mechanism between branched polyethyleneimines and zymosan is presently unclear. Further investigations are needed to unravel the underlying interaction mechanism.

Figure 7.

Figure 7.

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ELISA data show the amount of cytokine TNFα released by human monocyte cells (THP-1 cells) in responses to combinations of 600-Da BPEI and S. cerevisiae zymosan. Data are shown as an average of two biological replicates. Error bars denote standard deviation. Untreated cells were used as controls. All experiments were performed in triplicate and the presented data are representative results of the means ± standard error of the mean (SEM). A 95% confidence value with a p-value consisting of p < 0.05 was considered statistically significant.

Discussion

Lipopolysaccharide (LPS) is located in the outer membrane of Gram-negative bacteria. It plays a significant role in immune system interactions and the generation of immune responses. The LPS molecule is consisted of major components: lipid A, which is a hydrophobic portion responsible for anchoring LPS to the outer leaflet of the outer membrane; core oligosaccharide, which along with lipid A, helps maintain the integrity of the outer leaflet; and O-antigen polysaccharide, which is connected to the core and forms a polymer consisting of repeated oligosaccharide units that directly interact with the external environment (41,46–47). While LPS structures in various Gram-negative species share common characteristics, there are also variations that contribute to the unique properties and pathogenicity of each bacterium (42–45). These modifications, particularly in the Lipid A component, play a significant role in the bacteria’s pathogenicity and their interactions with the host immune system (40).

LPS molecules activate TLR4 receptor and induce the downstream signaling cascade that eventually produce inflammatory cytokines (15–16). TLR4 activation requires interaction with several co-receptors through a series of consecutive steps. LPS first combines with LPS-binding protein (LBP) and then LBP transfers LPS molecule to differentiation 14 (CD14) protein. CD14 in turn chaperones the formation of LPS and myeloid differentiation factor 2 (MD2) complex (15) (Figure 9). In this work, we demonstrated the ability of 600 Da BPEI to mitigate TNF-α cytokine production induced by Gram-negative LPS in THP-1 monocyte cells. We suggest a mechanism of interaction between the cationic BPEI with the anionic groups of LPS (Figure 9). BPEI, via cationic amines, can electrostatically bind with the anionic groups of LPS molecules and prevent subsequent binding with co-receptors. This results in lower cytokine production.

Figure 9.

Figure 9.

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Proposed mechanism of action of LPS and BPEI interactions. LPS molecules activate TLR4 receptor and induce the downstream signaling cascade that ultimately produces inflammatory cytokines. TLR4 activation requires interaction with several co-receptors through a series of consecutive steps. Cationic BPEI binds with LPS through electrostatic interactions and prevents it from combining with co-receptors. This eventually results in less TLR4 dimerization and mitigated cytokine production.

Overall, the results of this study represent a qualitative investigation of the effect of 600 Da BPEI against Gram-negative LPS. This is due to the presence of inherent complexities in this work which prevented a more quantitative assessment of BPEI neutralizing Gram-negative LPS induced-immune response. First, the molecular weight of LPS is not well understood in the literature which makes it impossible to measure the accurate molar ratio of LPS to BPEI. Also, the binding site of BPEI and LPS is unknown. We believe that the presence of LBP, CD4, and MD2 co-receptors that bind to LPS and prepare it to bind to TLR4 receptor are different than the BPEI binding site. These differences prevent BPEI from efficiently neutralizing LPS. Also, the various structural characteristics of LPS molecules isolated from different Gram-negative species create the possibility of different binding patterns with BPEI. This results in variations of the neutralizing efficiency of BPEI against the varied LPS molecules.

Nevertheless, the findings in this study demonstrate the ability of 600 Da BPEI to effectively reduce the production of inflammatory responses induced by S. cerevisiae zymosan (Figure 7). To the best of our knowledge, this is the first time that the release of inflammatory cytokines has been mitigated by combining zymosan glycoprotein with another compound. β-glucans possess multiple hydroxyl groups that facilitate bonding with reactive groups of other compounds, thereby modifying their water solubility, conformation, and capacity to form aggregates (48). Based on this, we propose three mechanisms of interactions between BPEI and β-glucans molecules. First of all, we suggest that the deionization of hydroxyl (OH) groups in zymosan occurs in the presence of amine groups of BPEI. Zymosan is composed of a mixture of carbohydrates, including β-glucans and mannans, as well as proteins (20–21). The β-glucan structure in zymosan contains hydroxyl (OH) groups attached to the glucose molecules which play a crucial role in stabilizing the overall structure through interstrand hydrogen bonding (49). Under physiological pH conditions, the majority of hydroxyl groups in the β-glucan backbone do not undergo significant deprotonation. This is because the pKa values of these hydroxyl groups are higher than the pH range typically found in physiological conditions. However, when cationic BPEI is present in the solution, the OH groups become deprotonated. The deprotonation of hydroxyl groups has the potential to influence the overall structure and properties of the β-glucan molecule, thereby potentially modifying its biological activity (49). Moreover, the interaction of zymosan with BPEI can be attributed to cooperative hydrogen bonding between the hydroxyl groups on the β-glucan of zymosan and the amine groups on the BPEI. Since solubility of β-(1→3)-glucans increases in basic media (50). The interaction between BPEI and zymosan can result in the formation of aggregates, which, in turn, can disrupt the organized structure of zymosan and impact its biological activity. Lastly, we propose the possibility that BPEI can form conjugates with β-glucan, resulting in modifications to the zymosan molecule. The presence of functional hydroxyl and aldehyde groups in β-glucan enables it to undergo conjugation with other biomolecules that have a strong affinity for binding to β-glucan, such as proteins and amino acids (48). Due to the shared presence of amine groups with amino acids, it is likely that the cationic compound BPEI utilizes a similar mechanism of interaction to form conjugates with β-glucan. Nevertheless, a comprehensive experimental investigation or specific understanding of the interactions involved is necessary to determine the precise behavior of β-glucan in the presence of cationic compounds like BPEI.

