WikiJournal Preprints/Inflammasomes: Role in health and disease

Discovery
The term "inflammasome" was introduced by Martinon et al. in 2002 that described the assembly of large complex structures in the cytoplasm of activated immune cells, leading to proteolytic activation of proinflammatory caspases, which drive immune response and inflammation.

Inflammasome assembly


Inflammation occurs when the immune system sends signaling molecules and white blood cells to the site of injury to fight bacteria, viruses, and other pathogens and help repair damaged tissue. During infection, the innate immune response employs a form of first defense through a cluster of pattern recognition receptors (PRRs) to recognize pathogen-associated molecules, pathogen-associated molecular patterns (PAMPs), and damage-associated molecular patterns (DAMPs) generated by the host. PRRs can be membrane-expressed receptors, such as a Toll-like receptor (TLR), or cytoplasmic NOD-like receptors (NLR). The inflammasome complex is activated by a subset of cytosolic PRRs that recognize various PAMPs and DAMPs.

PAMP and DAMP sensors in the cytoplasm are NLR, AIM2, and pyrin. Inflammasomes typically consist of sensors, adaptor molecule ASC, and procaspase-1. Upon detecting certain stimuli, the sensor recruits ASC to form a multimeric complex, which then recruits procaspase-1 into the complex. Subsequently, procaspase-1 is converted into active caspase-1 through proximity-induced cleavage. Following this, the active caspase-1 subunits, p20 and p10, cleave pro-IL-1β and pro-IL-18 to produce the cytokines IL-1β and IL-18, and activate gasdermin D, a pore-forming molecule, to induce a form of cell death called "pyroptosis."

All members of the NLR family contain a nucleotide-binding domain (NBD) and a C-terminal LRR domain. Based on the presence of N-terminal, pyrin (PYD) or CARD domains, NLRs can be further divided into NLRP and NLRC receptors. The human and mouse genomes encode 22 and 34 NLRs, respectively. Of these proteins, NLRP1, NLRP3 and NLRC4 are known to induce inflammasome formation to activate caspase-1. Other NLR members, namely NLRP12, NLRP6, and NLRP9b can also form inflammasomes, but research in their function as inflammasome sensors is ongoing.

Inflammasome assembly requires homotypic (binding of other proteins of the same type) CARD-CARD or PYD-PYD interactions between the constituent components. Furthermore, either CARD or PYD can induce oligomerization which is the basis of inflammasome assembly. When a ligand is detected, the sensor is released from the inhibitory state and oligomerizes ASC by inducing homotypic interactions between the PYD domain. Subsequently, ASC recruits pro-caspase-1 through interactions between its CARD domains.

The assembly of NLRP3, AIM2, and pyrin inflammasomes highly depend on the adaptor protein ASC. Since NLRP1 and NLRC4 have a CARD domain, they can directly recruit caspase-1. In other words, NLRP1 and NLRC4 can induce inflammasome assembly and pyroptosis independently of ASC.

Inflammasome family
Some inflammasomes have been described, including NLRP3, NLRP1, AIM2, NAIP-NLRC4, and pyrin. The NLRP3 inflammasome consists of the NLRP3 molecule sensor, the ASC protein adaptor, and pro-caspase-1. The AIM2 inflammasomes sense cytosolic DNA through its C-terminal HIN200 domain and can recruit pro-caspase-1 through ASC to form the AIM2-ASC-pro-caspase-1 complex. Unlike NLRP3 and AIM2, NLRP1 protein contains PYD and CARD domains, which interact directly with pro-caspase-1 without the adaptor protein ASC, but the presence of ASC can enhance NLRP1-mediated caspase-1 activation. NLRC4 has only a CARD domain, which recruits pro-caspase-1 directly in the absence of ASC to form NLRC4 inflammasomes.

Inflammasome NLRP3
By far the most studied NLR is NLRP3. NLRP3, also known as cryopyrin and NALP3, is upregulated in response to macrophage stimulation by PAMPs (e.g., lipopolysaccharide, LPS) or inflammatory cytokines (e.g., TNF-α).

