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Differential proinflammatory responses of colon epithelial cells to SARS-CoV-2 spike protein and Pseudomonas aeruginosa lipopolysaccharide

  • Aysegul Yılmaz ORCID logo EMAIL logo , Seyhan Turk ORCID logo , Ümit Yavuz Malkan ORCID logo , İbrahim Celalettin Haznedaroglu ORCID logo , Gulberk Ucar ORCID logo , Sukru Volkan Ozguven ORCID logo and Can Turk ORCID logo
Published/Copyright: October 24, 2024
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Turkish Journal of Biochemistry
From the journal Turkish Journal of Biochemistry Volume 49 Issue 6

Abstract

Objectives

The study aims to compare the proinflammatory responses of colon epithelial cells to two potent virulence factors: lipopolysaccharide (LPS) from Pseudomonas aeruginosa and spike (S) protein of SARS-CoV-2. Both agents are known to induce significant inflammatory responses, leading to severe clinical manifestations.

Methods

Human colon epithelial cells were treated with S protein and LPS at various time intervals (12, 24, 48, and 72 h). Cell viability was assessed, and the expression levels of key proinflammatory cytokines (IFN-γ, IL-1β, TNF-α, and IL-6) were measured using qRT-PCR. Statistical analyses were conducted to assess the data, incorporating t-tests and linear regression.

Results

The study found distinct patterns in cytokine expression in response to S protein and LPS. LPS treatment led to a rapid increase in cytokine expression at early time points (12 and 24 h), followed by a decline at later intervals. In contrast, S protein induced a more sustained proinflammatory response, with lower initial cytokine levels that persisted longer, particularly at 48 and 72 h.

Conclusions

The differential proinflammatory responses observed between S protein and LPS treatments highlight their unique impacts on colon epithelial cells. Specifically, LPS induced an early but transient spike in cytokine levels, suggesting a rapid but short-lived inflammatory response. Conversely, the S protein triggered a prolonged inflammatory reaction, which may contribute to the persistent symptoms seen in COVID-19. The findings provide insights into the molecular mechanisms underlying inflammatory responses in bacterial and viral infections. Understanding these differences can inform therapeutic strategies for conditions like sepsis and COVID-19, leading to targeted treatments that mitigate excessive inflammation and improve patient outcomes.

Keywords: spike; LPS; Pseudomonas aeruginosa ; proinflammatory genes; colon epithelial cell

Introduction

Pseudomonas aeruginosa (P. aeruginosa) is classified as a gram-negative bacterial species isolated from various environments such as plants, fruits, soil, rivers, and swimming pools. This bacterium is a significant opportunistic pathogen that commonly causes respiratory tract infections, urinary tract infections, and bacteremia. Infections acquired in hospital settings, especially from intensive care units, are among the most frequent causes of hot tub dermatitis and external otitis [1].

Gram-negative bacteria feature an outer membrane primarily comprised of lipopolysaccharide (LPS), exemplified by P. aeruginosa and Escherichia coli. LPS is a voluminous glycolipid that is segmented into three distinct domains: lipid A, core polysaccharide, and the O antigen [1]. LPS engages in a conformational link with myeloid differentiation factor 2 and is subsequently discerned by Toll-like receptor 4 (TLR4) as a pathogen-associated molecular pattern (PAMP). In addition, LPS has an affinity for the LPS-binding protein and the intermediary receptor CD14. This sequential interaction of proteins facilitates the transference of LPS, culminating as a robust instigator of the inflammatory cascade in septic conditions. Heightened inflammatory responses have the possibilities to induce acute respiratory distress syndrome (ARDS) as well as culminate in multi-organ failure [2]. Community-acquired P. aeruginosa has been identified as a causative agent of infectious diarrhea in immunocompromised adults and even immunocompetent children. In some cases, this infection leads to necrotizing bowel lesions, which are further complicated by fulminant septicemia and high mortality rates. This suggests that intestinal epithelial cells, which are the initial point of contact with the pathogen, play a crucial role in the innate immune response against P. aeruginosa infection [3].

