Cell type-specific roles of NLRP3, inflammasome-dependent and -independent, in host defense, sterile necroinflammation, tissue repair, and fibrosis

The nucleotide-binding oligomerization domain (NOD) - and leucine-rich repeat (LRR)-containing receptor pyrin domain-containing (NLRP)-3 have inflammasome-dependent and -independent functions. The NLRP3 inflammasome is a cytosolic pattern recognition platform that integrates infectious and non-infectious danger signals into the secretion of IL-1β and IL-18. The NLRP3 inflammasome is formed by the assembly of NLRP3, the adaptor molecule ASC, and caspase-1, which results in caspase-1 activation, which cleaves pro-IL-1β and pro-IL-18 into their active forms ( 1 ). IL-1β and IL-18 can be released actively through gasdermin pores in the plasma membrane or passively during pyroptosis, a type of inflammatory cell death ( 1 , 2 ). The activation of inflammasomes must be tightly regulated so as to prevent both systemic and tissue inflammation. For example, cryopyrin-associated periodic syndrome (CAPS) results from dysregulated NLRP3 activity due to gain-of-function mutations in NLRP3 , which leads to abnormal activation of the NLRP3 inflammasome ( 3 , 4 ). Overproduction of IL-1β has harmful effects in CAPS patients such as hearing loss, urticaria-like rash, joint pain, and inflammation of the eyes, bones, and central nervous system ( 5 , 6 ). IL-1 antagonists reverse systemic and local inflammation and protect organs in CAPS patients ( 7 – 9 ). Mice carrying the same gain-of-function mutations in the Nlrp3 gene develop systemic inflammation accompanied by growth retardation and early mortality ( 10 ). Conversely, mice lacking Nlrp3 , or molecules involved in inflammasome assembly, document the deleterious effect of NLRP3 inflammasome activation in sterile inflammation models ( 11 – 14 ). The vast majority of data on the NLRP3 inflammasome comes from cells of the myeloid lineage. Indeed, single-cell transcriptome data sets consistently report the absence of NLRP3, ASC, caspase-1, and IL-1β transcripts from cell types other than myeloid cells ( https://www.proteinatlas.org/ ). These data are consistent with the absence of IL-1β protein in parenchymal tissues. Cell type-specific modes of immune activation are an important element in limiting potentially harmful inflammation, cytokine storms, and immunopathology. Nevertheless, a surprising number of studies report NLRP3 expression and activity, and even IL-1β secretion, by human and mouse epithelial cells and other parenchymal tissue cells ( Table 1 ). This is not supported by many unbiased expression data sets. Some experimental studies reporting expression and function of the NLRP3 inflammasome in non-myeloid cells. IFN-γ (Interferon-gamma), Th2 (T helper 2), IgM (Immunoglobulin M), TGFβR (Transforming grow factor β) receptor). Here we review the published data on the inflammasome-dependent and inflammasome-independent roles of NLRP3 to better understand the function of NLRP3 in tissue-specific danger signaling and beyond the secretion of IL-1β ( Figure 1 ). Furthermore, we call for a critical discussion of the discrepancy between the apparent absence of transcripts of NLRP3 inflammasome components and of many cell types that are reported to carry out NLRP3 inflammasome functions. In addition, we discuss the experimental tools used in this context. Inflammasome-dependent and –independent roles of NLRP3 in myeloid cells. Left: Canonical activation of NLRP3 starts with TLR activation, mediate by Myd88 and NF-κB, which triggers the expression of NLRP3, pro-IL-1β and -IL-18. Several stimuli can trigger the inflammasome assembly, such as K + efflux, Ca +2 influx, crystals, phagosome rupture, mtROS and mtDNA. The conjunction of NLRP3, ASC and Caspase 1 lead to Caspase 1 activation and proteolytic cleavage of pro-IL-1β and -IL-18, as well GSDMD resulting in IL-1β and -IL-18 secretion and/or pyroptosis. Right: Non-canonical NLRP3 activation is triggered by TLR activation, via Myd88 and TRIF, or type I interferon receptor (IFRA), which lead to NF-κB activation and induction of NLRP3, pro-IL-1β and -IL-18 expression. The type I interferon triggers caspase-11, and caspase 1 expression. LPS and LPG can bind and directly activate caspase 11, which cleaves GSDMD in GSDMD-N driving pyroptosis. Nlrp3 can act as a transcription factor in T cells by binding directly to the Il4 , Il13 , Icos , Maf , Il1r1 promoter region. Nlrp3 can induce phosphorylation of Smad2/4 in the TGFβR pathway, thereby regulating the transcription of target genes. Created with BioRender.com .

