Inhibition of Bruton's TK regulates macrophage NF-kappa B and NLRP3 inflammasome activation in metabolic inflammation

Background and Purpose: There are no medications currently available to treat metabolic inflammation. Bruton's tyrosine kinase (BTK) is highly expressed in monocytes and macrophages and regulates NF-\u3baB and NLRP3 inflammasome activity; both propagate metabolic inflammation in diet-induced obesity. Experimental Approach: Using an in vivo model of chronic inflammation, high-fat diet (HFD) feeding, in male C57BL/6J mice and in vitro assays in primary murine and human macrophages, we investigated if ibrutinib, an FDA approved BTK inhibitor, may represent a novel anti-inflammatory medication to treat metabolic inflammation. Key Results: HFD-feeding was associated with increased BTK expression and activation, which was significantly correlated with monocyte/macrophage accumulation in the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD-fed mice inhibited the activation of BTK and reduced monocyte/macrophage recruitment to the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD-fed mice decreased the activation of NF-\u3baB and the NLRP3 inflammasome. As a result, ibrutinib treated mice fed HFD had improved glycaemic control through restored signalling by the IRS-1/Akt/GSK-3\u3b2 pathway, protecting mice against the development of hepatosteatosis and proteinuria. We show that BTK regulates NF-\u3baB and the NLRP3 inflammasome specifically in primary murine and human macrophages, the in vivo cellular target of ibrutinib. Conclusion and Implications: We provide \u201cproof of concept\u201d evidence that BTK is a novel therapeutic target for the treatment of diet-induced metabolic inflammation and ibrutinib may be a candidate for drug repurposing as an anti-inflammatory agent for the treatment of metabolic inflammation in T2D and microvascular disease

Variation of ribosomal DNA and inheritance of polymorphisms in 6 Petunia hybrida hort lines

Ribosomal DNA polymorphisms were studied in 6 lines of Petunia hybrida using EcoRI, BamHI, HindIII, KpnI, SacI or XhoI. Each line carries several unit types, and 13 types were found in lines, which was not expected. We characterized the unit types and we determined the number of loci. Two kinds of unit types carrying no or several HindIII sites were revealed. The longest EcoRI and BamHI fragments in St43 correspond to a 11.4 kb unit type. Moreover, a 2.6 kb EcoRI fragment cannot be mapped in the 11.4 kb unit. It was found to be equivalent to the 2.45 kb EcoRI fragment carrying the 25 S rRNA coding sequence. Consequently, it was mapped in another unit 11.7 kb long. In TIh1 the corresponding EcoRI and BamHI fragments enabled us to construct 8.8, 9.2 and 10.8 kb segments. These fragments are therefore considered to be part of the 11.4 kb unit length. Other lines display combinations of these length units. The inheritance of polymorphic fragments of lines (St43 and TIh1) for 50 individuals of the 2 possible backcrosses [(St43 x TIh1) x St43] and [(St43 x TIh1) x TIh1] indicated at least 2 loci. The presence in Sk176 of 6.2, 5.7 and 5.4 EcoRI fragments suggested 3 loci. The haploid plants obtained from the hybrid (St43 x TIh1) display 1 individual carrying the 3 unit types present in the hybrid which proves the presence of 3 rDNA loci in TIh1. Moreover, the segregation in the backcrosses corresponds to only 2 loci in St43. It carries a nulli-allele. Evidence for such hypotheses were obtained by in situ hybridization with a biotinylated probe. The TIh1 and TIh7 dihaploid lines display more unit types and, consequently, more polymorphisms than other lines.Dans 6 lignées de Petunia hybrida dont l’ADN a été hydrolysé par EcoRI, BamHI, HindIII, KpnI, SacI ou XhoI, l’ADN ribosomique est apparu très polymorphe. Chaque lignée porte plusieurs types d’unités ; ainsi 13 types différents sont révélés dans les lignées, ce qui est surprenant. Nous avons caractérisé les différents types et déterminé le nombre de loci. Deux types d’unités avec et sans sites HindIII sont révélés. Pour la lignée St43 les fragments EcoRI et BamHI permettent de construire une unité de 11,4 kb. En outre un fragment EcoRI de 2,6 kb ne peut être placé dans l’unité de 11,4 kb. II est en effet équivalent au fragment de 2,4 kb portant la séquence codante du gène 25 S. Il est donc placé dans une unité de 11,7 kb. Dans la lignée TIh1 les fragments correspondants ne permettent de construire que des unités de 8,8 kb, 9,2 kb, et 10,8 kb, donc considérées comme une partie d’unités de 11,4 kb. Les autres lignées montrent une combinaison des fragments précédents. L’hérédité du polymorphisme dans 50 descendants du couple de lignées (St43 et TIh1) et les 2 rétrocroisements possibles [(St43 x TIh1) x St43] et [(St43 x TIh1) x TIh1] indique au moins 2 loci. La présence dans Sk176 des fragments EcoRI 6,2, 5,7 et 5,4 suggère 3 loci. Parmi les plantes haploïdes obtenues de l’hybride F1 (St43 x TIh1), un descendant porte les 3 types d’unité présents dans l’hybride, ce qui ne peut s’expliquer que s’ils sont répartis sur 3 loci. De plus la ségrégation dans les rétrocroisements correspond à 2 loci pour St43, il porte donc un nuiliallèle. La confirmation des hypothèses est apportée par hybridation in situ avec une sonde ribosomique biotinylée. Les 2 lignées dihaploïdes TIh1 et TIh7 montrent le plus d’unités et par conséquent le polymorphisme le plus élevé

Variation of ribosomal DNA and inheritance of polymorphisms in 6 Petunia hybrida hort lines

