Turning down the heat: the potential role of RIP140 in inflammation

The control of inflammation is an essential process for maintaining immune homeostasis, but the regulatory mechanisms which govern this event remain poorly understood. Endotoxin tolerance is a convenient experimental model which can be used to elucidate potentially relevant mechanisms that control the host's inflammatory response. Endotoxin tolerance is marked by a transitory period of hyporesponsiveness to lipopolysaccharide (LPS, also known as endotoxin) stimulation following an initial exposure to LPS. Although the alteration of intracellular signaling has been well described, there are likely multiple mechanisms and pathways which contribute to the phenomenon called endotoxin tolerance. In the April issue of Nature Immunology, a role for the nuclear receptor corepressor, named receptor-interacting protein 140 (RIP140), is introduced as another potentially important molecular mechanism in modulating the inflammatory response.1 Using an experimental model of endotoxin tolerance, the authors show that RIP140 regulates the transcription and production of NF-κB-dependent pro-inflammatory cytokines. First, the authors demonstrated that conditions which induced endotoxin tolerance were associated with significantly lower levels of RIP140 protein. Meanwhile, specific knockdown of RIP140, using a transgenic mouse expressing a macrophage-specific RIP140-specific gene silencing mRNA, resulted in diminished cytokine (tumor-necrosis factor (TNF) and IL-1β) production and improved survival in response to LPS challenge. Furthermore, the decrease in RIP140 was dependent on the dose and time of LPS, and could also be elicited with the synthetic Toll-like receptor 2 (TLR2) and TLR3 agonists, Pam3CSK4 and poly (I:C). The authors next attempted to determine what was causing RIP140 to decrease after the initial LPS exposure. The LPS-mediated decrease in RIP140 was determined to be dependent on a SOCS1-associated SCF E3 ligase complex, called SOCS1-Rbx1, which promote ubiquitination and proteasome-mediated degradation. These findings were accomplished through blocking RIP140 degradation with the proteasome inhibitor MG132 and knockdown of Rbx1 and SOCS1 using Rbx1- and SOCS1-specific small interfering RNA, respectively. However, they found that RIP140 did not directly interact with the SOCS1–Rbx1 E3 ligase. On the other hand, the RelA subunit of NF-κB was determined to function as a co-adaptor of SOCS1–Rbx1 E3 ligase, to recruit RIP140 for degradation. When RelA was knocked down by small interfering RNA, RIP140 did not associate with SOCS1 and RIP140 was not degraded. Since the initiation of ubiquitin-mediated proteasome degradation hinges upon tyrosine phosphorylation events, the authors sought to identify a potential specific tyrosine kinase and its RIP140 targets. They showed that the non-receptor tyrosine kinase Syk phosphorylated RIP140, at residues Tyr364, Tyr418 and Tyr436. Furthermore, when those tyrosine residues were substituted with phenylalanine (Y3F-mutant RIP140), mutant RIP140 would no longer associate with SOCS1–Rbx1 E3 ligase and RIP140 became resistant to proteasome degradation. Interferon-γ (IFN-γ) is known to abrogate endotoxin tolerance and promote pro-inflammatory cytokine production. Using conditions that induce endotoxin tolerance, IFN-γ suppressed RIP140 degradation. And when RIP140 was knocked down or Y3F-mutant RIP140 was used, IFN-γ did not suppress the development of endotoxin tolerance or restore the production of TNF and IL-1β. Lastly, the authors performed gene expression experiments which showed that RIP140 affects the expression of specific genes, including Tnf and Il1b, and activated histone modifications on the promoters of these genes. Therefore, these data suggest that endotoxin tolerance is promoted by the degradation of RIP140, resulting in the loss of RelA binding and histone modifications, which contribute to the alteration of NF-κB transcriptional activity. Without tight regulatory control over the spectrum of a dynamic immune response, inflammation can rapidly spiral out of control, such as in sepsis. A burst of pro-inflammatory cytokines must be quickly balanced with mechanisms to dampen the response and return the host back to immune homeostasis. Endotoxin tolerance is a phenomenon which has been used to explain the prolonged immunosuppressed state and increased susceptibility to secondary infections of patients recovering from Gram-negative sepsis and other major proinflammatory events. Early studies starting from the 1940s first described that fever could be suppressed with repeated injections of typhoid vaccine.2 Subsequently, purified bacterial endotoxin pretreatment was also shown to reduce fever and LPS-induced lethality.3 Soon, endotoxin tolerance became a paradigm from which to explain resistance to inflammatory processes and infection, under many conditions. The broader concept of ‘microbial tolerance' has been espoused to support the blunted immune response induced by an infection and which is thought to originate from the host's attempt to prevent overwhelming cytokinemia. These states of tolerance have also been referred to as ‘desensitization', ‘immune paralysis' and ‘reprogramming'. Nonetheless, the mechanisms which are responsible for this tight control or which are perturbed during sepsis and other events remains incompletely defined (refer to the thorough reviews by Medvedev4 and Cavaillon5 for more information). The classical over simplified models of endotoxin tolerance can be a problem. There may be significant differences when studies are performed using single purified TLR agonists (such as LPS), in comparison to multiple different TLR agonists or whole bacteria. The in vitro experiments need be replicated with in vivo experiments and challenge experiments using live, replicating pathogenic bacteria. When conducting animal models of lethal infection, the choice of bacterial strain and animal species can be important determinants which can influence whether the model represents intoxication versus sepsis.6 Further complicating this field of research is the often forgotten counter-phenomenon of the Shwartzman-like reaction. In contrast to low doses of LPS (1–100 mg/ml) inducing a tolerant state, the Shwartzman-like reaction is elicited with ‘super low' doses of LPS (0.05–0.5 ng/ml) and results in the opposite effect, an exaggerated inflammatory response.7 The mechanisms which underlie this phenomenon are largely unexplored, yet should be important in teasing out the true significance of molecular mechanisms identified with the classical endotoxin tolerance models. The immune system is a complex, dynamic, highly redundant and decidedly orchestrated network. Events, such as sepsis, can cause a fatal disequilibrium of the pro-inflammatory immune response. This initial report on RIP140 adds another page to the growing text of knowledge on factors which potentially control inflammation. The clinical relevance of RIP140 will only be established by expanding and confirming these findings in relevant animal models. If true, future therapeutic approaches may be targeted to turn down the heat and control overwhelming inflammation.