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

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.

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

Bacterial sepsis characterised by an immunosuppressive and cytokine storm state is a challenge to treat clinically. While conventional antibiotics have been associated with exacerbating the cytokine storm, the role that bacteriophages may play in immune modulation of sepsis remains unclear. Bacteriophages are bacterial viruses that have the capacity to lyse specific bacteria and hence provide a natural alternative to antibiotics. K. pneumoniae is known to cause sepsis in humans, and in this study we isolated two lytic bacteriophages against this pathogen, one of which was a novel jumbo bacteriophage. We employed THP-1 monocyte cell lines, with different functional phenotypes for the interleukin-1 receptor associated kinase 3 (IRAK3- a cytoplasmic homeostatic mediator and prognostic marker of inflammation), to evaluate the role of the K. pneumoniae bacteriophages in modulating the immune response in-vitro. We showed for the first time that bacteriophages did not stimulate excessive production of tumour necrosis factor alpha, or interleukin-6, in THP-1 monocyte cell lines which displayed varying levels of IRAK3 expression.

Analysis of interleukin-1 receptor associated kinase-3 (IRAK3) function in modulating expression of inflammatory markers in cell culture models: A systematic review and meta-analysis.

BACKGROUND: IRAK3 is a critical modulator of inflammation in innate immunity. IRAK3 is associated with many inflammatory diseases, including sepsis, and is required in endotoxin tolerance to maintain homeostasis of inflammation. The impact of IRAK3 on inflammatory markers such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cell culture models remains controversial. OBJECTIVE: To analyse temporal effects of IRAK3 on inflammatory markers after one- or two-challenge interventions in cell culture models. METHODS: A systematic search was performed to identify in vitro cell studies reporting outcome measures of expression of IRAK3 and inflammatory markers. Meta-analyses were performed where sufficient data were available. Comparisons of outcome measures were performed between different cell lines and human and mouse primary cells. RESULTS: The literature search identified 7766 studies for screening. After screening titles, abstracts and full-texts, a total of 89 studies were included in the systematic review. CONCLUSIONS: The review identifies significant effects of IRAK3 on decreasing NF-κB DNA binding activity in cell lines, TNF-α protein level at intermediate time intervals (4h-15h) in cell lines or at long term intervals (16h-48h) in mouse primary cells following one-challenge. The patterns of TNF-α protein expression in human cell lines and human primary cells in response to one-challenge are more similar than in mouse primary cells. Meta-analyses confirm a negative correlation between IRAK3 and inflammatory cytokine (IL-6 and TNF-α) expression after two-challenges.

A novel p.A191D matrilin-3 variant in a Vietnamese family with multiple epiphyseal dysplasia: a case report.

BACKGROUND: Multiple epiphyseal dysplasia (MED) is a common skeletal dysplasia that is characterized by variable degrees of epiphyseal abnormality primarily involving the hip and knee joints. Mutations in a gene encoding matrilin-3 (MATN3) have been reported as disease causing of autosomal dominant MED. The current study identified a novel c.572 C > A variant (p.A191D) in exon 2 of MATN3 in a Vietnamese family with MED. CASE PRESENTATION: A standard clinical tests and radiological examination were performed in an 8-year-old Vietnamese girl patient. The clinical examination showed that patient height was under average, with bent lower limbs, limited mobility and dislocation of the joints at both knees. Radiological documentation revealed abnormal cartilage development at the epiphysis of the femur and patella. The patient has a varus deformity of the lower limbs. The patient was diagnosed with autosomal dominant MED using molecular testing in the order of the coding sequences and flanking sequences of five genes: COMP (exons 8-19), MATN3 (exon 2), COL9A2 (exon 3), COL9A3 (exon 3), COL9A1 (exon 8) by Sanger sequencing. A novel heterozygous missense variant (c.572 C > A, p.A191D) in MATN3 was identified in this family, which were not inherited from parents. The p.A191D was predicted and classified as a pathogenic variant. When the two predicted structures of the wild type and mutant matrilin-3 were compared, the p.A191D substitution caused conformational changes near the substitution site, resulting in deformity of the ß-sheet of the single A domain of matrilin- 3. CONCLUSIONS: This is the first Vietnamese MED family attributed to p.A191D matrilin-3 variant, and our clinical, radiological and molecular data suggest that the novel de novo missense variant in MATN3 contributed to MED.