COVID-19, engendered by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibits a pronounced propensity for rapid dissemination [4, 5]. Coronaviruses are composed of four principal structural proteins: E (envelope), S (spike), N (nucleocapsid), and M (membrane). The S protein is compounded of a transmembrane trimeric glycoprotein extending outward from the viral envelope. The structure is bifurcated into two distinct subunits: S1, which attaches to the cell receptor for virus entry, and S2, which facilitates the amalgamation of the viral and cellular membranes [6, 7]. Biochemical investigations have elucidated that the S protein instigates the activation of the NF-κB signaling cascade via an adaptor molecule termed myeloid differentiation primary response 88, which conveys extracellular signals to the cell’s interior. This activation triggers inflammation. In COVID-19 patients, an association has been observed between the viral RNA concentration of SARS-CoV-2 and the bacterial load of P. aeruginosa [1, 8]. During ARDS, the activation of TLR4 by LPS induces leukocyte migration to the lungs, intense proinflammatory cytokine synthesis, and subsequent lung damage seen in coronavirus infections [9].

The disease is known to affect not only alveolar epithelial cells but also intestinal epithelial cells. Fever and diarrhea are the most common symptoms observed in P. aeruginosa sepsis in infants and children. This suggests intestinal epithelial cells, which come into initial contact with the pathogen, may play a significant role in natural immunity against P. aeruginosa infection. The resulting cytokine storm exhibits striking similarities to the profile induced by LPS [10]. In COVID-19 infection, a cytokine storm occurs due to the uncontrolled inflammatory response of cellular immunity. This response involves a diverse array of cells, notably macrophages and neutrophils, which detect pathogens and damaged self-structures and subsequently induce inflammatory mediators. Additionally, epithelial and endothelial cells contribute to the inflammatory response and may die due to the resulting inflammation [11].

Proinflammatory cytokines are generated subsequent to the activation of pattern recognition receptors that recognize viral PAMPs or host-derived molecules signaling a disturbance in physiological equilibrium (danger-associated molecular patterns). This triggers a cytokine storm, leading to the viral sepsis observed in critically ill COVID-19 patients, characterized by multisystem immune dysregulation. SARS-CoV-2 instigated viral sepsis arises from the actions of a spectrum of proinflammatory cytokines, encompassing interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and IL-17. Cytokine levels have been associated with disease severity, and an inverse correlation has been demonstrated between IL-6, IL-8, TNF-α, and survival [12, 13]. These findings further emphasize the prospective pathogenic capacity of extreme proinflammatory responses in triggering the disease [14].

In individuals suffering from COVID-19, a range of gastrointestinal symptoms, including diarrhea, abdominal discomfort, emesis, and loss of appetite, have been documented alongside respiratory symptoms. In more than 10 % of patients, COVID-19 presents solely with gastrointestinal (GI) symptoms without pneumonia. Multiple studies have underscored the importance of GI symptoms in COVID-19 and their correlation with disease severity and systemic inflammation. The upregulation of ACE2 in the GI tract is believed to contribute to these symptoms in COVID-19 patients. Intestinal epithelial cells, which constitute the predominant cell type in the gut, possess a variety of RNA sensors to defend against viral invasion. Nonetheless, the precise pathogenesis and mechanisms underlying GI symptoms during SARS-CoV-2 infection remain incompletely elucidated [15, 16].

In light of this information, we aimed to determine the relationship of S protein, which is of great importance in COVID-19 infection in intestinal epithelial cells, with LPSs and the expression differences of inflammation-related genes.

Materials and methods

Reagents

All reagents for cell culture were acquired from Capricorn Scientific (Germany). LPS sourced from P. aeruginosa (catalog no. L9143, Sigma-Aldrich, USA) was procured in a desiccated form and conserved at temperatures ranging from 2 to 8 °C until utilized. The S protein was secured from Elabscience Bionovation Inc. (catalog no. PKSR030484, USA). The cell proliferation kit II (XTT) was obtained from Sigma-Aldrich, headquartered in the USA. Quantitative reverse transcription PCR (qRT-PCR) kits were employed in accordance with the accompanying protocol. The A.B.T.™ 2X qPCR SYBR-green mastermix was sourced from ATLAS Biotechnology in Türkiye. All substances and apparatuses employed in this investigation were either procured sterile or sterilized via autoclaving before usage.

Cell culture

Colon epithelial cells of human origin, catalog number CRL-1831, were procured from the American Type Culture Collection. These cells underwent incubation in a medium modified from Dulbecco’s eagle formulation, enriched with l-glutamine and augmented by 10 % fetal bovine serum, along with 100 μg/mL of penicillin and 100 μg/mL of streptomycin, all within a controlled environment maintaining 5 % CO2 at 37 °C. The experimental protocols commenced upon the cells attaining a confluence range of 70–80 % [17].

