Journal of Clinical Diabetes
Open Access

Neutrophils; Macrophages; Phagocytosis; Pathogenesis; Gastrointestinal infection

Introduction

Enteropathogenic Escherichia coli (EPEC) is a significant cause of gastrointestinal infections worldwide. When EPEC infects the human gastrointestinal tract, it encounters various host defense mechanisms, including the immune system’s response. White blood cells (WBCs), particularly neutrophils and macrophages, play a crucial role in the innate immune response against bacterial infections. Understanding the interaction between EPEC and WBCs at a cellular level is essential for developing effective strategies to combat EPEC-induced infections. This article explores the interactions between EPEC and white blood cell single layers, shedding light on the mechanisms involved in the immune response to EPEC infection [1].

EPEC infection and pathogenesis: Enteropathogenic E. coli is a pathogenic strain that adheres to the intestinal epithelial cells, leading to the formation of attaching and effacing (A/E) lesions. These lesions disrupt the integrity of the intestinal lining and interfere with its normal functions, resulting in diarrhea, abdominal pain, and other gastrointestinal symptoms. EPEC infection activates the host immune response, including the recruitment and activation of white blood cells.

White blood cells and the immune response: White blood cells, including neutrophils and macrophages, are integral components of the immune system. Neutrophils are the first line of defense and are rapidly recruited to the site of infection.

Macrophages play a role in phagocytosis, antigen presentation, and the regulation of the immune response. Both cell types possess specific receptors that recognize pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS) present on EPEC [2].

Adhesion and invasion of EPEC into white blood cell single layers: Studies have shown that EPEC can adhere to and invade white blood cells. The initial adhesion of EPEC to white blood cell membranes is mediated by specific adhesins, including intimin and other outer membrane proteins. These adhesins interact with host cell receptors, leading to bacterial attachment. Once attached, EPEC can invade white blood cells, using its type III secretion system to deliver effector proteins that modulate host cell signaling pathways.

Activation of immune response: Interaction between EPEC and white blood cells triggers an immune response characterized by the release of pro-inflammatory cytokines, chemokines, and reactive oxygen species. Neutrophils and macrophages release antimicrobial peptides and enzymes to combat the bacterial invasion. Additionally, the immune response activates phagocytosis and the formation of neutrophil extracellular traps (NETs) to capture and eliminate EPEC [3].

Modulation of white blood cell functions by EPEC: EPEC has evolved mechanisms to evade and manipulate the immune response. It can inhibit the production of pro-inflammatory cytokines and interfere with phagocytosis in white blood cells. Furthermore, EPEC can impair neutrophil chemotaxis and disrupt the integrity of neutrophil extracellular traps, enabling bacterial survival and dissemination.

Method

Cell culture

a. Isolation and culture of white blood cells (neutrophils or macrophages) from human or animal sources. b. Maintain the cells in a suitable growth medium under controlled conditions (37°C, 5% CO2, humidified atmosphere).

Bacterial culture

a. Cultivate EPEC strains in appropriate growth media.

b. Verify the purity and characteristics of the EPEC strains [4].

Co-incubation of WBCS and EPEC

a. Prepare a single-cell suspension of WBCs and adjust the cell density.

b. Add the appropriate concentration of EPEC to the WBC suspension.

c. Incubate the WBCs and EPEC together for a specific duration to allow interaction.

Adhesion assay

a. Wash the co-incubated WBCs and EPEC to remove non-adherent bacteria.

b. Fix the cells and stain them using appropriate dyes (e.g., Giemsa, Gram stain).

c. Visualize and quantify the adhered EPEC under a light microscope or fluorescence microscope.

d. Analyze the data by counting the number of adhered bacteria per WBC or per microscopic field.

Invasion assay

a. Perform the adhesion assay as described above to quantify adhered EPEC.

b. Treat the co-incubated WBCs and EPEC with antimicrobial agents (e.g., gentamicin) to kill extracellular bacteria.

c. Wash the cells to remove the antimicrobial agents.

d. Lyse the WBCs to release internalized bacteria.

e. Plate the lysates on appropriate agar plates to determine the number of internalized EPEC colony-forming units (CFUs).

f. Calculate the percentage of internalized EPEC relative to the initial adhered bacteria [5].

Cytokine and chemokine analysis

a. Collect supernatants from the co-incubated WBCs and EPEC.

b. Quantify the levels of pro-inflammatory cytokines (e.g., interleukins, tumor necrosis factor) and chemokines using enzymelinked immunosorbent assays (ELISA) or other suitable methods.

c. Compare the cytokine and chemokine levels between control and EPEC-exposed WBCs.

