Hepatitis E virus infection remodels the mature tRNAome in macrophages to orchestrate NLRP3 inflammasome response

Abstract

Hepatitis E virus (HEV) infection has been shown to activate NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in macrophages, a key mechanism of causing pathological inflammation, but the mechanisms regulating this response remain poorly understood. Here, we report that the mature tRNAome dynamically responds to HEV infection in macrophages. This directs IL-1β expression, the hallmark of NLRP3 inflammasome activation, at mRNA and protein levels. Conversely, pharmacological inhibition of inflammasome activation abrogates HEV-provoked tRNAome remodeling, revealing a reciprocal interaction between the mature tRNAome and the NLRP3 inflammasome response. Remodeling the tRNAome results in improved decoding of codons directing leucine- and proline synthesis, which are the major amino acid constituents of IL-1β protein, whereas genetic or functional interference with tRNAome-mediated leucine decoding impairs inflammasome activation. Finally, we demonstrated that the mature tRNAome also actively responds to lipopolysaccharide (a key component of gram-negative bacteria)-triggered inflammasome activation, but the response dynamics and mode of actions are distinct from that induced by HEV infection. Our findings thus reveal the mature tRNAome as a previously unrecognized but essential mediator of host response to pathogens and represent a unique target for developing anti-inflammatory therapeutics.

Keywords: NLRP3 inflammasome; amino acid deficiency; hepatitis E virus; macrophages; mature tRNAome.

Fig. 1.

The kinetics of IL-1β expression and the landscape of the mature tRNAome in macrophages upon HEV infection. (A) THP-1 and HL60 macrophages were inoculated with HEV particles and viral RNA levels were quantified by qRT-PCR (n = 4). IL-1β mRNA expression (n = 4) (B) and protein secretion (n = 6) (C) were quantified in THP-1 and HL60 macrophages after inoculation with HEV for 12, 24, or 48 h. (D) Accordingly, the mature tRNAome consisting of 57 species was quantified by qRT-PCR. Cluster analysis of the tRNAome and data were normalized to the control (CTR; set as 1). Data are means ± SEM. *P < 0.05; **P < 0.01.

Fig. 2.

Inhibition of NLRP3 inflammasome prevents HEV-induced tRNAome reprogramming. (A) THP-1 macrophages were inoculated with HEV for 12, 24, or 48 h. Mature IL-1β and cleaved caspase-1 in supernatant or pro-IL-1β and NLRP3 in lysates were determined by western blotting. THP-1 macrophages were pretreated with 10 μM NF-κB inhibitor (BAY11-7085), 10 μM NLRP3 inhibitor (MCC950), or 50 μM caspase-1 inhibitor (VX-765) for 2 h and then were incubated with HEV or HEV plus 10 μM BAY11-7085, 10 μM MCC950, or 50 μM of VX-765 for 12, 24, or 48 h. (B) Mature IL-1β in supernatant or pro-IL-1β and NLRP3 in lysates were determined by western blotting. (C) IL-1β mRNA and protein levels were quantified by qRT-PCR (n = 4 to 5) and ELISA (n = 4 to 6), respectively. (D) HEV mRNA levels were quantified by qRT-PCR (n = 3 to 5). (E) The mature tRNAome was quantified by qRT-PCR. Cluster analysis of the tRNAome and data were normalized to the control (CTR; set as 1). Data are means ± SEM. *P < 0.05; **P < 0.01.

Fig. 3.

Amino acid deprivation affects HEV-induced inflammatory gene expression. (A) THP-1 macrophages were cultured in medium containing 20 amino acids and inoculated with HEV for 12, 24, or 48 h. Gene expression of IL-1β, IL-6, IL-8, IL-12, TNFα, or GM-CSF was quantified by qRT-PCR (n = 3). (B) THP-1 macrophages were subjected to the deprivation of each of the 20 amino acids in RPMI 1640 and inoculated with HEV for 12, 24, or 48 h. The expression of inflammatory genes was quantified by qRT-PCR (n = 3), and data were normalized to the nonamino acid deprivation group (set as 1). (C) THP-1 macrophages were inoculated with HEV for 12, 24, or 48 h. The cognate tRNAs of leucine and proline were quantified by qRT-PCR, and data were normalized to the uninfected group (set as 1).

Fig. 4.

Leucine or proline deprivation abolishes HEV-induced inflammasome activation and tRNA reprogramming. THP-1 macrophages were subjected to 20 amino acid–containing, leucine deprivation, or proline deprivation RPMI 1640 medium and inoculated with HEV for 12, 24, or 48 h. (A) IL-1β protein levels were quantified by ELISA (n = 10 to 12). (B) Mature IL-1β and cleaved caspase-1 in the supernatant were determined by western blotting. (C) The mature tRNAome was quantified by qRT-PCR. Cluster analysis of the tRNAome and data were normalized to the control (CTR; set as 1). Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Knockdown of leucyl-tRNA-synthetase attenuates HEV-induced inflammatory response and tRNA reprogramming. (A) Schematic illustration of the process of aminoacylated tRNAs biogenesis. (B) LARS mRNA expression of THP-1 cells following shRNA mediated knockdown was quantified by qRT-PCR, and the shCTR control (cells transduced with a vector without targeting any host genes) was set as 1. Effects of LARS knockdown on inflammasome response measured after inoculation with HEV for 12, 24, or 48 h. (C) The expression levels of inflammatory genes were quantified by qRT-PCR (n = 4). (D) IL-1β protein levels were quantified by ELISA (n = 4). (E) Mature IL-1β and cleaved caspase-1 in the supernatant, and NLRP3, HEV ORF2, and pro-IL-1β in lysates were determined by western blotting. (F) The mature tRNAome was quantified by qRT-PCR. Cluster analysis of the tRNAome and data were normalized to the control (CTR; set as 1). Data are means ± SEM. *P < 0.05.