Ribosomal DNA polymorphisms were studied in 6 lines of Petunia hybrida using EcoRI, BamHI, HindIII, Kpnl, SacI or Xhol. Each line carries several unit types, and 13 types were found in lines, which was not expected. We characterized the unit types and we determined the number of loci. Two kinds of unit types carrying no or several HindIII sites were revealed. The longest EcoRI and BamHI fragments in St43 correspond to a 11.4 kb unit type. Moreover, a 2.6 kb EcoRI fragment cannot be mapped in the 11.4 kb unit. It was found to be equivalent to the 2.45 kb EcoRI fragment carrying the 25 S rRNA coding sequence. Consequently, it was mapped in another unit 11.7 kb long. In TIh1 the corresponding EcoRI and BamHI fragments enabled us to construct 8.8, 9.2 and 10.8 kb segments. These fragments are therefore considered to be part of the 11.4 kb unit length. Other lines display combinations of these length units. The inheritance of polymorphic fragments of lines (St43 and TIh1) for 50 individuals of the 2 possible backcrosses [(St43 x TIh1) x St43] and [(St43 x TIh1) x TIh1] indicated at least 2 loci. The presence in Sk176 of 6.2, 5.7 and 5.4 EcoRI fragments suggested 3 loci. The haploid plants obtained from the hybrid (St43 x TIh1) display 1 individual carrying the 3 unit types present in the hybrid which proves the presence of 3 rDNA loci in TIh1. Moreover, the segregation in the backcrosses corresponds to only 2 loci in St43. It carries a nulli-allele. Evidence for such hypotheses were obtained by in situ hybridization with a biotinylated probe. The TIh1 and TIh7 dihaploid lines display more unit types and, consequently, more polymorphisms than other lines.Variation de l'ADN ribosomique et hérédité du polymorphisme dans 6 lignées de Petunia hybrida Hort. Dans 6 lignées de Petunia hybrida dont l'ADN a été hydrolysé par EcoRI, BamHI, HindIII, KpnI, SacI ou XhoI, l'ADN ribosomique est apparu très polymorphe. Chaque lignée porte plusieurs types d'unités ; ainsi 13 types différents sont révélés dans les lignées, ce qui est surprenant. Nous avons caractérisé les différents types et déterminé le nombre de loci. Deux types d'unités avec et sans sites HindIII sont révélés. Pour la lignée St43 les fragments EcoRI et BamHI permettent de construire une unité de 11,4 kb. En outre un fragment EcoRI de 2,6 kb ne peut être placé dans l'unité de 11,4 kb. II est en effet équivalent au fragment de 2,4 kb portant la séquence codante du gène 25 S. Il est donc placé dans une unité de 11,7 kb. Dans la lignée TIh1 les fragments correspondants ne permettent de construire que des unités de 8,8 kb, 9,2 kb, et 10,8 kb, donc considérées comme une partie d'unités de 11,4 kb. Les autres lignées montrent une combinaison des fragments précédents. L'hérédité du polymorphisme dans 50 descendants du couple de lignées (St43 et TIh1) et les 2 rétrocroisements possibles [(St43 x TIh1) x St43] et [(St43 x TIh1) x TIh1] indique au moins 2 loci. La présence dans Sk176 des fragments EcoRI 6,2, 5,7 et 5,4 suggère 3 loci. Parmi les plantes haploïdes obtenues de l'hybride F1 (St43 x TIh1), un descendant porte les 3 types d'unité présents dans l'hybride, ce qui ne peut s'expliquer que s'ils sont répartis sur 3 loci. De plus la ségrégation dans les rétrocroisements correspond à 2 loci pour St43, il porte donc un nuiliallèle. La confirmation des hypothèses est apportée par hybridation in situ avec une sonde ribosomique biotinylée. Les 2 lignées dihaploïdes TIh1 et TIh7 montrent le plus d'unités et par conséquent le polymorphisme le plus élevé

Inhibition of Bruton's tyrosine kinase regulates macrophage NF-κB and NLRP3 inflammasome activation in metabolic inflammation

Background and Purpose Currently there are limited medicines available for the treatment of metabolic inflammation in diseases such as obesity and type 2 diabetes (T2D). Although initially associated with B‐ells, Bruton's tyrosine kinase (BTK) is present in a wide variety of cells including monocytes and macrophages, and has been implicated in the regulation of the NF‐κB and NLRP3 inflammasome activity. Experimental Approach Using in vivo models of chronic inflammation [high‐fat‐diet (HFD) feeding] and in vitro assays in primary murine and human macrophages we investigated if ibrutinib, an FDA approved medicine that targets BTK, may represent a novel anti‐inflammatory drug for the use in treating metabolic inflammation. Key results HFD feeding was associated with increased BTK expression and activation, which was significantly correlated with monocyte/macrophage accumulation in the liver, adipose tissue and kidney. Treatment of mice fed HFD with ibrutinib inhibited the activation of BTK and reduced monocyte/macrophage recruitment to the liver, adipose tissue and kidney. Reduced inflammatory gene expression associated with decreased activation of NF‐κB and the NLRP3 inflammasome in vivo. As a result, ibrutinib treated mice fed HFD had improved glycaemic control through restored signalling by the IRS‐1/Akt/GSK‐3β pathway; protecting mice against the development of hepatosteatosis and proteinuria. We show that inhibition of BTK reduces activation of NF‐κB and the NLRP3 inflammasome specifically in primary murine and human macrophages; which are the primary target of ibrutinib in vivo in the setting of metabolic inflammation. Conclusions and Implications In the present study we provide ‘proof of concept' evidence that BTK is a novel therapeutic target for the treatment of diet ‐metabolic inflammation. Ibrutinib may be a candidate for drug repurposing as an anti‐inflammatory for the treatment of metabolic inflammation in T2D and microvascular disease