CONCLUSION:

Although short-term activation of the innate immune system is beneficial and serves as crucial defensive mechanisms of the host against infection, excessive activation of Toll-like receptors (TLRs) is harmful as it disturbs immune homeostasis by causing prolonged production of pro-inflammatory cytokines and chemokines (51). Dysregulated activation of the inflammasome is implicated in a range of autoimmune and inflammatory diseases (52). However, dysregulated inflammatory response often results in impaired healing, poor tissue restoration and function, and increased scarring (53–55). We envision BPEI as a topical agent for acute and chronic wounds because it is a multi-functional agent that can address bacterial infection, biofilms, reduce inflammation. In this work, we continue to investigate 600 Da BPEI and demonstrate its anti-PAMP properties due to the electrostatic interactions between the cationic amines of 600 Da BPEI and the anionic phosphates and carboxylate groups of LPS molecules (34,39). In this manner, BPEI may help mitigate overproduction of inflammatory cytokines. The antagonistic effect of 600 Da BPEI molecules were more pronounced for intact bacterial cells and fungal zymosan. This builds on previous work where 600 Da BPEI was shown to be a broad-spectrum antibiotic potentiator that can overcome antibiotic resistance and eradicate biofilms of multidrug resistant strains of Gram-positive and Gram-negative bacteria including methicillin resistant Staphylococcus aureus (MRSA), methicillin resistant Staphylococcus epidermidis (MRSE), Pseudomonas aeruginosa, and carbapenem resistant Enterobacteriaceae (CRE) (27–36).

Value proposition

BPEI offers a two-pronged value proposition to healthcare providers and life sciences organizations alike.

For life sciences organizations specializing in wound care, BPEI offers a low-cost, off-the-shelf technology that can be easily incorporated into wound lavage solutions, gels, creams, and foams, reducing the cost and complexity of manufacturing wound care products.

For healthcare providers treating patients with slow-healing wounds, BPEI provides broad-spectrum properties that can target multiple factors that inhibit healing, simplifying wound care treatment while also improving patient outcomes.

Competitive advantage

Existing wound care products often contain alcohol, iodine, silver, chlorinated compounds, peptides, QACs, enzymes, and/or other antimicrobials. Numerous products exist because any one of them cannot address the diverse causes of slow wound healing. By comparison, BPEI is a singular active ingredient with broad-spectrum properties. It simultaneously targets many factors that inhibit healing, including Gram-positive pathogens, Gram-negative pathogens, their bacterial biofilms, fungi, and pro-inflammatory toxins. To the best of our knowledge, there are no existing devices, pharmaceuticals, or OTC products that incorporate these features into one product.

The novelty of the science or the invention

As a small-molecule, low molecular-weight compound, cationic BPEI binds to anionic targets found within pathogens, biofilms, and toxins. Since BPEI is not a peptide or an enzyme, it remains stable in the wound environment. BPEI also functions as a buffer to modulate pH within the wound environment, accelerating wound healing.

Acknowledgements

This work was made possible due to the guidance and contributions of Prof. Christina Bourne and Dr. Yunpeng Lan in the Department of Chemistry and Biochemistry at the University of Oklahoma. We would also like to thank Dr. Phil Bourne and the Protein Production Core (PPC) at the University of Oklahoma. The PPC is supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103640.

Funding Sources

Funding was provided by the National Institutes of Health (CVR, R03AI142420) and The University of Oklahoma. This study was also sponsored by Grant W81XWH2210047 from the Department of Defense/USAMRAA (PRMRP #PR210140). The content is solely the responsibility of the authors and does not necessarily represent the official views of the Department of Defense.

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