Hepatocytes are the main parenchymal cells in the liver involved in detoxification, lipid metabolism, albumin synthesis, and the secretion of coagulation factors. The Human Protein Atlas shows that under homeostatic conditions, hepatocytes do not express NLRP3 and ASC , although they express low levels of CASP1 . Some studies show that diabetes, liver inflammation, and steatosis induce the expression of Nlrp3 , Casp1 , and Il1b in the liver tissue, including immune and non-immune cells ( 46 , 124 , 125 ). Activation of the NLRP3 inflammasome by ROS induces the release of IL-1β and IL-18 by hepatocytes, which then undergo pyroptosis ( 130 ). Hepatocytes carrying gain-of-function mutations were sufficient to drive spontaneous collagen deposition in the liver ( 131 ). Conversely, hepatocytes with Nlrp3 -deficiency and/or pharmacological inhibition of Nlrp3 ameliorate liver inflammation by reducing IL-1β, IL-6, and TNF-α production; they also ameliorate liver fibrosis by reducing Col1a and aSma. Together, this information shows that canonical inflammasome activation in hepatocytes is sufficient to propagate liver injury and fibrosis.

There is a discrepancy between transcriptional data obtained from RNAseq analysis and functional studies of the NLRP3 inflammasome in non-immune cells. One of the possible explanations is the low sensitivity of single-cell RNA sequencing analysis, which may miss low levels of transcripts under homeostatic conditions. However, different RNA sequencing data sets and data available on the Human Protein Atlas from certain disease conditions, such as diabetes, ischemia-reperfusion, or tissue remodeling, still do not report NLRP3 inflammasome-related transcripts in epithelial cells ( GSE131882 , GSE119531 , and GSE206084 ). The most common form of functional evidence is provided by in vitro studies employing immortalized cell lines that may have undergone somatic mutations and phenotypic changes over time. In this regard, primary cell cultures would seem to provide more solid evidence. However, such primary isolates are frequently contaminated by tissue-resident immune cells, leading to erroneous conclusions. The use of non-specific antibodies in immunohistochemistry and Western blotting may also be one of the reasons for this discrepancy ( 132 ). In fact, less than half of the routinely used antibodies bind to their specific targets ( 133 ). Therefore, the specificity of the NLRP3 antibody should be questioned, especially when used as a negative control, when documenting tissue samples as NLRP3-deficient. The use of constitutive NLRP3 knockout animals has greatly contributed to several discoveries about the canonical role of the NLRP3 inflammasome, mainly in immune cells. However, such animals cannot determine the cell type-specific effects of the NLRP3 inflammasome. Similarly, NLRP3 inhibitors or whole-body knockout mice cannot attribute an improvement of the condition to the sole and exclusive inhibition of the inflammasome in parenchymal cells without considering the participation of immune cells ( 27 , 29 , 109 , 122 ). Thus, studies concluding on NLRP3 inflammasome activity in non-immune cells have to be carefully evaluated for methodological consistency and possible involvement of myeloid cells. The use of cell type-specific knockout mice, e.g., with a floxed NLRP3 motif under the control of cell type-specific expression of Cre recombinase, may help to determine the role of the NLRP3 inflammasome in parenchymal cells. However, such experiments need a series of controls to validate the cell type-specific depletion and exclude Cre leakage or epigenetic silencing of Cre recombinase ( 134 – 136 ). Another approach to evaluating the role of NLRP3 in specific cell types is through tools that induce NLRP3 expression, such as the use of animals with gain-of-function mutations and the transfection of cells with the gene of interest. For example, animals with the human A350V gain-of-function mutation and/or deletion of NLRP3 in podocytes have shown that activation of this inflammasome is sufficient to promote the glomerular damage observed in animals with diabetic kidney disease ( 115 ). In a similar manner, the specific deletion of Nlrp3 in epithelial cells demonstrated the importance of this inflammasome in promoting TGF-β signaling ( 31 ).

NLRP3 is involved in multiple cellular mechanisms. The canonical activation of NLRP3 is well-known and understood. However, inflammasome-independent functions of NLRP3 may play various roles in different cell types.Here we show that NLRP3 can act to induce tissue fibrosis by enhancing TGF-β receptor signaling and fibrosis-associated markers ( 31 , 32 , 44 ). NLRP3 can also regulate the translocation of intestinal microorganisms and enhance endothelial cell damage by increasing the expression of selectins, which promote neutrophil recruitment and thereby NETosis and granule secretion. However, many of the cell-specific functions attributed to NLRP3 are inconsistent with the expression of its transcripts in various tissues and/or cell types, such as lung and kidney epithelial cells. It is likely that many of these conflicting results are due to problematic in vitro tools, such as immortalized cell lines of untested nature or, in the case of primary cells, possible immune cell contamination. Thus, the use of tools to delete or induce NLRP3 or its related molecules in specific cell types, e.g., knockout, knockin, should be better explored so that new therapeutic alternatives can be created and even expand beyond merely immune-mediated diseases.