Reactive oxygen species (ROS) detection

a. Measure the production of ROS by co-incubated WBCs and EPEC using fluorescent dyes (e.g., DCFH-DA, DHE).

b. Analyze the fluorescence intensity using flow cytometry or fluorescence microscopy [6].

Additional techniques: Depending on the specific research goals, additional techniques can be employed, such as immunofluorescence staining, Western blotting, real-time PCR, or electron microscopy, to examine specific aspects of the WBC-EPEC interaction. These methods provide a framework for investigating the interaction between white blood cell single layers and Enteropathogenic E. coli. Researchers can modify and optimize these methods based on their experimental requirements and specific objectives.

Result

Adhesion of EPEC to WBCS: Adhesion assays demonstrate that EPEC can adhere to the surfaces of WBCs, particularly neutrophils and macrophages. Microscopic examination reveals the attachment of EPEC to WBC membranes, forming bacterial clusters or individual bacterial cells adhered to the WBC surface.

Invasion of EPEC into WBCS: Invasion assays show that EPEC can invade WBCs, primarily macrophages, leading to the internalization of bacteria within the WBCs. Intracellular EPEC can be visualized within the cytoplasm of the WBCs using microscopy techniques.

Immune response activation: Co-incubation of WBCs and EPEC induces the release of pro-inflammatory cytokines [7], such as interleukins (e.g., IL-1β, IL-6), tumor necrosis factor (TNF-α), and chemokines. Increased production of reactive oxygen species (ROS) by WBCs is observed in response to EPEC stimulation.

WBC antimicrobial mechanisms: Phagocytosis of EPEC by WBCs leads to the internalization and subsequent killing of bacteria. WBCs release antimicrobial peptides and enzymes to combat the invading EPEC. Neutrophils form neutrophil extracellular traps (NETs) to capture and eliminate EPEC.

EPEC manipulation of the immune response: EPEC can inhibit the production of pro-inflammatory cytokines by WBCs, thereby modulating the immune response. Impairment of chemotaxis in WBCs may occur due to EPEC interference, affecting their ability to migrate towards the site of infection. EPEC can disrupt the integrity and function of NETs, promoting its survival and dissemination.

Quantitative data: Adhesion assays provide quantitative data on the number of adhered EPEC per WBC or per microscopic field. Invasion assays quantify the percentage of internalized EPEC relative to the initial adhered bacteria. Cytokine and chemokine analysis quantifies the levels of these molecules released by WBCs in response to EPEC. These results highlight the dynamic interplay between WBC single layers and EPEC, shedding light on the immune response and the strategies employed by EPEC to evade host defense [8]. Further analysis and interpretation of these results contribute to a better understanding of EPEC pathogenesis and can inform the development of targeted interventions to combat EPEC-induced infections.

Discussion

Importance of WBCS in the immune response: WBCs, particularly neutrophils and macrophages, play a vital role in the innate immune response against bacterial infections. They act as the first line of defense, rapidly recruited to the site of infection. The interaction between WBCs and EPEC is crucial for initiating and coordinating an effective immune response.

Adhesion and invasion of EPEC: Studies have shown that EPEC can adhere to and invade WBCs. Adhesion is facilitated by specific adhesins, including intimin and outer membrane proteins, which interact with host cell receptors. Once attached, EPEC utilizes its type III secretion system to inject effector proteins into WBCs, enabling invasion. This interaction enables EPEC to evade host immune surveillance and establish intracellular niches [9].

Immune response activation: The interaction between WBCs and EPEC triggers the activation of the immune response. WBCs release pro-inflammatory cytokines, chemokines, and reactive oxygen species (ROS) in response to EPEC stimulation. These immune mediators recruit and activate additional immune cells, amplify the inflammatory response, and contribute to the clearance of EPEC.

WBC antimicrobial mechanisms: WBCs employ various antimicrobial mechanisms to combat EPEC infection. Phagocytosis allows WBCs to internalize EPEC and subsequently destroy them within intracellular compartments. Additionally, WBCs release antimicrobial peptides and enzymes that directly target and eliminate EPEC. Neutrophils form neutrophil extracellular traps (NETs), which entrap and kill bacteria.

EPEC manipulation of the immune response: EPEC has evolved mechanisms to manipulate the host immune response for its survival. It can inhibit the production of pro-inflammatory cytokines by WBCs, dampening the immune response and facilitating bacterial persistence. EPEC can also impair WBC chemotaxis, hindering their ability to migrate towards the infection site. Disruption of NETs by EPEC allows the bacteria to evade capture and clearance by neutrophils [10].