Cell proliferation

For the assessment of cell proliferation, an XTT assay based on the sodium salt of benzene sulfonic acid hydrate was utilized. Cells were distributed into 96-well plates at a concentration of 104 cells per well, where they were cultivated over a 24 h incubation duration. Subsequent to this incubation phase, CRL-1831 colon epithelial cells were treated with P. aeruginosa LPS and S protein at the determined doses (0.1, 1, 5, 10, 50, and 100 ng/mL) for 12, 24, 48, and 72 h in conjunction with the assay’s reagents. Following the treatment, the cell cultures were subjected to incubation with 50 µL of XTT reagent at 1 mg/mL for a duration of 4 h. Subsequently, absorbance was quantified utilizing an ELISA microplate reader (Biotek Synergy H1, USA) at a wavelength of 450 nm [18].

Gene expression analysis

Gene expression analysis was conducted to assess the impact of S protein and LPS on the immune response in CRL-1831 cells. Cells were exposed to 100 ng/mL LPS and 100 ng/mL S protein for durations of 12, 24, 48, and 72 h. Following treatment, RNA isolation was performed using TRIzol (ABP Biosciences, China). Intestinal epithelial cells treated with 100 ng/mL LPS and 100 ng/mL of S protein underwent mRNA isolation at specified time intervals, and TNF-α, IFN-γ, IL-1β, and IL-6 gene expression levels were determined using the qRT-PCR method. Gene expression was evaluated using real-time PCR with 100 ng of RNA on both the Lightcycler® 96 system (Roche Diagnostic Systems, USA) and A.B.T.TM 2X qPCR SYBR-green mastermix for analysis of qRT-PCR. Primers targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH), IL-1β, IL-6, TNF-α, and IFN-γ were utilized. The amplification protocol commenced with an initial preheating stage at 55 °C for 5 min and 95 °C for 5 min, succeeded by 40 cycles consisting of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min. Melting curve analyses were conducted to ensure specificity, and the expression data were normalized to the threshold cycle values of the GAPDH gene [19]. Primer sequences are detailed in Supplementary Data 1.

Statistical analysis

Statistical analyses were meticulously carried out for each experiment, which was replicated a minimum of three times. Experiment results were expressed as the mean ± SD. Graphs and statistical analyses were performed with GraphPad Prism (version 8.0.1, USA). Using linear regression analysis, the changes in IFN-γ, IL-1β, TNF-α, and IL-6 expression levels within 12, 24, 48, and 72 h after treatment with LPS and S protein were determined. Also, IL-1β, TNF-α, IFN-γ, and IL-6 levels in CRL-1831 cells following treatment with S protein and LPS were compared to the control group at specified time intervals. The data were scrutinized using two-tailed t-tests, and optical density values between treated and untreated cells were compared with statistical significance set at p<0.05.

Results

Cell viability in CRL-1831 cells treatment with LPS and S protein

Figure 1A depicts the dose-dependent cell viability modulation at each temporal juncture post-LPS treatment. Figure 1B presents the correlation between cell viability and escalating concentrations at each measured interval following S treatment. In LPS-treated cells, no significant change in viability levels was detected at 12 and 24 h. At 48 h, cell viability rates decreased significantly. At 72 h, a decrease in viability was also observed. When the dose ratios were examined, it was seen that with the increase in dose treatments in 12 and 24 h, especially 50 ng/mL and 100 ng/mL LPS application, caused a decrease in the viability level compared to other ratios. When S protein treatment was applied, cell viability decreased in the first 12 h, and this decrease continued until 24 h. At 48 h, there was a decrease in viability at rates similar to 24 h, while the lowest percentage of viability was detected with 72 h of treatment. In both S protein and LPS treatment, a decrease in viability rates was detected with time as well as dose increase. At 72 h, a greater decrease was observed in the viability of S-treated cells compared to other time periods. When S protein dose ratios were analyzed, it was observed that similar to LPS, especially 50 and 100 ng/mL S treatment caused a significant decrease in viability levels at 12 and 24 h. At 48 h, a similar decrease was observed, while at 72 h, 100 ng/mL S protein application caused a remarkable decrease. However, although viability levels decreased, it was determined that both LPS and S protein did not cause a cytotoxic effect compared to the control group at all doses.

