A systematic review and meta-analyses of interleukin-1 receptor associated kinase 3 (IRAK3) action on inflammation in in vivo models for the study of sepsis

Abstract

Background: Interleukin-1 receptor associated kinase 3 (IRAK3) is a critical modulator of inflammation and is associated with endotoxin tolerance and sepsis. Although IRAK3 is known as a negative regulator of inflammation, several studies have reported opposing functions, and the temporal actions of IRAK3 on inflammation remain unclear. A systematic review and meta-analyses were performed to investigate IRAK3 expression and its effects on inflammatory markers (TNF-α and IL-6) after one- or two-challenge interventions, which mimic the hyperinflammatory and immunosuppression phases of sepsis, respectively, using human or animal in vivo models.

Methods: This systematic review and meta-analyses has been registered in the Open Science Framework (OSF) (Registration DOI: 10.17605/OSF.IO/V39UR). A systematic search was performed to identify in vivo studies reporting outcome measures of expression of IRAK3 and inflammatory markers. Meta-analyses were performed where sufficient data was available.

Results: The search identified 7778 studies for screening. After screening titles, abstracts and full texts, a total of 49 studies were included in the systematic review. The review identified significant increase of IRAK3 mRNA and protein expression at different times in humans compared to rodents following one-challenge, whereas the increases of IL-6 and TNF-α protein expression in humans were similar to rodent in vivo models. Meta-analyses confirmed the inhibitory effect of IRAK3 on TNF-α mRNA and protein expression after two challenges.

Conclusions: A negative correlation between IRAK3 and TNF-α expression in rodents following two challenges demonstrates the association of IRAK3 in the immunosuppression phase of sepsis. Species differences in underlying biology affect the translatability of immune responses of animal models to human, as shown by the dissimilarity in patterns of IRAK3 mRNA and protein expression between humans and rodents following one challenge that are further influenced by variations in experimental procedures.

Fig 1. PRISMA flowchart.

PRISMA flowchart depicting the process of search and selection of studies for the systematic review.

Fig 2. IRAK3 mRNA expression outcome: One-challenge intervention group versus control group.

Two independent meta-analyses were conducted for human studies (1.1.1) and rodent studies (1.1.2). In human in vivo studies, the subjects were administered with LPS via intravenous injection [45] or instillation into the contralateral lung [56], and IRAK3 mRNA expression in whole blood [45] or alveolar macrophages [56] was measured. Mice or rats underwent CLP [36, 38] or were inoculated intranasally with K. pneumoniae [40], or inoculated or injected with LPS [29, 43], and IRAK3 mRNA expression was measured in samples taken from peritoneum [29], lung [36, 38, 40], or eye [43] at long term (LT; 16h – 72h).

Fig 3. TNF-α and IL-6 protein expression outcome in humans: One-challenge intervention group versus control group.

In one-challenge intervention, human subjects were administered with LPS [, –52, 54, 55, 57, 60]. TNF-α and IL-6 protein level was measured at short term (ST; 1h – 3h), intermediate term (IT; 4h – 15h), or at long term (LT; 16h – 72h).

Fig 4. TNF-α and IL-6 mRNA or protein expression outcome in mice: IRAK3 knockout group versus IRAK3 wildtype group after one-challenge intervention.

In one-challenge intervention, mice inhaled LPS [37], or were inoculated with H. influenzae [32], or K. pneumoniae [41], or S. pneumoniae [40]. TNF-α or IL-6 mRNA or protein expression was measured at short term (ST; 1h – 3h) or long term (LT; 16h - 72h) after one-challenge.

Fig 5. IRAK3 mRNA and protein expression outcome: One-challenge intervention group versus two-challenge intervention group.