NLRP3 inflammasome activation is preceded by the canonical and non-canonical pathways. Both pathways initiate the induction of inflammasome components, and their substrates, following cytokine or Toll-like receptor (TLR) activation. This increases the transcription of pro-IL-1β, pro-IL-18, and other NLRP3-related molecules via the translocation of NF-κB into the nucleus. In the canonical pathway, danger signals promote the oligomerization of NLRP3, the adaptor protein ASC, and pro-caspase-1. Once activated, caspase-1 cleaves pro-IL-1β and pro-IL-18 at aspartate residues to generate the effector forms of these cytokines ( 15 ). Furthermore, caspase-1 activation leads to the cleavage of gasdermin D (GSDMD), which can disrupt inner membrane lipids (phosphatidylserine and phosphatidylinositol) and cell osmolality, triggering pyroptosis and releasing alarmins such as IL-1α, IL-1β, and IL-18 ( 15 ). Non-canonical NLRP3 inflammasome activation involves activation of caspases 4 and 5 in humans and caspase-11 in mice ( 15 ). Prior to this, NF-κB translocates to the nucleus, where it increases the transcription of interferon regulatory factors (IRF) 3 and 7, and induces the expression of IFN-α/β ( Figure 2 ). These IFNs trigger the expression of caspase-11, which cannot cleave pro-IL-1β to IL-1β, but through the induction of caspase-1, the active form of IL-1β can be generated, i.e., the non-canonical pathway to induce IL-1β ( 38 ). In addition, caspase-11 can be activated by physically binding to LPS ( 39 ) or lipophosphoglycan (LPG) ( 40 ). Once activated, caspase-11 can also induce pyroptosis by cleaving GSDMD. The main role of the non-canonical NLRP3 is to protect against Gram-negative bacteria that evade the phagosome and invade the cytosol ( 41 ). In this way, Cheng et al. showed that LPS can activate lung endothelial cells towards pyroptosis, which is abolished in Casp11 -deficient endothelial cells ( 42 ). Reported inflammasome-dependent and -independent functions of NLRP3 in non-immune cells. In T cells, canonical activation of NLRP3 has been described to favor differentiation of naive T cells into Th17 cells, as well as to increase IFN-γ production in the face of viral infection. In an inflammasome independent way, the NLRP3 acts as a transcription factor in T cells, favoring a Th2 profile. In epithelial cells, canonical activation of NLRP3 is related to the production and release of IL-1β and IL-18 during inflammatory processes, whereas the non-canonical pathway favors the maintenance of the intestinal epithelial barrier. In epithelial cells the inflammasome independent function is related with TGF-β pathway activation and fibrosis, as well acting as a transcription factor controlling the expression of IL-33. The role of NLRP3 in endothelial cells has been pointed out during inflammatory processes, in which IL-1 β production favors endothelial dysfunction and increased expression of selectins, acting on the recruitment of inflammatory cells to the injured site. In B cells NLRP3 stimulates the IgM production, as well the expression of CXCR4 and CCR7, homing related chemokines. *EpC (epithelial cell). *EnC (endothelial cell). Created with BioRender.com . An inflammasome-independent function of NLRP3 is the promotion of fibrosis through regulation of the TGF-β signaling pathway ( 31 , 32 , 37 , 43 ). The absence of Nlrp3 in epithelial cells, but not caspase 1, IL-1β, or IL-18, reduced their ability to produce αSma and Mmp9, and decreased epithelial-mesenchymal transition when stimulated with TGF-β ( 31 ). Numerous studies demonstrate that Nlrp3 regulates TGF-β through the phosphorylation of Smad2/3 ( 31 , 37 , 44 – 46 ). Furthermore, NLRP3 acts as a transcription factor in the nucleus of T cells, promoting Th2 polarization through its ability to bind directly to DNA, specifically in the promoter regions of Il-4 , Il-13 , Il1r1 , Icos , and Maf ( 19 ). Similarly, Nlrp3 acts as a transcription factor and regulates the expression of IL-33 in epithelial cells ( 47 ). In addition, Nlrp3 deficiency or inhibition prevented the production of IL-33 in a model of atopic dermatitis ( 47 ). We will discuss in more detail the functions of NLRP3 that are not related to inflammasome activation in specific cell types.

All authors contributed equally to this review. All authors contributed to the article and approved the submitted version.