Figure 1: Dose-response curves of cytotoxicity assessment in CRL-1831 cells. The cells were treated to various doses of LPS and S protein (0.1, 1, 5, 10, 50, and 100 ng/mL). Dose-response curves of cytotoxicity of LPS (A) and S protein (B) in CRL-1831 cells after 12, 24, 48, and 72 h-incubation time. It is indicated that both LPS and S protein are not in cytotoxic doses. LPS, lipopolysaccharide.
Figure 1:

Dose-response curves of cytotoxicity assessment in CRL-1831 cells. The cells were treated to various doses of LPS and S protein (0.1, 1, 5, 10, 50, and 100 ng/mL). Dose-response curves of cytotoxicity of LPS (A) and S protein (B) in CRL-1831 cells after 12, 24, 48, and 72 h-incubation time. It is indicated that both LPS and S protein are not in cytotoxic doses. LPS, lipopolysaccharide.

Time-dependent effects of LPS on immune genes’ expressions

The fold change ratio was calculated by comparing TNF-α, IFN-γ, IL-1β, and IL-6 levels between the LPS or S protein-treated groups and the control group at each time point. Genes with fold change ratios below one were considered downregulated, while those above one were considered upregulated. Examination of IFN-γ levels in LPS-treated cells revealed an initial increase in gene expression at 12 h, followed by a gradual decrease in expression levels in subsequent time intervals. In TNF-α levels, it was observed that expression levels increased within the first 12 and 24 h. However, expression levels decreased at 48 h and beyond. Analysis of IL-1β levels showed a pattern similar to TNF-α, with a significant increase in expression levels observed within the first 24 h, followed by a substantial decrease by the 48 h. While IL-6 expression levels were found to be significantly high within the first 12 h, a gradual decrease in expression levels was observed at 24, 48, and 72 h (Figure 2A).

Figure 2: Time dependent changes in inflammatory cytokine expression induced by LPS (A) and S protein (B). This figure illustrates the fold changes in TNF-α, IFN-γ, IL1β, and IL-6 gene expression levels. Relative mRNA expressions were quantified using qRT-PCR. The data were normalized to GAPDH and presented as fold changes relative to untreated cells. Control values are represented as dashed lines. Above the dashed line indicates upregulation, below it indicates downregulation. LPS, lipopolysaccharide.
Figure 2:

Time dependent changes in inflammatory cytokine expression induced by LPS (A) and S protein (B). This figure illustrates the fold changes in TNF-α, IFN-γ, IL1β, and IL-6 gene expression levels. Relative mRNA expressions were quantified using qRT-PCR. The data were normalized to GAPDH and presented as fold changes relative to untreated cells. Control values are represented as dashed lines. Above the dashed line indicates upregulation, below it indicates downregulation. LPS, lipopolysaccharide.

Time-dependent effects of S protein on immune genes’ expressions

It was observed that at 48 h, the expression of IFN-γ gene began to increase, and the increase continued at 72 h. An increase in TNF-α and IL-1β expression was observed from 12 h onwards, and the expression increase persisted until 72 h. The expression level of IL-6 showed an initial increase within the first 12 h, followed by a decrease at 24 h, an increase at 48 h, and another decrease at 72 h (Figure 2B).

Correlation analysis of S protein and LPS treatment on CRL-1831 cells

It was found that two of these genes (TNF-α and IFN-γ) exhibited a significant correlation with time. Specifically, a significant decrease in TNF-α expression values over time was observed in cells treated with LPS. In contrast, although no significant differences were observed over time in other groups following LPS treatment, a decrease in all gene expressions compared to the control group was evident (Figure 3A). Conversely, after treatment with S protein, an increase in gene levels was observed throughout the specified time intervals. A significant increase was only detected in IFN-γ (p=0.008), while values very close to the significance level were obtained for IL-6, IL-1β, and TNF-α (p=0.056, p=0.081, p=0.094, respectively) (Figure 3B).

Figure 3: Validation of qPCR assay using standard curves. DNA from CRL-1831 cells was subjected to qPCR amplification in threefold reactions. Standard curves were then constructed by charting the relative quantification outcomes against the time points of LPS and S protein exposure, specifically at 12, 24, 48, and 72 h. RQ, relative quantification.
Figure 3:

Validation of qPCR assay using standard curves. DNA from CRL-1831 cells was subjected to qPCR amplification in threefold reactions. Standard curves were then constructed by charting the relative quantification outcomes against the time points of LPS and S protein exposure, specifically at 12, 24, 48, and 72 h. RQ, relative quantification.

