Effects of Klebsiella pneumoniae Bacteriophages on IRAK3 Knockdown/Knockout THP-1 Monocyte Cell Lines

Schubert, Bryce Dylan; Ku, Heng; Kabwe, Mwila; Nguyen, Trang Hong; Irving, Helen Ruth; Tucci, Joseph

doi:10.3390/v14112582

Cited by 3 publications

(1 citation statement)

References 56 publications

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“…Further validation of simpler methods to remove endotoxins from bacteriophages is likely to enhance their ability to be used therapeutically especially if combined with rapid, accurate and robust endotoxin detection tests. Several studies have illustrated that purified bacteriophages do not cause an excessive immune response [26,39], but these studies employed different purification methods and distinct bacteriophages. While our work focused on F. nucleatum bacteriophage FNU1, other bacteriophages that target diverse species of bacteria should be tested to determine their effects on immune cells and the degree of purification required.…”

Section: Discussionmentioning

confidence: 99%

IRAK3 Knockout and Wildtype THP-1 Monocytes as Models for Endotoxin Detection Assays and Fusobacterium nucleatum Bacteriophage FNU1 Cytokine Induction

Binte Mohamed Yakob Adil,

Kabwe,

Cianciarulo

et al. 2023

IJMS

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Microbial resistance to antibiotics poses a tremendous challenge. Bacteriophages may provide a useful alternative or adjunct to traditional antibiotics. To be used in therapy, bacteriophages need to be purified from endotoxins and tested for their effects on human immune cells. Interleukin-1 Receptor Associated Kinase-3 (IRAK3) is a negative regulator of inflammation and may play a role in the modulation of immune signalling upon bacteriophage exposure to immune cells. This study aimed to investigate the immune effects of crude and purified bacteriophage FNU1, a bacteriophage that targets the oral pathobiont Fusobacterium nucleatum, on wildtype and IRAK3 knockout THP-1 monocytic cell lines. The IRAK3 knockout cell line was also used to develop a novel endotoxin detection assay. Exposure to crude FNU1 increased the production of pro-inflammatory cytokines (Tumour necrosis factor – alpha (TNF-α) and Interleukin 6 (IL-6)) compared to purified FNU1 in wildtype and IRAK3 knockout THP-1 monocytes. In the IRAK3 knockout THP-1 cells, exposure to crude FNU1 induced a higher immune response than the wildtype monocytes, supporting the suggestion that the inhibitory protein IRAK3 regulates reactions to endotoxins and impurities in bacteriophage preparations. Finally, the novel endotoxin detection assay generated here provides a robust and accurate method for determining endotoxin concentrations.

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Section: Discussionmentioning

confidence: 99%

IRAK3 Knockout and Wildtype THP-1 Monocytes as Models for Endotoxin Detection Assays and Fusobacterium nucleatum Bacteriophage FNU1 Cytokine Induction

Binte Mohamed Yakob Adil,

Kabwe,

Cianciarulo

et al. 2023

IJMS

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Therapeutic Phages as Modulators of the Immune Response: Practical Implications

Górski,

Międzybrodzki,

Jończyk-Matysiak

et al. 2023

Clinical Infectious Diseases

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While the medical community awaits formal proof of the efficacy of phage therapy, as is required by evidence-based medicine, existing data suggest that phages could also be applied based on their non-antibacterial action, especially phage-mediated immunomodulation. Promising avenues have been revealed by findings indicating that phages may mediate diverse actions in the immune system, while the list of phages able to dampen the aberrant immunity associated with a variety of disorders continuously grows. Here we summarize what is known in this field and possible options for the future. While available data are still scarce and preliminary, it appears that “phage repurposing” is worthy of more research, which could reveal new perspectives on applying phage therapy in contemporary medicine.

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Evaluation of effectiveness of bacteriophage purification methods

Binte Mohamed Yakob Adil,

Tucci,

Irving

et al. 2024

Virol J

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The use of bacteriophages for therapy has increased over the last decade. While there is need for clear regulatory pathways for bacteriophage approval for mainstream use in clinical practice, practitioners and patients have been able to access bacteriophage therapy under compassionate grounds and through magistral preparations. However, there is currently no standard for purifying these bacteriophages to ensure safety, and good manufacturing practice certification may not be achieved in these emergency uses. In this study, we employed an Interleukin Receptor Associated Kinase (IRAK) 3 knockout monocyte-based assay to evaluate the endotoxin removal efficacy of three common bacteriophage purification methods: Triton X-100 exposure, CsCl density gradient ultracentrifugation, and Pierce™ High-Capacity Endotoxin Removal Resin spin columns. In our experiments we tested these purification methods on three different bacteriophage morphotypes: siphovirus, podovirus and myovirus. We showed that the lowest endotoxin levels and immune responses were achieved when purifying bacteriophages with Triton-X treatment. The results from purifying with CsCl density gradient ultracentrifugation were comparable, and these were both significantly better than purification with Pierce™ High-Capacity Endotoxin Removal Resin spin columns. We also showed that Triton X-100 purification resulted in the lowest loss of bacteriophage titres. Finally, of the bacteriophages tested here, it did not appear that virus morphology affected efficacy of endotoxin removal.

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Effects of Klebsiella pneumoniae Bacteriophages on IRAK3 Knockdown/Knockout THP-1 Monocyte Cell Lines

Cited by 3 publications

References 56 publications

IRAK3 Knockout and Wildtype THP-1 Monocytes as Models for Endotoxin Detection Assays and Fusobacterium nucleatum Bacteriophage FNU1 Cytokine Induction

IRAK3 Knockout and Wildtype THP-1 Monocytes as Models for Endotoxin Detection Assays and Fusobacterium nucleatum Bacteriophage FNU1 Cytokine Induction

Therapeutic Phages as Modulators of the Immune Response: Practical Implications

Evaluation of effectiveness of bacteriophage purification methods

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