Inflammasome

The principle of the inflammasome was defined in 2002 by Jürg Tschopp at the University of Lausanne (Martinon F et al. 2002). In 2002, his team discovered that a subgroup of NLRs (NLRP1) is able to assemble and oligomerize into a common structure in vitro. This oligomerization leads to the activation of the caspase-1 cascade and thus to the production of proinflammatory cytokines, in particular IL-1β and IL-18. This multimolecular NLRP1 complex was term ed the "inflammasome" (NLRTP inflammasome) . Later, other inflammasome types were discovered with different compositions and different activation mechanisms.

The biological relevance of inflammasomes was recognized in 2006, when the role of the INLRP1 inflammasome in various infections, toxin exposure, and other diseases was discovered. infections, toxin exposure, gout and type 2 diabetes (Martinon F et al. 2006; Kanneganti TD et al. 2006). Viral DNA, muramyl dipeptide (MDP), asbestos and silicon dioxide can also trigger activation of inflammasomes.

In addition to NLR proteins, other PRRs can also form inflammasomes. In 2009, an inflammasome of the PYHIN family (pyrin and HIN domain-containing protein) was described with the designation "absent in melanoma 2(AIM2)" (Hornung V et al. 2009), which forms upon recognition of foreign cytoplasmic double-stranded DNA (dsDNA).

Inflammasomes are instruments of the innate immune system that are responsible for the activation of inflammatory reactions. Inflammasomes are cytosolic (intracellular) multi-protein complexes(Martinon F et al. 2002), which are predominantly found in immune cells, such as dendritic cells and macrophages, but also in epithelia of the skin and mucous membranes (intestinal and urinary bladder epithelia).

Activation of the inflammasome promotes the proteolytic cleavage, maturation and secretion of the pro-inflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18) as well as the cleavage of gas dermin D. The N-terminal fragment resulting from gasdermin D cleavage, triggers a pro-inflammatory form of programmed cell death of pyroptosis and is responsible for the secretion of mature cytokines, presumably through the formation of pores in the plasma membrane (Broz P et al. 2016). In addition, inflammasomes can also trigger a special form of programmed cell death, PANOptosis, which has essential features of apoptosis, pyroptosis/necroptosis(Zhuang L et al.2023).

Activation of inflamm asomes is triggered by different types of cytosolic pattern recognition receptors (PRRs), either microbe-derived (pathogen-associated molecular patterns/PAMPs) or host cell-derived (damage-associated molecular patterns/DAMPs). The pattern recognition receptors involved in inflammasomes include the so-called NLRs (nucleotide-binding oligomerization domain and leucine-rich repeat-containing receptors) as well as AIM2 (absent in melanoma 2), IFI16 (IFN-inducible protein 16) and pyrin (Broz P et al. 2016).

The inflammasome receptors interact with the adaptor protein ASC via their caspase activation and recruitment domain (CARD) or via the pyrin domain (PYD), which then activates caspase-1 via its CARD domain by proteolytic cleavage (Broz P et al. 2016). Finally, the activated caspase-1 cleaves the immature pro-inflammatory cytokines pro-IL-1beta and pro-IL-18 as well as gas dermin D into their mature and effective end stages (interleukin-1alpha/interleukin-18). The interleukins IL 1beta and IL 18 in particular are responsible for inflammation-induced inflammatory signaling and pyroptotic cell death.

In addition to these so-called canonical inflammasomes, non-canonical inflammasome complexes have also been described that act independently of caspase-1. In animal experiments, non-canonical inflammasomes can be activated by direct recognition of cytosolic bacterial lipopolysaccharide (LPS). In human cells, the corresponding caspases of non-canonical inflammasomes are caspase-4 and caspase-5 (Broz P et al. 2016).

So far, inflammasomes have mainly been detected in professional immune cells of the innate immune system, such as macrophages and neutrophils. However, it is now known that inflammasome components are expressed to a relevant extent in epithelial barrier tissues (Winsor N et al. 2019).