The impact of S protein and LPS treatment on immune gene expression in CRL-1831 cells

Figure 4 illustrates only the cytokine groups where significant differences were observed. No significant differences were observed in either the LPS or S protein groups after 12 h of treatment. However, following 24 and 48 h of LPS treatment, a significant difference was observed in IL-1β expression levels. These levels increased significantly at 24 h (p=0.035) and decreased significantly at 48 h (p=0.001). Additionally, TNF-α levels significantly decreased compared to the control group after 72 h of LPS treatment (p<0.001). Furthermore, levels of IFN-γ significantly decreased after 48 h of LPS treatment (p=0.006), and IL-6 levels significantly decreased after 72 h of LPS treatment (p=0.004). Following treatment with S protein, although borderline results were observed, no significant differences were detected at any time point (Supplementary Data 2). Specifically, TNF-α levels at 12 h (p=0.063) and IL-1β levels at 24 h (p=0.073) were found to be close to significance, although not statistically significant. Other data that were not statistically significant are presented in Supplementary Data 2.

Figure 4: Differential gene expression in CRL-1831 cells at 24, 48, and 72 h following LPS and S protein treatment. This figure shows only significantly differently expressed genes. With LPS treatment, IL-1β at 24 h (p=0.035) and at 48 h (p=0.001), IFN-γ at 48 h (p=0.006), TNF-α at 72 h (p<0.001) and IL-6 at 72 h (p=0.004) have shown statistically significant changes. RQ, relative quantification; LPS, lipopolysaccharide.
Figure 4:

Differential gene expression in CRL-1831 cells at 24, 48, and 72 h following LPS and S protein treatment. This figure shows only significantly differently expressed genes. With LPS treatment, IL-1β at 24 h (p=0.035) and at 48 h (p=0.001), IFN-γ at 48 h (p=0.006), TNF-α at 72 h (p<0.001) and IL-6 at 72 h (p=0.004) have shown statistically significant changes. RQ, relative quantification; LPS, lipopolysaccharide.

Discussion

Our study results have demonstrated that the application of spike protein and LPS, which induce a superantigen-like effect, affects proinflammatory gene expression in colonic epithelial cells over specified time intervals. Specifically, the S protein leads to an upregulation of these genes over progressive time intervals, while LPS application results in their downregulation. Superantigens are potent antigens that can cause overly rapid responses in the immune system and affect a significant portion of the pure T cell population (sometimes up to 30 %). Interactions between superantigens and T cells can lead to various outcomes, including anergy (functional inactivation of T cells), inflammation, cytotoxicity, and autoimmunity. Contrary to conventional antigens, superantigens bypass the need for processing by antigen-presenting cells. A functional superantigen directly interacts with the variable region of the beta chain of the T cell receptor (TCR) and major histocompatibility complex class II (MHC-II) molecules. Consequently, it accelerates the T cell selection process by bypassing antigen presentation and rapidly activating substantial proportion of T cells, potentially encompassing up to 20 % [20, 21].

There are various studies regarding the superantigenic activity of the S protein. Cheng et al., using computational models based on molecular structure, demonstrated that the SARS-CoV-2 S glycoprotein binds to TCRs with high affinity and can form a ternary complex with class II MHC. They suggested that the S protein could directly interact with the variable region of the TCR β chain through the putative superantigen motif (T678 to Q690), thereby activating T cells and triggering hyperactive adaptive immune responses [22]. Studies have also investigated COVID-19-associated diseases, such as multisystem inflammatory syndrome in children, characterized by persistent fever and hyperinflammation in various organs, including the heart, GI system, and kidneys 22], [23], [24.

P. aeruginosa, an opportunistic pathogen, interacts with host receptors to critically influence its pathogenesis. LPS can play a pivotal role in P. aeruginosa pathogenesis. Known as a potent endotoxin, LPS triggers a robust inflammatory response in the immune system. This strong response supports the superantigen-like properties of LPS. By inducing widespread inflammation in the immune system, LPS can lead to overstimulation of T cells. This condition can result in serious health issues such as septic shock [25]. P. aeruginosa LPS was used in this study due to its role as a key molecule on the bacterial cell wall recognized by the host to neutralize pathogens. In mammals, LPS serves as a molecular pattern associated with microorganisms that can induce activation of signaling pathways leading to the production of proinflammatory cytokines, thus acting as a potent activator of the host’s innate immune response [1].

