Physiological role of the Inflammasomes

An inflammasome represents a high molecular weight complex that activates inflammatory caspases and activates cytokines of the IL-1 family. Several inflammasomes have been described and were so far defined by the NLR protein that they contain – the NLRP1 (NALP1) inflammasome, the NLRP3 (NALP3) inflammasome and the IPAF (NLRC4) inflammasome.

• Introduction 
• Inflammasomes – Activity Regulation
• Inflammasomes – Therapeutics Implications
• Measuring the Inflammasomes

Introduction

Interestingly, the AIM2 (absent in melanoma 2) inflammasome, which has been described recently, does not contain any member of the NLR family. Inflammasomes can be activated through multiple signals including live bacteria, microbial toxins, xeno-compounds, PAMPs and DAMPs. It is suggested that the LRR domains of NLRP3 mediate autorepression, probably by SGT1 and HSP90 chaperones that maintain NLRP3 in an inactive, but signal-competent state. Upon sensing of their respective ligands, NLRP1 or NLRP3 oligomerize via the NACHT domain, what leads to PYD clustering and to the recruitment of the adapter protein ASC (apoptosis-associated speck-like protein containing a CARD) due to homotypic PYD-PYD interactions. In the case of AIM2, oligomerization is likely mediated by clustering upon multiple binding sites within dsDNA and not by a central oligomerization domain like NACHT.

However, AIM2 oligomerization also leads to the recruitment of ASC via homotypic PYD-PYD interactions. ASC contains an N-terminal PYD domain and a C-terminal CARD domain that allows the recruitment of inflammatory caspases through homotypic CARD-CARD interactions. Thus inflammatory caspases are brought into close proximity what permits autoactivation and formation of the active caspase. In case of procaspase-1, a p10/p20 tetramer is formed after autocleavage. In addition to caspase-1, NLRP1 can also recruit caspase-5 to the complex but the role of caspase-5 is still under debate. Contrary to NLRP1, NLRP3 and AIM2, IPAF does not recruit an adapter molecule but directly interacts with procaspase-1 via its CARD domain (see Figure below). However, depending on the IPAF stimulus, maximal caspase-1 activation downstream of IPAF can require ASC or NAIP. The assembly of the different inflammasomes elicits a common downstream cascade, namely the activation of inflammatory caspases. These include caspase-1, -4 and -5 in human and caspase-1, -11 and -12 in mouse. However, caspase-1 appears to be the most dominant inflammatory caspase associated with inflammasomes. The inflammatory caspases all have a CARD domain followed by a domain containing the catalytic residue cysteine and are called inflammatory caspases because their main substrates are cytokines (such as pro-IL-1β, pro-IL-18 and eventually pro-IL-33) which are cleaved to their active and secreted form. In addition, inflammasome activation can lead to host cell death in certain cell types, known as pyroptosis. Pyroptosis is thought to be important in restricting the intracellular replication of invasive pathogens.

Inflammasomes – Activity Regulation

Even though mechanisms that regulate inflammasome activity remain elusive, various proteins were identified that may interfere with inflammasome activation and inflammasome dependent inflammatory caspase processing. In general, two subtypes of inflammasome regulators can be distinguished – those containing a CARD domain and those with a PYD domain. Such proteins encompass not only host derived inflammasome regulators, but also various bacterial virulence factors inhibiting caspase-1 activation and viral PYD proteins.

Inflammasomes – Therapeutics Implications

As IL-1β and other cytokines are key players in the inflammatory response, it is tempting to speculate that IL-1β, inflammatory caspases and inflammasomes play an important role in several diseases (see Figure below). Indeed, some human hereditary or acquired diseases have been linked to elevated IL-1β, some of which can be treated by antagonists against IL-1β or its receptor. A number of diseases, known as cryopyrin-associated periodic syndroms (CAPS), have been directly linked to NLRP3 mutations. Furthermore, gout, an autoinflammatory disease characterized by severe joint inflammation, is associated with the deposition of MSU crystals in joints, among other characteristics. As MSU is a potent NLRP3 inflammasome agonist, it is believed inflammasome-regulated IL-1β exerts a pathogenic role in gout.

Furthermore, IL-1β secretion by the NLRP3 inflammasome is triggered by high extracellular glucose in b-cells. Elevated IL-1β is a risk factor for the development of Type 2 Diabetes Mellitus (T2DM) and contributes to insulin resistance.Thus by functioning as a sensor for metabolic stress, like in the form of monosodium urate (MSU) or hyperglycemia, the NLRP3 inflammasome likely contributes to the pathogenesis of gout or T2DM, respectively.In addition, several inflammasome regulators have a significant relevance in diseases. In patients with FMF (familial Mediterranean fever), pyrin was shown to be mutated. Elevated IL-1β levels in the autoinflammatory disease PAPA (pyogenic arthritis, pyoderma gangrenosum, and acne) are associated with mutations in PSTPIP1, a pyrin interacting protein. Together, this indicates the importance of pyrin and inflammasome regulators in autoinflammatory diseases and may allow novel entry points for disease treatment.

• • Measuring the Inflammasomes

    Inflammasomes are multi-protein complexes implicated in physiological and pathological inflammation. Immunoblotting for caspase-1 is the gold-standard method of detecting inflammasome activation, but also presents certain technical difficulties, particularly with the choice of protein precipitation, protein separation and transfer protocols. Priming and Activation steps are crucial for successful immunoblotting as well. Listed below are Spinger Protocols from the Series of Methods and Protocols using Adipogen’s Standard Antibodies for Inflammasomes detection.