The Extra(cellular) Function of Inflammasomes

By Sashini Ranawana

The main research questions in the field of immunology tend to focus on why the immune system sometimes fails to defend against infectious diseases and cancers. It is hard to imagine that the same protective response can have equally disastrous effects when it is overactive. This is the case in the immune disorders of autoinflammation (characterised by recurrent episodes of innate immune system activation), and the hypersensitivity conditions of autoimmunity (the targeting of antigens found on ‘self’ cells by lymphocytes) and allergy (inappropriate reactions to harmless environmental molecules). A component of the innate immune system, the inflammasome, has been extensively studied for its role in such inflammatory disorders.1,2 Both the dysregulation and normal activation of this protein complex have been found to exacerbate the immune response when it isn’t required, implicating it as a promising target in the treatment of these diseases.

Inflammation is the immune system’s immediate and effective response to eliminate pathogenic organisms and maintain cellular function after tissue damage. The crucial molecules needed to initiate it, interleukins IL-1β and IL-18, are therefore under tight control by multiple signalling pathways. The production of immature pro-IL-1β by the transcription factor NF-κB, and the increased transcription of constitutively expressed pro-IL-18, are both controlled by the stimulation of innate immune receptors. Inflammasomes provide the next level of regulation by controlling the caspase-1 dependent cleavage, and hence activation, of these molecules. This process is dependent on the detection of infection or damage signals by sensor molecules of the complex.3 NLRP1 sensors detect double-stranded viral RNA, while numerous other sensors are present in the cytosol to recognize different by-products of pathogenic infections or tissue destruction.3,4 The inflammasome ultimately functions to indirectly activate the molecule gasdermin D, to bring about the cellular death ‘pyroptosis’ and release pro-inflammatory interleukins to the surrounding environment.5

The apoptosis-associated speck like protein containing a caspase recruitment domain (ASC) adaptor is a key link between sensors and pro-caspase-1.3 Initially thought to only play a role in the assembly of inflammasomes in the cytosol, this molecule has now also been implicated in the further propagation of inflammation through its function in the extracellular space.6 Individual molecules accumulate to form ASC ‘specks’ after pyroptosis, stimulating the maturation of more IL-1β and inducing inflammasome formation in cells they are phagocytosed by. Franklin et al. revealed that these protein aggregates, if not efficiently cleared after infection, become antigens that are opsonised by antibodies, act as communication molecules to recruit more immune cells and contribute to the increased severity of certain inflammatory diseases.6 Thus, a natural response to protect cells, tissues and organs from further damage can be quickly and damagingly intensified.

To understand the dynamic interactions between ASC molecules, Franklin et al. first had to develop a means to visualise them. They created a mouse macrophage cell line expressing the fusion gene product of ASC combined with a fluorescence protein, which could then be visualised by confocal microscopy. They discovered the presence of ASC specks in the extracellular space after initial activation of the intracellular NLRP3, NLRP1 and AIM2 inflammasomes. By using antibodies against green fluorescence protein in the cell-containing media, which had access to the cytosol through membrane pores created by gasdermin D, they deduced that these specks form even before pyroptosis of the cell is complete. Pure ASC specks from wild-type and caspase-1 knockout macrophages were then added to supernatants from cells that had died by inflammasome-mediated pyroptosis. These extracellular specks were found to function as normal to recruit pro-caspase-1 and activate the pro-IL-1β released from the cells.6

In a series of experiments to investigate the inflammation-promoting properties of these extracellular inflammasomes, ASC specks were found to be phagocytosed by macrophages. When experimentally introduced alongside the fluorescent molecule dextran to phagolysosomes, these ASC specks damaged the intracellular membrane and enabled the release of dextran. Specks that escaped to the cytosol were then found to recruit non-aggregated ASC molecules from the host cell, catalysing the formation of an intracellular structure with the potential to indefinitely propagate inflammation.6

The mode of action of ASC aggregates draws clear parallels with the pathophysiology of various inflammatory disorders. To establish this link, researchers used a mouse model of chronic obstructive pulmonary disease (COPD), a condition known to be associated with abnormal NLRP3 inflammasome activity.7 Using antibodies against ASC, they noted a markedly higher concentration of specks in the lung fluid of COPD mice continuously exposed to cigarette smoke (an aggravator of the condition) compared to control COPD mice exposed to normal air. In human volunteers with the same disease, active specks were also detected at a significantly higher level than in both individuals with non-inflammatory pulmonary hypertension and healthy controls. The ability of these active extracellular inflammasomes to last for longer periods of time in inflammatory disorders could provide further insight into their pathologies. Interestingly, it could also help explain the link between ASC specks and autoimmune disorders. After an analysis of blood samples from 80 patients with various autoimmune conditions, Franklin et al. found that 18% had antibodies targeted against ASC aggregates.6 Whether these antibodies are merely by-products of autoimmunity, or whether they contribute themselves to the inflammatory responses observed in autoimmune disorders, remains to be determined.

Chronic inflammation is a health risk that is expected to increase within the next few years.8 Diseases such as asthma, allergy, diabetes and COPD are increasing in frequency and intensity, as air pollution, urbanisation, climate change and obesity become more pressing problems worldwide. However, due to the work done by Franklin et al., the additional extracellular function of inflammasomes can now be considered as a possible molecular mechanism contributing to this persistent and exacerbated immune response. Experiments to determine why the concentration of these ASC specks increase, above the level where macrophages can efficiently clear them, in certain individuals, might be the first step towards gaining a better understanding of their role in inflammation. This opens up the possibility for inhibitors of inflammasome formation or ASC oligomerization to be used therapeutically against these increasingly common and life-threatening illnesses.

References:

(1) Wilson S P, Cassel S L. Inflammasome mediated autoinflammatory disorders. Postgraduate Medicine. 2010;122(5): 125-133. Available from: doi: 10.3810/pgm.2010.09.2209.

(2) Sönmez H E, Özen S. A clinical update on inflammasomopathies. International Immunology. 2017;29(9): 393-400. Available from: doi: 10.1093/intimm/dxx020.

(3) Latz E, Xiao T S, Stutz A. Activation and regulation of the inflammasomes. Nature Reviews Immunology. 2013;13(6): 397-411. Available from: doi: 10.1038/nri3452.

(4) Bauernfried S, Scherr M J, Pichlmair A, Duderstadt K E, Hornung V. Human NLRP1 is a sensor for double-stranded RNA. Science. 2021;371(6528): eabd0811. Available from: doi: 10.1126/science.abd0811.

(5) Lieberman J, Wu H, Kagan J C. Gasdermin D activity in inflammation and host defence. Science Immunology. 2019;4(39): eaav1447. Available from: doi: 10.1126/sciimmunol.aav1447.

(6) Franklin B S, Bossaller L, Nardo D D, Ratter J M, Stutz A, Engels G, et al. ASC has extracellular and prionoid activities that propagate inflammation. Nature Immunology. 2014;15(8): 727-737. Available from: doi: 10.1038/ni.2913.

(7) Colarusso C, Terlizzi M, Molino A, Pinto A, Sorrentino R. Role of the inflammasome in chronic obstructive pulmonary disease (COPD). Oncotarget. 2017;8(47): 81813-81824. Available from: doi: 10.18632/oncotarget.17850.

(8) Furman D, Campisi J, Verdin E, Carrera-Bastos P, Targ S, Franceschi C. Chronic inflammation in the etiology of disease across the life span. Nature Medicine. 2019;25(12): 1822-1832. Available from: doi: 10.1038/s41591-019-0675-0.

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