Inflammasome family: NLRP1, NLRP3, NLRP6 and NLRC4 are subgroups of the NLR family. They have two common features: Firstly, the nucleotide-binding oligomerization domain(NOD), which is bound by ribonucleotide phosphates (rNTP). It is essential for self-oligomerization (Ye Z et al. 2008). On the other hand, a C-terminal leucine-rich repeat (LRR), which serves as a ligand recognition domain for other receptors or for microbial ligands. NLRP1 has been found in neurons, while both NLRP3 and NLRC4 (IPAF) have been identified in microglial cells.

Inflammasomes can be subdivided depending on the interaction partner:

• NLRP1 inflammasome(NLRP is an acronym for: "NACHT domain, leucine-rich repeat, pyrin domains" a key protein of the inflammasome. In addition to NOD and LRR, NLRP1 contains a pyrin domain (PYD) at its N-terminus and a FIIND motif and a CARD at its C-terminus, which distinguishes it from the other inflammasomes. Upon activation, the C-terminal CARD interacts with the CARD of procaspase-1 or procaspase-5, while its N-terminal PYD interacts homotypically with the PYD of the adapter protein ASC, whose CARD can then recruit another pro-caspase-1. The general recruitment and cleavage of procaspase-1 can then activate all downstream caspase-1 signaling pathways.
• NLRP3- Inflammasome: see also NLRP. In addition to the NOD and LRR domains, NLRP3 contains a PYD domain like NLRP1 and therefore activates caspase-1 in the same way by using its PYD to recruit ASC. It forms only one oligomer per cell and its oligomer consists of seven NLRP3 molecules. The NLRP3 inflammasome is the largest inflammasome with a diameter of about 2 µm (Stutz A et al. 2009). The oligomerization of NL RP3 is activated by a large number of stimuli, including both PAMPs and DAMPs. Examples of such stimuli from the DAMP group are crystalline substances such as urate crystals, alum or asbestos. The NLRP3 inflammasome also reacts to PAMPs from various pathogens, such as viruses. Influenza A, bacteria, e.g. Neisseria gonorrhoeae and various bacterial toxins are proven activators (Duncan JA et al. 2009). All NLRP3 activators induce the efflux of cytosolic potassium from the cells. Activation of the NLRP3 inflammasome by cholesterol crystals and MSU crystals increases NLRP3-induced IL-1beta production (Jamilloux Y et al. 2014). This process is probably abolished in arteriosclerosis and gout, allowing these crystals to form in the cell. Inorganic particles such as titanium dioxide, silicon dioxide and asbestos can also trigger activation of the inflammasome (Yazdi AS et al. 2010).
• NAIP/NLRC4 inflammasome: NLRC4 forms the subgroup of the NLRC family. In addition to NOD and LRR, the inflammasome formed by the NLRC4 protein contains only one CARD domain, which it uses to directly recruit the adapter protein ASC or pro-caspase-1. The NAIP/NLRC4 inflammasome is involved in host defense (Liu L et al. 2014). In humans, NAIPs are activated by binding to the bacterial PAMPs in the cytosol, which are provided by the needle components (NAIP1) of the bacterial type 3 secretion system, flagella. After ligand binding, NAIPs interact with NLRC4 to initiate the assembly of the NAIP/NLRC4 inflammasome. The NAIP/NLRC4 inflammasome is the best described epithelial inflammasome and plays an important role in restricting intraepithelial bacterial populations in early stages of enterobacterial infection (Sellin ME et al. 2015: Sellin ME et al. 2018). Intracellular bacteria trigger activation of the inflammasome, leading to targeted expulsion of infected epithelial cells from the epithelium to reduce the bacterial load.
• AIM2 inflammasome: AIM2 stands for "Absent In Melanoma 2". AIM2 is a member of the IFI20X/IFI16 family. It plays a putative role in the control of cell proliferation. The AIM2 inflammasome is a detector of cytosolic double-stranded DNA (dsDNA) and plays an important role in the coordination of immune defense against DNA viral infections as well as intracellular bacterial infections (Broz P et al. 2016). AIM2 is activated by viral dsDNA, bacterial dsDNA and is therefore associated with various diseases. For example, it has been postulated that auto-inflammation in psoriasis could be related to the recognition of its own DNA by AIM2 (Broz P et al. 2016). In addition, the activation of AIM2 could play an additive role in autoimmunological diseases such as systemic lupus erythematosus. The AIM2 inflammasome is also activated by pharmacological disruption of nuclear envelope integrity (Di Micco A et al. 2016). The PYD domain of AIM2 interacts homotypically with ASC through PYD-PYD interactions. The ASC-CARD domain recruits pro-caspase-1 into the complex. Caspase-1 ultimately leads to the release of the proinflammatory cytokines IL1-beta and IL18.

