Rongbin Zhou

A role for mitochondria in NLRP3 inflammasome activation

An inflammatory response initiated by the NLRP3 inflammasome is triggered by a variety of situations of host ‘danger’, including infection and metabolic dysregulation. Previous studies suggested that NLRP3 inflammasome activity is negatively regulated by autophagy and positively regulated by reactive oxygen species (ROS) derived from an uncharacterized organelle. Here we show that mitophagy/autophagy blockade leads to the accumulation of damaged, ROS-generating mitochondria, and this in turn activates the NLRP3 inflammasome. Resting NLRP3 localizes to endoplasmic reticulum structures, whereas on inflammasome activation both NLRP3 and its adaptor ASC redistribute to the perinuclear space where they co-localize with endoplasmic reticulum and mitochondria organelle clusters. Notably, both ROS generation and inflammasome activation are suppressed when mitochondrial activity is dysregulated by inhibition of the voltage-dependent anion channel. This indicates that NLRP3 inflammasome senses mitochondrial dysfunction and may explain the frequent association of mitochondrial damage with inflammatory diseases.

Thioredoxin-interacting protein links oxidative stress to inflammasome activation

The NLRP3 inflammasome has a major role in regulating innate immunity. Deregulated inflammasome activity is associated with several inflammatory diseases, yet little is known about the signaling pathways that lead to its activation. Here we show that NLRP3 interacted with thioredoxin (TRX)-interacting protein (TXNIP), a protein linked to insulin resistance. Inflammasome activators such as uric acid crystals induced the dissociation of TXNIP from thioredoxin in a reactive oxygen species (ROS)-sensitive manner and allowed it to bind NLRP3. TXNIP deficiency impaired activation of the NLRP3 inflammasome and subsequent secretion of interleukin 1β (IL-1β). Akin to Txnip−/− mice, Nlrp3−/− mice showed improved glucose tolerance and insulin sensitivity. The participation of TXNIP in the NLRP3 inflammasome activation may provide a mechanistic link to the observed involvement of IL-1β in the pathogenesis of type 2 diabetes.

The NLRP3 Inflammasome: A Sensor for Metabolic Danger?

Interleukin-1β (IL-1β), reactive oxygen species (ROS), and thioredoxin-interacting protein (TXNIP) are all implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). Here we review mechanisms directing IL-1β production and its pathogenic role in islet dysfunction during chronic hyperglycemia. In doing so, we integrate previously disparate disease-driving mechanisms for IL-1β, ROS, and TXNIP in T2DM into one unifying model in which the NLRP3 inflammasome plays a central role. The NLRP3 inflammasome also drives IL-1β maturation and secretion in another disease of metabolic dysregulation, gout. Thus, we propose that the NLRP3 inflammasome contributes to the pathogenesis of T2DM and gout by functioning as a sensor for metabolic stress.

ER stress activates the NLRP3 inflammasome via an UPR-independent pathway

Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1β. This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.

RNA viruses promote activation of the NLRP3 inflammasome through a RIP1-RIP3-DRP1 signaling pathway

The NLRP3 inflammasome functions as a crucial component of the innate immune system in recognizing viral infection, but the mechanism by which viruses activate this inflammasome remains unclear. Here we found that inhibition of the serine-threonine kinases RIP1 (RIPK1) or RIP3 (RIPK3) suppressed RNA virus–induced activation of the NLRP3 inflammasome. Infection with an RNA virus initiated assembly of the RIP1-RIP3 complex, which promoted activation of the GTPase DRP1 and its translocation to mitochondria to drive mitochondrial damage and activation of the NLRP3 inflammasome. Notably, the RIP1-RIP3 complex drove the NLRP3 inflammasome independently of MLKL, an essential downstream effector of RIP1-RIP3–dependent necrosis. Together our results reveal a specific role for the RIP1-RIP3-DRP1 pathway in RNA virus–induced activation of the NLRP3 inflammasome and establish a direct link between inflammation and cell-death signaling pathways.

Recognition of double-stranded RNA by TLR3 induces severe small intestinal injury in mice.

The role of TLRs on intestinal epithelial cells (IECs) is controversial, and the mechanisms by which TLRs influence mucosal homeostasis are obscure. In this study, we report that genomic dsRNA from rotavirus, and its synthetic analog polyinosinic-polycytidylic acid (poly(I:C)), induce severe mucosal injury in the small intestine. Upon engaging TLR3 on IECs, dsRNA triggers IECs to secrete IL-15, which functions to increase the percentage of CD3+NK1.1+ intestinal intraepithelial lymphocytes (IELs) and enhances the cytotoxicity of IELs. Moreover, The CD3+NK1.1+ IELs are proved as CD8αα+ IELs. These results provide direct evidence that abnormal TLR3 signaling contributes to breaking down mucosal homeostasis and the first evidence of pathogenic effects mediated by CD8αα+ IELs. The data also suggest that genomic dsRNA may be involved in the pathogenesis of acute rotavirus gastroenteritis.