The NLRP3 gene encodes the protein cryopyrin. NLRP3 lacks a caspase recruitment domain (CARD) and thus cannot recruit procaspase-1 except in the presence of the adaptor molecule ASC. Cryopyrin is a member of a family of proteins called intracellular "NOD-like" receptor (NLR) proteins. Cryopyrin is found mainly in white blood cells and cartilage-forming cells (chondrocytes). Cryopyrin recognizes bacteria; chemicals such as asbestos, silica, and uric acid crystals; and compounds released by injured cells.

NLRP3 inflammasomes can be activated by the pore-forming activity of a broad spectrum of Gram-positive and Gram-negative bacteria, especially by triggering potassium (K+) secretion.

Inflammasome NLRP1
NLRP1 is expressed in adaptive immune cells and tissues as well as in non-hematopoietic tissues. Unlike other NLRs, the NLRP1 sensor contains function-to-find (FIIND) and CARD domains, in addition to PYD, NBD, and LRR domains. Oligomerized NLRP1 can directly recruit caspase-1 through its CARD domain and can be further enhanced by binding to ASC through its pyrin domain (PYD). The mouse genome encodes three NLRP1 paralogs (NLRP1a, NLRP1b, NLRP1c), all of which lack the PYD. NLRP1b is activated by cleavage at the N terminus and FIIND domain by lethal factor (LF), a component of anthrax lethal toxin (LeTx) produced by Bacillus anthracis. This activation of NLRP1b by LeTx has been shown to protect mice from Bacillus anthracis infection.

Inflammasome AIM2
AIM2 also known as PYHIN4, is a member of the PYHIN family (containing a Pyrin domain and a HIN200 domain). AIM2 is an interferon-inducible gene. AIM2 (absent in melanoma 2) was initially identified during functional screening for tumor suppressor genes in melanoma. AIM2 does not belong to the NLR proteins, but several research groups identified AIM2 as a component of the inflammasomes.

AIM2 can form inflammasomes whose assembly is stimulated by cytosolic DNA recognition from bacteria or viruses or DNA from apoptotic cells. The binding of DNA to the HIN domain results in a conformational change and oligomerization of AIM2 around the DNA molecule, which further enables recruitment of ASC and caspase-1 to form stable inflammasomes. Similar to NLR inflammasomes, AIM2 inflammasomes result in IL-1β and IL-18 secretion, as well as cell death.

Inflammasome NAIP-NLRC4
NLRC4 is expressed mainly in hematopoietic tissues and cells. NLRC4 has a CARD domain so that it can interact directly with procaspase-1 through homotypic CARD-CARD binding leading to caspase-1 processing, and this process can be independent of ASC. NLRC4 is activated in response to many pathogenic bacteria, including Salmonella typhimurium and Pseudomonas aeruginosa. NLRC4 acts as a sensor for bacterial flagellin or structural components of type III bacterial secretion system (T3SS). However, NLRC4 is not a direct sensor of these ligands. The system uses NAIP (NLR-family apoptosis-inhibiting protein) in the cytosol as a sensor of NLRC4 ligands, so NAIP is essential for NLRC4 inflammasome activation. In humans, one NAIP has been identified.

Inflammasome Pyrin
In humans, pyrin consists of an N-terminal PYD, a central B-box, a coiled-coil domain, and a C-terminal B30.2/SPRY domain. Study shows that pyrin inflammasomes are assembled on modified cytoskeletal proteins. Toxins produced by various bacterial species, such as Clostridium difficile (TcdB), Clostridium botulinum (C3), Vibrio parahemolyticus (VopS), Burkholderia cenocepacia, and Bordetella pertussis (PT), induce covalent modifications of Rho family members. These modifications include glycosylation, adenylation, and ribosylation of ADP, and lead to the assembly of pyrin inflammasomes.

Inflammasome activation
Some pathways for inflammasome activation have been identified.

NLRP3 inflammation activation
NLRP3 inflammasomes are activated by diverse endogenous and exogenous agonists, including PAMPs and toxins from bacterial, fungal, and protozoan pathogens, as well as DAMPs such as ATP, uric acid crystals, and β-amyloid fibrils. For NLRP3 inflammasome activation, a two-signal model has been proposed; the first signal is referred to as priming, while the second is aimed at inflammasome assembly. In the model, the first signal is provided by microbial components or endogenous cytokines, while the second signal is from extracellular ATP, pore-forming toxins, or particulate matter.