The S protein and LPS both possess superantigen-like properties that elicit robust reactions in the immune system and lead to the release of proinflammatory cytokines. Hence, in this study, the S protein and P. aeruginosa LPS molecule were preferred for investigating their effects on intestinal epithelial cells. Considering their similar clinical presentation and cytokine release effects, the aim was to examine the effects induced by treatment with S protein and LPS at specified intervals in intestinal epithelial cells, particularly in relation to proinflammatory cytokines.

TNF-α is a proinflammatory cytokine produced by macrophages and monocytes during acute inflammation, responsible for various intracellular signaling events that can lead to target cell necrosis or apoptosis under specific conditions. TNF-α has been shown to promote the production of inflammatory cytokines, enhance the adhesion and permeability of endothelial cells, and recruit immune cells such as neutrophils, monocytes, and lymphocytes to sites of inflammation. These activities facilitate both acute and chronic systemic inflammatory responses in the context of infection or autoimmunity. Additionally, TNF-α exhibits selective cytotoxicity towards transformed cells. Elevated levels of TNF-α have been associated with cachexia and endotoxin-induced septic shock. TNF-α initiates a rapid and robust immune response, limiting the extent and duration of inflammation once the invasion has been resolved. Furthermore, as a co-stimulator, TNF-α enhances the responses of neutrophils, monocytes, and lymphocytes, thereby defending against microbial infections [26].

Interleukins orchestrate both pro-inflammatory and anti-inflammatory responses, as well as the differentiation and activation of immune cells. Initially believed to mediate communication solely between leukocytes (hence the term interleukin), it is now recognized that a diverse array of cell types produce these cytokines. IL-1 is pivotal in T-cell-mediated immunity, facilitating the secretion of IL-2, which is crucial for T-cell homeostasis, and enhancing the expression of IL-2 receptors. IL-1β amplifies acute-phase signaling, directs the migration of immune cells to the primary infection site, activates epithelial cells, and stimulates the production of secondary cytokines. The acute-phase response to infection manifests through a variety of local and systemic effects that are predominantly proinflammatory, such as the elevated production of specific cytokines, which can contribute to viral clearance [27].

IL-6 is a significant proinflammatory cytokine with diverse inflammatory functions. IL-6 influences the activity of various cell types; thus, it is characterized as a pleiotropic cytokine. It functions both as a proinflammatory cytokine and an anti-inflammatory myokine. A diverse array of immune cells, including macrophages, neutrophils, dendritic cells, and lymphocytes, secrete IL-6. Notably, the release of IL-6 in an inflammatory environment is attributed to the numerous cells that produce it, which are structural components of the infected tissue rather than solely immune cells. These include mesenchymal cells, endothelial cells, and fibroblasts, among others. These observations underscore the abundance and significant potential of IL-6 in inflammatory conditions. Centrally, IL-6 facilitates the differentiation of naive CD4 T cells into effector and helper cells. By bridging innate immunity with adaptive immune responses, IL-6 promotes TH7 differentiation, as well as the activation and differentiation of cytotoxic CD8 T lymphocytes [28]. From a biological perspective, IFN-γ is a pleiotropic cytokine with antiviral, antitumor, and immunomodulatory properties. Consequently, it plays a crucial role in orchestrating both innate and adaptive immune responses. In an inflammatory milieu, IFN-γ activates the immune response and promotes the eradication of pathogens, while also preventing excessive immune activation and subsequent tissue damage [28].

The clinical presentation of COVID-19 is distinguished by elevated levels of IL-2, IL-6, IL-8, TNF-α, IFN-γ, monocyte chemoattractant protein-1, macrophage inflammatory protein-1 alpha (MIP1α), interferon gamma-induced protein 10, and granulocyte-macrophage colony-stimulating factor in the circulatory system of affected individuals [29, 30]. The gravity of COVID-19 and the resultant mortality rates have been related to elevated expression levels of IL-6 and TNF-α [31]. The referenced study noted that a low-grade inflammatory response was sustained in an elevated state for a minimum duration of 24 h subsequent to the exposure to SARS-CoV. Therefore, it has been suggested that virus-infected epithelial cells or macrophages activated by the S protein could serve as sources of proinflammatory mediators contributing to hyperinflammation in COVID-19 patients 32], [33], [34. Khan et al. investigated the levels of IL-6, IL-1β, and TNF-α in lung cells treated with S protein for 12 and 24 h, finding that these cytokine levels increased progressively over the 12 to 24 h period [35]. Similar results were obtained in our study, and an increase was also detected at 48–72 h, including IFN-γ. The fact that a different picture was observed against S protein compared to LPS may be due to its protein structure which provides a strong immune response for a longer period of time.