Mice underwent CLP or sham procedure; at 24h (LT) after CLP sham macrophages or monocytes were extracted and ex vivo treated with LPS. At 4h (IT) [38], 6h (IT) [36] and 24h (LT) [39] post-ex vivo treatment outcomes were measured. In one-challenge intervention, IRAK3 mRNA or protein expression was measured in cells extracted from sham-treated mice, and then ex vivo treated with LPS. In two-challenge intervention, IRAK3 mRNA or protein expression was measured in cells extracted from CLP-treated mice, and ex vivo treated with LPS. Alternatively, mice or rats were injected with LPS; at 24h (LT) after first LPS challenge mice or rats were treated again with LPS; and following the second challenge at 3h (ST) [64], at 24h (LT) [43] IRAK3 mRNA or protein expression in hepatic tissues [64] or enucleated eyes [43] were measured. In one-challenge intervention group, mice or rat were pre-treated with phosphate buffered saline or sterilized physiological saline for 24h [64] or 5d [43], and then treated with LPS, for 3h [64] or 24h [43] when outcomes were measured.

Fig 6. TNF-α and IL-6 protein expression outcome: One-challenge intervention group versus two-challenge intervention group.

Independent meta-analyses of TNF-α and IL-6 protein expression were conducted for human studies (8.1.1 and 8.1.2) or rodent studies (8.1.3). In human one-challenge intervention, human subjects were administered with LPS and at ST following LPS administration. TNF-α and IL-6 protein level in whole blood was measured. In human two-challenge intervention, human subjects were administered with LPS on 5 consecutive days and TNF-α and IL-6 protein level was measured at ST on day 5 [52]; or human subjects were administered with LPS twice with washout period of 1–2 weeks and TNF-α and IL-6 were quantified at ST following LPS administration [54, 55, 60]; or after the first in vivo LPS administration, whole blood extracted from the subjects was ex vivo treated with LPS and TNF-α and IL-6 were measured at 24h (LT) following ex vivo treatment [45, 51]. In mouse in vivo models, mice underwent CLP or sham procedure; at 24h (LT) [36] or 72h (LT) [39] after treatment macrophages or monocytes were extracted and ex vivo treated with LPS. At 16h (LT) [36] or 24h (LT) [39] post-ex vivo treatment outcomes were measured. In mouse one-challenge intervention, TNF-α protein level was measured in cells extracted from sham-treated mice, and ex vivo treated with LPS. In mouse two-challenge intervention, TNF-α protein expression was measured in cells extracted from CLP-treated mice, and ex vivo treated with LPS. Alternatively, mice were pre-treated with LPS [64] or lipoteichoic acid [61]; at 24h (LT) after first LPS or lipoteichoic acid challenge mice were treated again with LPS, and at 3h (ST) [64], 24h (LT) [61] serum or plasma TNF-α protein levels were measured for two-challenge intervention group. In one-challenge intervention group, mice were pre-treated with phosphate buffered saline for 24h, and then treated with LPS for 3h [64] or 24h [43] when outcomes were measured.

Fig 7. TNF-α mRNA and protein expression outcome after two-challenge intervention: IRAK3 knockout group versus IRAK3 wildtype group.

Mice underwent CLP; at 24h (ST) after CLP mice were inoculated with P. aeruginosa, and at 6h (IT) after second challenge TNF-α mRNA expression was measured, and at 24h (LT) for protein expression measured [36]. Alternatively, mice underwent CLP; at 24h (LT) after CLP macrophages were extracted and ex vivo challenged with LPS, and at 4h (IT) after second challenge TNF-α mRNA expression was measured [38]. Similarly, mice underwent CLP; at 72h (LT) after CLP mice were injected with LPS, and at 24h (LT) after second challenge TNF-α protein expression was measured [39].

Fig 8. Scheme of IRAK3 and cytokine expression in humans following the first and second in vivo challenge.

During ST (1h – 3h) after the first stimulation of TLR/IL-1R with LPS, NF-κB transcription factor is activated to induce the production of cytokines (TNF-α and IL-6), which leads to “cytokine storm”. IRAK3 expression is induced by transcription factors including activator protein-1 (AP1), hypoxia-inducible factor-α (HIF-α), glucocorticoid receptor (GR); and the peak increase of IRAK3 expression is observed at IT (4h – 15h) following the first challenge to suppress cytokine release for maintaining homeostasis. After the second challenge in human, IRAK3 expression is further increased to limit cytokine production, thus the extent of the cytokine increase after second challenge is significantly lower than the increase level after the first challenge.