RBM3 regulates temperature sensitive miR-142-5p and miR-143 (thermomiRs), which target immune genes and control fever

Abstract

Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40 °C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142-5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40 °C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142-5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142-5p and miR-143.

Figure 1.

Differential gene expression in febrile and non-febrile patients. (A) Heatmap showing top 50 most upregulated (red) and downregulated (blue) genes in 30 febrile patients compared to 35 non-febrile controls. (B) Re-analysis of RBM3 expression from Hu et al., 2013 (18) in 30 febrile and 35 non-febrile patients.

Figure 2.

Expression of the cold-shock protein RBM3 is altered between 37°C and 40°C in THP-1-derived macrophages. (A) Confirmation of THP-1 differentiation into macrophages as shown by increased expression of CD11b and CD44 using flow cytometry. (B) Viability of THP-1 cells incubated at 40°C compared to controls (37°C) as measured by flow cytometry following Annexin V and propidium iodide (PI) staining. Ultraviolet-exposed cells (UV+) were used as positive control. (C) Gene function enrichment of genes overexpressed at 40°C. (D) Top ten most downregulated genes at 40°C. (E) Western blot for RBM3 expression following incubation at 37°C and 40°C with actin used as the loading control. PMA, phorbol-12-myristate 13-acetate.

Figure 3.

ThermomiR expression increases at 40°C in THP-1-derived macrophages. (A) Expression of temperature-sensitive miRNAs (thermomiRs) measured by small RNA sequencing at 37°C and after 2, 8 and 24 h of incubation at 40°C. (B) Expression of thermomiRs and controls measured by RT-qPCR after incubation for 24 h. Data are from three independent experiments each in triplicate and show mean ± SEM. Two-tailed Student's t-test was used to determine significance, denoted by * (P < 0.05) and ** (P < 0.001). ns, not significant.

Figure 4.

RBM3 siRNA knockdown increases the expression of thermomiRs. (A) Expression of RBM3 mRNA by RT-qPCR, and (B) RBM3 protein levels by Western blot in THP-1-derived macrophages nucleofected with negative control siRNA and each of the three siRNAs against RBM3: #58, #59 and #60. (C) Average counts of individual miRNAs from the three RBM3 knockdown experiments as measured using NanoString nCounter. Two miRNAs miR-142 and miR-143 showed the greatest change in expression. (D) Expression of pyrogens in THP-1-derived macrophages: IL6ST, TNF and TLR-2 following inhibition of miR-142–5p and miR-143 (relative to control inhibitor). (E) Expression of miR-142 and miR-143 in THP-1-derived macrophage at 24 h after nucleofection with respective miRNA mimics (relative to control). (F) Expression of IL6ST, TNF and TLR- in THP-1-derived macrophage at 24 h following nucleofection of miR-142 and miR-143 mimic (relative to control). Data are from three independent experiments each in triplicate and show mean ± SEM. Two-tailed Student's t-test was used to determine significance, denoted by * (P < 0.05) and ** (P < 0.001). inh, inhibitor; ns, not significant.

Figure 5.

Temporal changes of RBM3, miR-142–5p and miR-143 expression in PBMCs exposed to 40°C over a 24 h time course. (A) Expression of RBM3 mRNA in PBMCs by RT-qPCR (n = 5). (B) Expression of miR-142–5p and (C) miR-143 in PBMCs by Taqman miRNA assays (n = 5). Fold change was measured relative to 0 h. N1–N5 denote five healthy individuals recruited for this experiment.

Figure 6.

Proposed negative feedback mechanism regulating body temperature when fever occurs. Triggering an immune response (e.g. via infection), increases the expression of endogenous pyrogens, leading to fever. Fever will result in decreased expression of RBM3, which in turn leads to increased expression of thermomiRs normally targeted by RBM3, thereby fine-tuning expression of endogenous pyrogens. The thermomiRs themselves play integral roles in coordinating the response to fever and infection. This negative feedback loop attenuates excessive increases in body temperature.