Dhar et al. collected samples at different time points (4, 24, 48, 96, and 144 h) in LPS-treated mouse groups and reported increased levels of IL-1β, CXCL1/KC, MIP-2 and TNF-α in bronchoalveolar lavage fluid and serum [36]. The zenith of cytokine secretion subsequent to LPS administration was observed at 4 h; this was followed by a discernible diminution in levels, persisting from 96 to 144 h post-treatment [32]. In our study, an increase occurred at 12 h and a decrease in cytokine levels after 24 h. Immediately after exposure to LPS, a rapid inflammatory response begins. This response entails a swift unleashing of proinflammatory cytokines, including IL-6, TNF-α, and IL-1β. Such immunomodulators facilitate intercellular communication at the onset of the immune response and trigger the inflammatory cascade. Subsequent to the initial hours, the inflammatory response persists, and typically, cytokine secretion culminates in a peak. During this period, immune cells (macrophages, dendritic cells, etc.) are activated against LPS and maintain the inflammatory response. However, some anti-inflammatory mechanisms kick in and help stabilize the inflammatory response. During this period, the immune system tries to contain the potential damage of an excessive inflammatory response, after which the inflammatory response usually slows down, and cytokine levels begin to normalize. Enhanced release of anti-inflammatory cytokines, such as IL-10, may contribute to dampening inflammation and fostering tissue healing [36].

Another study delineated the parallels between COVID-19 and sepsis induced by bacterial LPS by comparing their immunopathogenesis and pathophysiology [9]. In addition to similar symptoms such as multiple organ failures, immunosuppression, hypoalbuminemia, and cytokine storm, differences were also noted, such as COVID-19 can cause sepsis, but sepsis is not a causative agent of SARS-CoV-2 infection [8, 9, 36]. Petruk et al. investigated the interplay between S protein and LPS, assessing their influence on NF-κB activation and cytokine production in monocytes, peripheral blood mononuclear cells, and human blood specimens [2]. Their findings demonstrated that the S protein, in conjunction with low concentrations of LPS, enhanced NF-κB activation in monocytic THP-1 cells and induced cytokine responses in both human blood and peripheral blood mononuclear cells. This observation suggests that the interaction between the S protein and LPS could represent a promising therapeutic target, potentially leading to the development of drugs capable of mitigating the hyperinflammatory response observed during COVID-19 infection [2, 34].

The limitation of our study is that S protein and LPS could not be evaluated together. The use of a single cell line is another limitation of this study, as potential variations were not observed. In the present investigation, our objective was to ascertain the variances in immunological reactions of colon epithelial cells consequent to the administration of specified dosages and time periods of LPS and the S protein. As a result, an increase in immune response was observed in the initial time intervals (12–24 h) following LPS administration, followed by a decrease in subsequent time intervals. Conversely, immune-related expressions in cells treated with S protein remained consistently elevated across all time intervals. S protein triggers immunological responses through a mechanism distinct from the inflammation induced by LPS in bacterial infections, particularly evident in viral infections such as COVID-19. While clinical manifestations of immune response may appear similar in COVID-19 and P. aeruginosa infections, significant molecular differences are observed [37].

Additional research is required to clarify the fundamental processes responsible for diarrhea caused by LPS-induced bacterial infections and viral infections, as well as to determine the relationship between respiratory and GI symptoms [16]. Further studies leveraging the pathophysiology of LPS-induced sepsis are expected to aid in coping with COVID-19. It can be argued that insights from COVID-19 management could also be beneficial in addressing sepsis, which remains a significant global health burden [38]. Besides, there are many therapeutic approaches to overcome SARS-CoV-2 from a biochemical perspective [39]. A detailed study of bacterial and viral-induced proinflammatory responses may also help to prevent the development of secondary infections. In addition, the adaptation of established treatment protocols for diseases sharing similar pathogenic pathways could enhance treatment effectiveness and reduce mortality associated with SARS-CoV-2 infection and sepsis.