Signal 1 involves activation of the MyD88 activating pathway or other transcription factors (e.g., NF-κB), which upregulates the expression of Nlrp3 and other inflammasome components. Signal 2 involves multiple molecular signaling events induced by NLRP3 stimuli, including potassium ion efflux, mitochondrial dysfunction, and reactive oxygen species (ROS) production. Cytosolic potassium ion efflux is a common trigger in NLRP3 inflammasome activation. NEK7 (NIMA-related kinase 7), a mitosis-associated serine-threonine kinase, acts as an NLRP3-binding protein through interactions between their respective LRR domains. NEK7 acts downstream of potassium ion release to regulate oligomerization and activation of NLRP3 inflammasomes. Mitochondrial dysfunction is another trigger in NLRP3 activation through mitochondrial DNA (mtDNA) shedding, mitophagy, and apoptosis. Meanwhile, ROS induces the binding of thioredoxin-interacting proteins to NLRP3, which is important for NLRP3 inflammasome activation.

Non-canonical activation
A common and accepted (canonical) pathway in inflammasome activation is the recruitment of caspase-1 in response to PAMPs or DAMPs. However, there is also a non-canonical pathway where NLRP3 inflammasomes activation occurs downstream of the cleavage of caspase-11 (or caspase-4 and caspase-5 in humans) and gasdermin D. In non-canonical inflammasomes signaling, caspase-11 acts as an LPS sensor in the cytosol. Upon recognizing LPS, caspase-11 initiates IL-1β proteolytic maturation and gasdermin D-dependent pyroptotic cell death. Caspase-11 directly binds to LPS via the CARD domain. Caspase-11 signaling engages the NLRP3 inflammasomes, thereby cross-recruiting caspase-1 signaling and inducing the maturation of IL-1β and IL-18.

Regulator of inflammation activation
Activation of NLRP3 inflammasomes is mostly beneficial for host immunity, but excessive production of IL-1β and IL-18 can result in sterile inflammation that may increase the risk of metabolic diseases and autoinflammation. Therefore, activation of the NLRP3 inflammasome must be appropriately controlled through binding proteins and post-translational modifications.

Members of the POP (pyrin-only protein) family, including POP116 and POP216, block inflammasome assembly by binding to ASCs and inhibiting ASC recruitment to NLRP3. Negative regulatory molecules that target ion depletion, mitochondrial function, and ROS signaling can also block NLRP3 inflammasome activation. For example, the ketone body compound β-hydroxybutyrate can inhibit NLRP3 inflammasome-mediated inflammation by preventing the release of potassium ions.

Post-translational modifications of NLRP3, including ubiquitination and deubiquitination, can also suppress or activate inflammasome activation. During the activation step, phosphorylation of NLRP3 by Golgi-mediated protein kinase D (PKD) at Ser293 (or human Ser295) can trigger inflammasome assembly. In contrast, phosphorylation of NLRP3 by PKA at Ser 291 of mice mediates the negative regulation of bile acids-induced NLRP3 inflammasomes. In addition to phosphorylating enzymes, deubiquitinating enzymes are also involved in inflammasome regulation. For example, BRCC3 promotes inflammasome activation by deubiquitinating NLRP3 at the LRR domain. ABRO1, a component of the BRCC3 complex, can enhance NLRP3 inflammasome activation by regulating NLRP3 deubiquitination after LPS induction.

Antimicrobial immunity
The main effects of inflammasome activation are pyroptosis and/or secretion of IL-1β and IL-18, which protect against microorganisms invading the body. Inflammasome proteins are expressed mainly by macrophages and dendritic cells. The intestines and lungs are the main tissues and cells involved in inflammasome-mediated immunity against microbial infection. Epithelial cells are essential lining cells on these body surfaces, and inflammasomes are known to be involved in defense there. In the case of antiviral defense, NLRP3 inflammasomes have been known to be involved in inhibiting viral replication.

Comensal microbial
Inflammasomes may also play a role in shaping gut microbiota composition. NLRP6-deficient mice have altered gut microbiota composition characterized by the presence of Prevotellaceae species and become more susceptible to colitis. These changes in microbial composition and susceptibility to colitis can be transmitted to normal mice, indicating that changes in the gut microbiota mediate this phenotype. NLRP6 inflammasome assembly and IL-18 secretion are required to maintain gut hemostasis through the regulation of microbiota composition, and the absence of NLRP6 inflammasomes leads to the expansion of potentially pathogenic microbiota members.

