Nucleotide-binding Domain-like Receptor Family Research Articles

The GTPase Rab1 Is Required for NLRP3 Inflammasome Activation and Inflammatory Lung Injury.

Uncontrolled inflammatory response during sepsis predominantly contributes to the development of multiorgan failure and lethality. However, the cellular and molecular mechanisms for excessive production and release of proinflammatory cytokines are not clearly defined. In this study, we show the crucial role of the GTPase Ras-related protein in brain (Rab)1a in regulating the nucleotide binding domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation and lung inflammatory injury. Expression of dominant negative Rab1 N124I plasmid in bone marrow-derived macrophages prevented the release of IL-1β and IL-18, NLRP3 inflammasome activation, production of pro-IL-1β and pro-IL-18, and attenuated TLR4 surface expression and NF-кB activation induced by bacterial LPS and ATP compared with control cells. In alveolar macrophage-depleted mice challenged with cecal ligation and puncture, pulmonary transplantation of Rab1a-inactivated macrophages by expression of Rab1 N124I plasmid dramatically reduced the release of IL-1β and IL-18, neutrophil count in bronchoalveolar lavage fluid, and inflammatory lung injury. Rab1a activity was elevated in alveolar macrophages from septic patients and positively associated with severity of sepsis and respiratory dysfunction. Thus, inhibition of Rab1a activity in macrophages resulting in the suppression of NLRP3 inflammasome activation may be a promising target for the treatment of patients with sepsis.

Characterization of NLRP3-like gene from Apostichopus japonicus provides new evidence on inflammation response in invertebrates

Inflammatory/defensive response after pathogen invasion is considered a local defense reaction in vertebrates. Inflammation response in Apostichopus japonicus was hardly determined due to scarce information available for nucleotide binding domain-like receptor family, pyrin domain-containing (NLRP) family. In the present study, invertebrate NLRP homologue was identified from A. japonicus (designated as AjNLRP3-like) by rapid amplification of cDNA ends. Full-length cDNA of AjNLRP3-like measured 2970 bp with 2265 bp open reading frame encoding a 754-amino acid (aa) residue protein. Structural analysis revealed that AjNLRP3-like processed characteristic domains of pyrin (32–102aa) and NACHT (183–339aa). Multiple sequence alignment and phylogenetic analysis supported that AjNLRP3-like belongs to a new member of NLRP3 protein subfamily. Spatial expression analysis revealed that AjNLRP3-like was ubiquitously expressed in all examined tissues with larger magnitude in coelomocytes. Both Vibrio splendidus challenge in vivo and lipopolysaccharide stimulation in vitro significantly upregulated mRNA expression of AjNLRP3-like when compared with the control group. NLRP3-mediated inflammation response depended on release of lysosomal cathepsin B (CTSB) and subsequent activation of high-mobility group box (HMGB) in vertebrates. We investigated expression profiles of AjNLRP3-like and AjHMGB after AjCTSB knock-down and discovered that AjNLRP3-like was depressed by 0.66-fold and 0.47-fold, whereas AjHMGB was depressed by 0.70-fold and 0.50-fold at 24 and 48 h in AjCTSB-silenced group, respectively. Similarly, down-regulation of AjHMGB was also observed after AjNLRP3-like knock-down. This study therefore suggests that A. japonicus feature similar inflammatory events as those in vertebrates, and activation of AjNLRP3-like depends on AjCTSB expression and release of AjHMGB.

A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-κB activation

Nod1 and Nod2 are intracellular proteins that are involved in host recognition of specific bacterial molecules and are genetically associated with several inflammatory diseases. Nod1 and Nod2 stimulation activates NF-kappaB through RICK, a caspase-recruitment domain-containing kinase. However, the mechanism by which RICK activates NF-kappaB in response to Nod1 and Nod2 stimulation is unknown. Here we show that RICK is conjugated with lysine-63-linked polyubiquitin chains at lysine 209 (K209) located in its kinase domain upon Nod1 or Nod2 stimulation and by induced oligomerization of RICK. Polyubiquitination of RICK at K209 was essential for RICK-mediated IKK activation and cytokine/chemokine secretion. However, RICK polyubiquitination did not require the kinase activity of RICK or alter the interaction of RICK with NEMO, a regulatory subunit of IkappaB kinase (IKK). Instead, polyubiquitination of RICK was found to mediate the recruitment of TAK1, a kinase that was found to be essential for Nod1-induced signaling. Thus, RICK polyubiquitination links TAK1 to IKK complexes, a critical step in Nod1/Nod2-mediated NF-kappaB activation.