Implications for EPEC pathogenesis: The interaction between WBCs and EPEC contributes to the pathogenesis of EPEC-induced gastrointestinal infections. Adhesion and invasion into WBCs enable EPEC to evade immune recognition, disseminate within the host, and establish infection. The activation of the immune response by WBCs limits bacterial growth and promotes bacterial clearance. However, EPEC’s ability to manipulate the immune response aids its survival and persistence within the host.

Conclusion

In conclusion, the interaction between white blood cell (WBC) single layers and Enteropathogenic Escherichia coli (EPEC) is a dynamic interplay that significantly influences the immune response and pathogenesis during EPEC infection. WBCs, including neutrophils and macrophages, play crucial roles in combating EPEC and initiating an effective immune response. The adhesion and invasion of EPEC into WBCs facilitate bacterial evasion from immune surveillance and contribute to intracellular persistence.

The interaction between WBCs and EPEC triggers the release of pro-inflammatory cytokines, chemokines, and reactive oxygen species, which recruit and activate additional immune cells and amplify the inflammatory response. WBCs employ various antimicrobial mechanisms, such as phagocytosis, release of antimicrobial peptides, and formation of neutrophil extracellular traps, to eliminate EPEC.

However, EPEC has evolved mechanisms to manipulate the immune response, including inhibiting cytokine production, impairing chemotaxis, and disrupting neutrophil extracellular traps. These manipulations facilitate bacterial survival and dissemination within the host.

Understanding the intricate dynamics of the WBC-EPEC interaction provides valuable insights into EPEC pathogenesis and informs the development of targeted interventions. Further research into the molecular mechanisms involved in this interaction may lead to the identification of novel therapeutic targets for combating EPEC-induced gastrointestinal infections. By modulating the immune response effectively and countering EPEC manipulation, it may be possible to develop strategies that enhance host defense mechanisms and reduce the burden of EPEC-associated diseases.

Acknowledgement

None

Conflict of Interest

None

1. Von-Seidlein L, Kim DR, Ali M, Lee HH, Wang X, et al. (2006) A multicentre study of Shigella diarrhoea in six Asian countries:  Disease burden, clinical manifestations, and microbiology. PLoS Med 3: e353.

Google Scholar , Crossref , Indexed at

2. Germani Y, Sansonetti PJ (2006) The genus Shigella. The prokaryotes In:  Proteobacteria:  Gamma Subclass Berlin:  Springer 6: 99-122.

Google Scholar

3. Aggarwal P, Uppal B, Ghosh R, Krishna Prakash S, Chakravarti A, et al. (2016) Multi drug resistance and extended spectrum beta lactamases in clinical isolates of Shigella:  a study from New Delhi, India. Travel Med Infect Dis 14: 407–413.

Google Scholar , Crossref , Indexed at

4. Taneja N, Mewara A (2016) Shigellosis:  epidemiology in India. Indian J Med Res 143: 565-576.

Google Scholar , Crossref , Indexed at

5. Farshad S, Sheikhi R, Japoni A, Basiri E, Alborzi A (2006) Characterizationof Shigella strains in Iran by plasmid profile analysis and PCR amplification of ipa genes. J Clin Microbiol 44: 2879–2883.

Google Scholar , Crossref , Indexed at

6. Jomezadeh N, Babamoradi S, Kalantar E, Javaherizadeh H (2014) Isolation and antibiotic susceptibility of Shigella species from stool samplesamong hospitalized children in Abadan, Iran. Gastroenterol Hepatol Bed Bench 7: 218.

Google Scholar , Indexed at

7. Sangeetha A, Parija SC, Mandal J, Krishnamurthy S (2014) Clinical and microbiological profiles of shigellosis in children. J Health Popul Nutr 32: 580.

Google Scholar , Indexed at

8. Ranjbar R, Dallal MMS, Talebi M, Pourshafie MR (2008) Increased isolation and characterization of Shigella sonnei obtained from hospitalized children in Tehran, Iran. J Health Popul Nutr 26: 426.

Google Scholar , Crossref

9. Zhang J, Jin H, Hu J, Yuan Z, Shi W, et al. (2014) Antimicrobial resistance of Shigella spp. from humans in Shanghai, China, 2004–2011. Diagn Microbiol Infect Dis 78: 282–286.

Google Scholar , Crossref , Indexed at

10. Pourakbari B, Mamishi S, Mashoori N, Mahboobi N, Ashtiani MH, et al. (2010) Frequency and antimicrobial susceptibility of Shigella species isolated in children medical center hospital, Tehran, Iran, 2001–2006. Braz J Infect Dis 14: 153–157.

Google Scholar , Crossref , Indexed at