• IFI16 inflammasome: Like AIM2, IFI16 (IFN-inducible protein 16) belongs to the PYHIN family (containing pyrin and HIN domains). IFI16 plays an important role in humans (IFI204 is the mouse ortholog) in the regulation of IFN production in both bacterial and viral infections. In contrast to AIM2, IFI16 is a nuclear DNA sensor (Winsor N et al. (2019). Upon interaction with viral DNAs, IFI16 was shown to recruit caspase-1 through interaction with ASC, leading to apoptosis of CD4+ T cells in response to HIV infection.

• Pyrin inflammasome: The assembly of the pyrin inflammasome is triggered by bacterial toxins as well as effector proteins by recognizing pathogen-induced perturbations in cytoskeletal dynamics. Pyrin recognizes the inactivation of the Rho-GTPase RHOA by bacterial toxins (Broz P et al. 2016). Upon detection of RHOA inactivation, pyrin interacts with ASC via its N-terminal PYD domain to induce caspase-1 activation.

• Non-canonical inflammasomes: These are independent of caspase-1. In humans, non-canonical inflammasomes rely on caspase 4 and caspase 5. All of these caspases are able to directly bind intracellular LPS and subsequently form macromolecular complexes that mediate the cleavage of gas dermin D and the induction of pyroptotic cell death.

Activation of the multimeric inflammasome complexes occurs through proteases (e.g. caspase-1 = cysteinyl-aspartate specific protease; Martinon F et al. 2002). By binding to the adapter molecule ASC (apoptosis-associated speck-like protein), caspase-1 can be bound and activated. The activated inflammasome complex is formed.

The NLRP3 inflammasome is best characterized. NLRP3 is activated by a variety of bacteria (including Staphylococcus, Listeria, or Neisseria), viruses (including EMCV, Sendai, and influenza viruses), and by fungi (including Candida spec. and Saccharomyces) (Kanneganti TD et al. 2006).

Furthermore, endogenous factors such as cellular stress can also lead to activation of NLRP3. This applies, for example, to released uric acid crystals (in gout), to cholesterol crystals (in atherosclerosis), or to environmental pathogens such as silicon oxide, asbestos, and UV-B radiation (Hornung V et al. 2008; Feldmeyer L et al. 2007).

Inflammasome activation is triggered by different types of cytosolic pattern recognition receptors (PRRs) that respond to either microbe-derived pathogen-associated molecular patterns (PAMPs) or host cell-generated damage-associated molecular patterns (DAMPs).

PRRs involved in inflammasomes include NLRs (nucleotide-binding oligomerization domain and leucine-rich repeat-containing receptors) as well as AIM2 (absent in melanoma 2), IFI16 (IFN-inducible protein 16) and pyrin (Broz P et al. 2016). The inflammasome receptors interact with the adaptor protein ASC via their caspase activation and recruitment domain (CARD) or via the pyrin domain (PYD), which then recruits pro-caspase-1 via its CARD domain and activates the effector caspase by proteolytic cleavage (Broz P et al. 2016). Finally, the activated caspase-1 cleaves the immature pro-inflammatory cytokines pro-IL-1β and pro-IL-18 as well as gasdermin D, which are responsible for inflammatory signaling and pyroptotic cell death, respectively.