Corresponding author: Aysegul Yılmaz, MSc and Research Assistant, Department of Medical Microbiology, Faculty of Medicine, Lokman Hekim University, Söğütözü Mah. 2179. Sk. No:6, 06510 Çankaya/Ankara, Ankara, Türkiye, E-mail:

Funding source: Türkiye Bilimsel ve Teknolojik Araştırma Kurumu

Award Identifier / Grant number: 222S896

  1. Research ethics: Not applicable.

  2. Informed consent: Not applicable.

  3. Author contributions: The authors have accepted responsibility for the entire content of this manuscript and approved its submission. Concept – AY, CT, and SVO; supervision – AY, GU, İCH, CT; materials – AY and ST; data collection and/or processing – AY, ÜYM; analysis and/or Interpretation – AY, CT; writing – AY and CT.

  4. Use of Large Language Models, AI and Machine Learning Tools: None declared.

  5. Conflict of interests: The authors state no conflict of interest.

  6. Research funding: This work was supported by TUBITAK Project number 222S896.

  7. Data availability: The raw data can be obtained on request from the corresponding author.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/tjb-2024-0144).

Received: 2024-06-23
Accepted: 2024-08-09
Published Online: 2024-10-24

© 2024 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

  1. Frontmatter
  2. Research Articles
  3. Can different formulae be used in the diagnosis and staging of chronic kidney disease?
  4. 25-Hydroxyvitamin-D levels in Sjögren’s syndrome: is it the right time to dismiss the case or not?
  5. Ektacytometric examination of red blood cells’ morphodynamical features in diabetic nephropathy patients
  6. Evaluation of the relationship between serum and humor aqueous raftlin (Rftn1) levels and diabetic retinopathy
  7. Toll-like receptor 2-mediated ERK activation significantly upregulates interleukin-6 expression in M2-polarized macrophages
  8. Expression levels of some genes in the MAPK pathway (DUSP1, DUSP2, DUSP4, DUSP6 and DUSP10) in eyelid tumor tissue
  9. Decreased plasma gelsolin in the COVID-19-related acute respiratory distress syndrome
  10. Differential proinflammatory responses of colon epithelial cells to SARS-CoV-2 spike protein and Pseudomonas aeruginosa lipopolysaccharide
  11. Screening for creatine transporter deficiency in autism spectrum disorder: a pilot study
  12. An in vitro assessment of ionizing radiation impact on the efficacy of radiotherapy for breast cancer
  13. Possible protective effect of remifentanil against testicular ischemia-reperfusion injury
  14. Comparison effect of hyperglycaemia induced mixed meal tolerance and oral glucose tolerance test on body oxidative stress
  15. Is serum hornerin a potential biomarker in fibromyalgia? A pilot study
  16. Collaborative online international learning (COIL): an engaging strategy for narrowing learning distances between two continents
  17. Reviewer Acknowledgment
  18. Reviewer Acknowledgment
https://doi.org/10.1515/tjb-2024-0144
Keywords for this article
spike; LPS; Pseudomonas aeruginosa; proinflammatory genes; colon epithelial cell
Creative Commons
BY 4.0

Articles in the same Issue

  1. Frontmatter
  2. Research Articles
  3. Can different formulae be used in the diagnosis and staging of chronic kidney disease?
  4. 25-Hydroxyvitamin-D levels in Sjögren’s syndrome: is it the right time to dismiss the case or not?
  5. Ektacytometric examination of red blood cells’ morphodynamical features in diabetic nephropathy patients
  6. Evaluation of the relationship between serum and humor aqueous raftlin (Rftn1) levels and diabetic retinopathy
  7. Toll-like receptor 2-mediated ERK activation significantly upregulates interleukin-6 expression in M2-polarized macrophages
  8. Expression levels of some genes in the MAPK pathway (DUSP1, DUSP2, DUSP4, DUSP6 and DUSP10) in eyelid tumor tissue
  9. Decreased plasma gelsolin in the COVID-19-related acute respiratory distress syndrome
  10. Differential proinflammatory responses of colon epithelial cells to SARS-CoV-2 spike protein and Pseudomonas aeruginosa lipopolysaccharide
  11. Screening for creatine transporter deficiency in autism spectrum disorder: a pilot study
  12. An in vitro assessment of ionizing radiation impact on the efficacy of radiotherapy for breast cancer
  13. Possible protective effect of remifentanil against testicular ischemia-reperfusion injury
  14. Comparison effect of hyperglycaemia induced mixed meal tolerance and oral glucose tolerance test on body oxidative stress
  15. Is serum hornerin a potential biomarker in fibromyalgia? A pilot study
  16. Collaborative online international learning (COIL): an engaging strategy for narrowing learning distances between two continents
  17. Reviewer Acknowledgment
  18. Reviewer Acknowledgment
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