Alternative pre-mRNA splicing of Toll-like receptor signaling components in peripheral blood mononuclear cells from patients with ARDS

Abstract

A key physiological feature of acute respiratory distress syndrome (ARDS) is inflammation. Toll-like receptor (TLR) signaling is required to combat the infection that underlies many ARDS cases but also contributes to pathological inflammation. Several TLR signaling pathway genes encoding positive effectors of inflammation also produce alternatively spliced mRNAs encoding negative regulators of inflammation. An imbalance between these isoforms could contribute to pathological inflammation and disease severity. To determine whether splicing in TLR pathways is altered in patients with ARDS, we monitored alternative splicing of MyD88 and IRAK1, two genes that function in multiple TLR pathways. The MyD88 and IRAK1 genes produce long proinflammatory mRNAs (MyD88_L and IRAK1) and shorter anti-inflammatory mRNAs (MyD88_S and IRAK1c). We quantified mRNA encoding inflammatory cytokines and MyD88 and IRAK1 isoforms in peripheral blood mononuclear cells (PBMCs) from 104 patients with ARDS and 30 healthy control subjects. We found that MyD88 pre-mRNA splicing is altered in patients with ARDS in a proinflammatory direction. We also observed altered MyD88 isoform levels in a second critically ill patient cohort, suggesting that these changes may not be unique to ARDS. Early in ARDS, PBMC IRAK1c levels were associated with patient survival. Despite the similarities in MyD88 and IRAK1 alternative splicing observed in previous in vitro studies, there were differences in how MyD88 and IRAK1 alternative splicing was altered in patients with ARDS. We conclude that pre-mRNA splicing of TLR signaling genes is altered in patients with ARDS, and further investigation of altered splicing may lead to novel prognostic and therapeutic approaches.

Keywords: IRAK1; MyD88; acute respiratory distress syndrome; alternative pre-mRNA splicing; inflammation; peripheral blood mononuclear cell.

Fig. 1.

Increased expression of MyD88_L in peripheral blood mononuclear cells (PBMCs) from patients with acute respiratory distress syndrome (ARDS). The boxplots depict the ln-transformed mRNA distribution of IL-6, TNF-α, MyD88_S, MyD88_L, IRAK1, and IRAK1c in PBMCs from patients with ARDS and healthy control subjects. Mean differences and P values (independent-sample t-test) are shown below for each variable.

Fig. 2.

MyD88 but not IRAK1 pre-mRNA splicing is altered in patients with acute respiratory distress syndrome (ARDS). Left: ln-transformed IRAK1 mRNA was compared with ln-transformed IRAK1c mRNA where the solid line displays predicted values from the linear regression model adjusted for mean age. Right: ln-transformed MyD88_L mRNA levels were compared with ln-transformed MyD88_S mRNA levels. Lines corresponding to predicted values from the final model for patients with ARDS (red) and healthy controls (blue) adjusted for mean age. Further statistical information relevant to this figure is presented in Table 3.

Fig. 3.

Altered MyD88 pre-mRNA splicing and IRAK1 expression in peripheral blood mononuclear cells (PBMCs) from patients with interstitial lung disease (ILD). The boxplots depict the ln-transformed mRNA distribution of MyD88_S, MyD88_L, IRAK1, and IRAK1c in PBMCs from patients with ILD (I), patients with ARDS (A), and healthy control subjects (H). One-way ANOVA P values are shown below for each variable. The ILD cohort included 22 patients of 63.9 ± 14.5 (means ± SD) yr of age; 16 of the 22 (72.7%) patients with ILD were male. Data for healthy subjects and patients with ARDS are the same as in Fig. 1.

Fig. 4.

Decreased IRAK1c mRNA is associated with worse clinical outcomes in patients with acute respiratory distress syndrome (ARDS). Left: boxplot of ln-transformed IRAK1c mRNA levels compared with survival at day 28. Right: ln-transformed IRAK1c mRNA levels were compared with APACHE III scores with the solid line displaying the predicted values from single variable linear regression. Estimates reflect changes in clinical outcomes with corresponding 95% confidence intervals and P values reported below each graph.

Fig. 5.

The interaction between MyD88_S and IL-6 is opposite between peripheral blood mononuclear cells (PBMCs) of patients with acute respiratory distress syndrome (ARDS) and healthy subjects. The graphs depict ln-transformed IL-6 mRNA levels compared with either ln-transformed MyD88_S mRNA levels (A), ln-transformed MyD88_L mRNA levels (B), ln-transformed IRAK1c mRNA levels (C), and ln-transformed IRAK1 mRNA levels (D). Lines corresponding to predicted values from the final model (see materials and methods) for patients with ARDS (red) and healthy control subjects (blue) are adjusted for mean age. Further statistical information relevant to A is presented in Table 5.