Showing papers by "Shizuo Akira published in 2023"

Breaking self‐regulation of Regnase‐1 promotes its own protein expression

Abstract: The RNA‐binding protein (RBP) Regnase‐1 is an endonuclease that regulates immune responses by modulating target mRNA stability. Regnase‐1 degrades a group of inflammation‐associated mRNAs, which contributes to a balanced immune response and helps prevent autoimmune diseases. Regnase‐1 also cleaves its own mRNA by binding stem‐loop (SL) RNA structures in its 3′UTR. To understand how this autoregulation is important for immune responses, we generated mice with a 2‐bp genome deletion in the target SL of the Regnase‐1 3′‐untranslated region (3′UTR). Deletion of these nucleotides inhibited SL formation and limited Regnase‐1‐mediated mRNA degradation. Mutant mice had normal hematopoietic cell differentiation. Biochemically, mutation of the 3′UTR SL increased Regnase‐1 mRNA stability and enhanced both Regnase‐1 mRNA and protein levels in mouse embryonic fibroblasts (MEFs). The expression of Il6, a Regnase‐1 target gene, was constitutively suppressed at steady‐state in mutant MEFs. Additionally, Regnase‐1 protein expression in mutant MEFs was significantly elevated compared to that in wild‐type MEFs at steady state and upon proinflammatory cytokine stimulation. These data suggest a negative feedback mechanism for Regnase‐1 expression and represent a unique mouse model to probe Regnase‐1 overexpression in vivo.

TDAG51 promotes transcription factor FoxO1 activity during LPS‐induced inflammatory responses

Abstract: Tight regulation of Toll‐like receptor (TLR)‐mediated inflammatory responses is important for innate immunity. Here, we show that T‐cell death‐associated gene 51 (TDAG51/PHLDA1) is a novel regulator of the transcription factor FoxO1, regulating inflammatory mediator production in the lipopolysaccharide (LPS)‐induced inflammatory response. TDAG51 induction by LPS stimulation was mediated by the TLR2/4 signaling pathway in bone marrow‐derived macrophages (BMMs). LPS‐induced inflammatory mediator production was significantly decreased in TDAG51‐deficient BMMs. In TDAG51‐deficient mice, LPS‐ or pathogenic Escherichia coli infection‐induced lethal shock was reduced by decreasing serum proinflammatory cytokine levels. The recruitment of 14‐3‐3ζ to FoxO1 was competitively inhibited by the TDAG51‐FoxO1 interaction, leading to blockade of FoxO1 cytoplasmic translocation and thereby strengthening FoxO1 nuclear accumulation. TDAG51/FoxO1 double‐deficient BMMs showed significantly reduced inflammatory mediator production compared with TDAG51‐ or FoxO1‐deficient BMMs. TDAG51/FoxO1 double deficiency protected mice against LPS‐ or pathogenic E. coli infection‐induced lethal shock by weakening the systemic inflammatory response. Thus, these results indicate that TDAG51 acts as a regulator of the transcription factor FoxO1, leading to strengthened FoxO1 activity in the LPS‐induced inflammatory response.

Platelet TLR7 is essential for the formation of platelet-neutrophil complexes and low-density neutrophils in lupus nephritis.

Abstract: OBJECTIVES Platelets and low-density neutrophils (LDNs) are major players in the immunopathogenesis of systemic lupus erythematosus (SLE). Despite evidence showing the importance of platelet neutrophil complexes (PNCs) in inflammation, little is known about the relationship between LDNs and platelets in SLE. We sought to characterize the role of LDNs and TLR7 in clinical disease. METHODS Flow cytometry was used to immunophenotype LDNs from SLE patients and controls. The association of LDNs with organ damage was investigated in a cohort of 290 SLE patients. TLR7mRNA expression was assessed in LDNs and high-density neutrophils (HDNs) using publicly available mRNA sequencing datasets, and our own cohort using RT-PCR. The role of TLR7 in platelet binding was evaluated in platelet: HDN mixing studies using TLR7 deficient mice and Klinefelter syndrome patients. RESULTS SLE patients with active disease have more LDNs, which are heterogeneous and more immature in patients with evidence of kidney dysfunction. LDNs are platelet bound, in contrast to HDNs. LDNs settle in the PBMC layer due to the increased buoyancy and neutrophil degranulation from platelet binding. Mixing studies demonstrated that this PNC formation was dependent on platelet-TLR7, and that the association results in increased NETosis. The neutrophil-to-platelet ratio (NPR), is a useful clinical correlate for LDNs, and a higher NPR is associated with past and current flares of lupus nephritis. CONCLUSIONS LDNs sediment in the upper PBMC fraction due to PNC formation, which is dependent on the expression of TLR7 in platelets. Collectively, our results reveal a novel TLR7-dependent crosstalk between platelets and neutrophils, which may be an important therapeutic opportunity for lupus nephritis.

MGIT-seq for the Identification of Nontuberculous Mycobacteria and Drug Resistance: a Prospective Study

Abstract: Because nontuberculous mycobacterial pulmonary disease is a considerable health burden, a simple and clinically applicable analytical protocol enabling the identification of subspecies and drug-resistant disease is required to determine the treatment strategy. We aimed to develop a simplified workflow consisting only of direct sequencing of mycobacterial growth indicator tube cultures (MGIT-seq). ABSTRACT Because nontuberculous mycobacterial pulmonary disease is a considerable health burden, a simple and clinically applicable analytical protocol enabling the identification of subspecies and drug-resistant disease is required to determine the treatment strategy. We aimed to develop a simplified workflow consisting only of direct sequencing of mycobacterial growth indicator tube cultures (MGIT-seq). In total, 138 patients were prospectively enrolled between April 2021 and May 2022, and culture-positive MGIT broths were subjected to sequencing using MinION, a portable next-generation sequencer. Sequence analysis was conducted to identify species using core genome multilocus sequence typing and to predict macrolide and amikacin (AMK) resistance based on previously reported mutations in rrl, rrs, and erm(41). The results were compared to clinical tests for species identification and drug susceptibility. A total of 116 patients with positive MGIT cultures were included in the analysis. MGIT-seq yielded 99.1% accuracy in species-level identification and identified 98 isolates (84.5%) at the subspecies level. Macrolide and AMK resistance were detected in 19.4% and 1.9% of Mycobacterium avium complex (MAC) and Mycobacterium abscessus isolates. The predicted macrolide and AMK resistance was consistent with the results of conventional drug susceptibility tests, with specificities of 97.6% and 100.0%, respectively. Direct MGIT-seq has achieved comprehensive identification and drug resistance detection of nontuberculous mycobacteria, which could be applicable to determine the treatment strategy by a single test in clinical practice.