In addition to these so-called canonical inflammasomes, various studies have also described non-canonical inflammasome complexes that act independently of caspase-1. In animal experiments, the non-canonical inflammasome can be activated by direct recognition of cytosolic bacterial lipopolysaccharide (LPS) by caspase-11, which subsequently triggers pyroptotic cell death. In human cells, the corresponding caspases of the non-canonical inflammasome are caspase 4 and caspase 5 (Broz P et al. 2016).

Inflammasomes have been detected primarily in professional immune cells of the innate immune system (e.g. in macrophages). However, recent studies indicate a high expression of inflammasome components in epithelial barrier tissues, where they have been shown to represent an important first line of defense (Winsor N et al. 2019).

NLRP3 inflammasome-associated diseases:

With the Toll-like receptors and the cytosolic NLRs, the innate immune system has two pathogen recognition systems for the rapid and non-specific defense against pathogens.

DAMPs (Danger-associated molecular pattern): The binding of non-microbial pathogens -DAMPs- can also lead to the release of inflammatory mediators such as IL-1beta and IL-18. These can be different pathogens such as extracellular ATP (from destroyed or activated cells), crystalline structures (asbestos, silicon oxide, uric acid, calcium pyrophosphate or cholesterol crystals) or reactive oxygen species (ROS).

What all substances have in common is that they lead to NLRP3 activation and thus induce the release of IL-1beta. After IL-1beta has been released in bioactive form, IL-1beta binds to its receptor (IL-1R) and initiates inflammatory reactions of various kinds (Dinarello CA 2009).

NLRP3 mutations: Some genetically fixed diseases are associated with elevated serum levels of interleukin-1β. These autoimmune diseases are summarized under the generic term"hereditary periodic fever syndromes" (cryopyrin-associated-periodic syndromes - CAPS). These are syndromes with periodic episodes of fever and inflammation.

Gout and pseudogout: In recent years, gout and pseudogout have been linked to NLRP3 (Martinon F et al. 2006). For both diseases, the pathognetic principle is the precipitation of uric acid or calcium pyrophosphate crystals in the joints, strong activators of NLRP3. The result is a marked release of IL-1beta.

Silicosis/asbestosis: An analogous mechanism applies to other crystalline substances such as silicon and asbestos dust. The uptake of these substances by alveolar macrophages leads to NLRP3-dependent increased production of IL-1β (Rimal B et al. 2005). Diesel soot particles or cigarette smoke also mediate NLRP3-dependent chronic pulomnal inflammatory processes.

Alzheimer's disease: Alzheimer's disease as a progressive, neurodegenerative disease of the cranial nerves is characterized by the deposition of misfolded beta-amyloid peptide chains (Alzheimer's plaques) in the central nervous system. Phagocytizing cells of the microglia are stimulated to phagocytosis by beta-amyloid-containing plaques and release inflammatory cytokines (Simard AR et al. 2006).

Other NLRP3-associated diseases/processes:

Contact allergy: This type IV reaction has been associated with activation of IL-1β and IL-18 by the NLRP3 inflammasome. An example of this reaction mechanism has been demonstrated in contact sensitization with 2,4-dinitrochlorobenzene (Watanabe H et al. 2007).

Psoriasis: In psoriasis, the body's own cytosolic DNA activates the AIM2 inflammasome in the epithelial cells of the skin and thus initiates the inflammatory cascade.

Imiquimod activates the NLRP3 inflammasome: Imiquimod interferes with the respiratory chain of the cells, releasing massive amounts of oxygen radicals. A critical threshold is apparently exceeded; NLRP3 is activated.

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