NKG2D–retinoic acid early inducible‐1 recognition between natural killer cells and kupffer cells in a novel murine natural killer cell–dependent fulminant hepatitis

Increasing evidence suggests the contribution of natural killer (NK) cells to pathogenesis of human hepatitis, but the detailed mechanisms have yet to be clearly elucidated. In this study, injection of polyinosinic:polycytidylic acid (poly I:C) and D‐galactosamine (D‐GalN) was used to establish a novel murine fulminant hepatitis model: results showed that predepletion of either NK cells or Kupffer cells could completely abolish the liver injury. Injection of poly I:C/D‐GalN into mice could promote tumor necrosis factor‐α production and surface retinoic acid early inducible‐1 (Rae1) protein expression by Kupffer cells, which then activated NK cells to produce interferon‐γ via NKG2D‐Rae1 recognition. NK cell–derived interferon‐γ and Kupffer cell–derived tumor necrosis factor‐α synergistically mediated the severe liver injury. Moreover, Kupffer cell–derived interleukin‐12 and interleukin‐18 were also found to improve cross talk between NK cells and Kupffer cells. Conclusion: These results provide the first in vivo evidence that NKG2D/ligand interaction is involved in the synergic effects of NK cells and Kupffer cells on acute liver injury. (HEPATOLOGY 2009.)

NKG2D recognition mediates Toll-like receptor 3 signaling-induced breakdown of epithelial homeostasis in the small intestines of mice.

Toll-like receptors (TLRs) and NK receptors are the two most important receptor families in innate immunity. Although it has been observed that TLR signaling can induce or up-regulate the expression of the ligands for stimulatory NK receptors on monocytes or muscle cells, there is not yet a report indicating whether TLR signaling can break down self-tolerance through NK receptors. The present work reports that TLR3 signaling by polyinosinic–polycytidylic acid stimulation induces intestinal epithelial cells (IECs) to express retinoic acid early inducible-1 (a ligand for NKG2D) and to induce NKG2D expression on CD8αα intestinal intraepithelial lymphocytes by IL-15 derived from TLR3-activated IECs. The blockade of interaction between NKG2D and Rae1 inhibits the cytotoxicity of intraepithelial lymphocytes against IECs in a cell–cell contact-dependent manner and therefore alleviates polyinosinic–polycytidylic acid-induced epithelial destruction and acute mucosal injury of small intestine. These results demonstrate that TLR signaling induces tissue injury through the NKG2D pathway, suggesting that TLR signaling may break down self-tolerance through induction of abnormal expression of ligands for stimulatory NK receptors.

TLR-9 Activation Aggravates Concanavalin A-Induced Hepatitis via Promoting Accumulation and Activation of Liver CD4+ NKT Cells

Increasing evidence suggests that TLRs are involved in the pathogenesis of liver diseases; however, the underlying mechanisms remain obscure. In this study, we found that treatment with CpG-oligodeoxynucleotide (ODN) promoted the accumulation and activation of murine hepatic NKT cells. Additional experiments showed that CpG-ODN preferred to act on CD4+ NKT cells, while having less effect on CD4− NKT cells. The effect of CpG-ODN on liver NKT cells depended on the presence of Kupffer cells and IL-12. Meanwhile, CpG-ODN pretreatment aggravated liver injury and promoted the production of inflammatory cytokines in a Con A-induced fulminant hepatitis model via TLR9 activation. Collectively, our data demonstrate that TLR9 stimulation prefers to promote the accumulation and activation of hepatic CD4+ NKT cells and suggest that TLR9 signaling might be involved in the pathogenesis of human hepatitis.

Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2D57N mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.Summary Mitochondria need to be juxtaposted to phagosomes to synergistically produce ample reactive oxygen species (ROS) in phagocytes for pathogens killing. However, how phagosomes transmit signal to recruit mitochondria remains unclear. Here, we report that the kinases Mst1 and Mst2 function to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activate Rac GTPase to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for mitochondrial recruitment to phagosomes. Inactive forms of Rac, including the human Rac2D57N mutant, disrupt the TRAF6-ECSIT complex by sequestering TRAF6, and severely dampen ROS production and greatly increase susceptibility to bacterial infection. These findings demonstrate the TLR-Mst1-Mst2-Rac signalling axis to be critical for effective phagosome-mitochondrion function and bactericidal activity.