Pyroptosis cell death
Pyroptosis is a form of cell death induced by inflammasome activation, characterized by cell swelling followed by lysis and release of intracellular contents. Gasdermin D has been identified as a link between cell death and activation of caspase-1 and/or caspase-11. Pyroptotic caspases 1 and 11 (caspase-4 and caspase-5 in humans) cleave Gasdermin D at D276 in the binding region, thereby eliminating the intramolecular inhibitory effect of the C-terminus domain. Upon binding to membrane lipids, the N end region of Gasdermin D (GSDMD-N) oligomerizes to form pores with an inner diameter of 10 to 18 nm. The formation of these pores disrupts the osmotic potential of the cell, thereby causing swelling and lysis. Expression of the N end fragment is also sufficient to kill bacterial cells.

CAPS disease
Mutations in NLRP3 cause cryopyrin-associated periodic syndromes (CAPS), a disease mediated mainly by cytokines from the innate immune system, especially IL-1β. The signs and symptoms of CAPS affect several body systems. CAPS is commonly characterized by episodes of skin rash, fever, and joint pain. These episodes may be triggered by exposure to cold temperatures, fatigue, other stressors, or may arise spontaneously. Episodes may last from a few hours to several days. These episodes usually begin in infancy or early childhood and persist throughout life. CAPS patients respond positively when treated with IL-1 blockers, suggesting that inappropriate IL-1β production by the NLRP3 inflammasome is a driver of CAPS development.

Gout
Gout, also called uric acid disease, is when crystals of monosodium urate (MSU) accumulate in the joints and cause painful inflammation. Studies show that the mechanism of the MSU-induced inflammatory response depends on the proinflammatory cytokine IL-1β. This IL-1-dependent inflammatory phenotype is now understood to depend on the formation of NLRP3 inflammasomes in response to the 'danger signal' of uric acid crystals. In the absence of NLRP3 inflammasome components, macrophages cannot secrete active IL-1β following stimulation with MSU and calcium pyrophosphate crystal dihydrate.

During the initiation phase, MSU crystals are deposited within the joint stimulate extracellular TLRs expressed by the monocytes resident, leading to the transcription of pro-IL-1β. MSU crystals are also phagocytosed by resident monocytes, which is positively regulated by TLR activation, resulting in oligomerization of the NLRP3 inflammasome, activation of caspase-1, and cleavage of pro-IL-1β into active IL-1β.

NLRP3 inflammasome inhibitor
The involvement of NLRP3 inflammasomes in various diseases encourages researchers to find NLRP3-targeted drugs. Clinical treatment of NLRP3-related diseases has only used IL-1β antibodies or recombinant IL-1β receptor antagonists, such as canakinumab and anakinra. However, these inhibitors have the disadvantage that inflammatory diseases do not only involve IL-1β as a cause. In addition, IL-1β can be produced by an inflammasome-independent pathway, and this cytokine can also be produced from other types of inflammasomes. Therefore, inhibitors targeting IL-1β may cause unwanted immunosuppressive effects.

Several small molecule compounds have shown anti-inflammatory effects on NLRP3 inflammasome activation in vitro, including MCC95090, β-hydroxybutyrate, Bay 11-7082, dimethyl sulfoxide (DMSO), and type I interferon.

Dapansutril (OLT1177), an orally active β-sulfonyl nitrile compound, is a newly developed drug that inhibits the NLRP3 inflammasome. Nanomolar concentrations of OLT1177 reduced the release of IL-1β and IL-18 after activation of canonical and non-canonical NLRP3 inflammasomes. The molecule showed no effect on NLRC4 and AIM2 inflammasomes, suggesting specificity for NLRP3. In phase 2a clinical trials, dapansutril demonstrated a good safety profile and efficacy in reducing target joint pain.

Acknowledgements
The author acknowledge the support of Dicky Rizky Febrian during preparing this manuscript.

Competing interests
None.

Ethics statement
No animal or human was involved as research subject.