The inflammasome in biomaterial‐driven immunomodulation

Abstract Inflammasomes are intracellular structures formed upon the assembly of several proteins that have a considerable size and are very important in innate immune responses being key players in host defense. They are assembled after the perception of pathogens or danger signals. The activation of the inflammasome pathway induces the production of high levels of the pro‐inflammatory cytokines Interleukin (IL)‐1β and IL‐18 through the caspase activation. The procedure for the implantation of a biomaterial causes tissue injury, and the injured cells will secrete danger signals recognized by the inflammasome. There is growing evidence that the inflammasome participates in a number of inflammatory processes, including pathogen clearance, chronic inflammation and tissue repair. Therefore, the control of the inflammasome activity is a promising target in the development of capable approaches to be applied in regenerative medicine. In this review, we revisit current knowledge of the inflammasome in the inflammatory response to biomaterials and point to the yet underexplored potential of the inflammasome in the context of immunomodulation.

(NLR), the adaptor molecule apoptosis-associated speck like protein containing a card (ASC) and caspase-1. The NLRs have the important function of surveying the intracellular microenvironment for the presence of metabolic perturbations, toxic substances and infection.
When NLRs sense danger signals they undergo oligomerization and form macromolecules that have the ability of activating different inflammatory pathways (Zhong et al., 2013). The assembly of the NLRs, ASC and caspase-1 induces the formation of a penta-or heptamer structure: the inflammassome (Strowig et al., 2012). The assembly of the inflammasome causes the activation of inflammatory caspases that will lead to the cleavage of pro-interleukin (IL)-1β and pro-IL-18 into IL-1β and IL-18, and also a type of cell death, pyroptosis (Lamkanfi & Dixit, 2014). Different NLRs, upon activation, form inflammasomes as for example, NLRP1, NLRP3, NLRC4, NLRC5 and NAIP2 (Jha et al., 2017). The NLR protein 3 inflammasome (NLRP3 inflammasome), also recognized as cryopyrin or NALP3, is currently the better described inflammasome, being present predominantly in myeloid cells. The NRLP3 inflammasome is activated in response to stimulation by PAMPs, DAMPs and/or LAMPs of macrophages that will secrete extensive amounts of IL-1β and IL-18 through caspase-1 activation (Latz, 2010;Ogura et al., 2006).
The process of NLRP3 inflammasome assembly is not completely appreciated, but it is known that it is tightly regulated and its activation requires two-step signals-first, it must be primed, and then activated ( Figure 2) (Lamkanfi & Dixit, 2014). The first activation signal will cause de enhancement of the expression of the inflammasome constituents and will target proteins through the activation of the nuclear factor kappa light chain enhancer of activated B cells (Bauernfeind et al., 2009). The second activation signal will promote the oligomerization of the different components of the inflammasome, leading procaspase-1 to an autocatalytic processing causing caspase-1 activation that will in turn cleave pro-IL-1β and pro-IL-18 into the mature and active forms. Caspase-1 will also cleave gasdermin D that inserts its N-terminal domain (GSDMD Nterm ) into cellular membranes, permeabilizing the membrane to the release of the mature cytokines and to pyroptotic cell death (Evavold et al., 2018;Sborgi et al., 2016). The second NLRP3 activating signal comprises three main mechanisms: (i) formation of reactive oxygen species; (ii) lysosomal damage; and (iii) intracellular potassium efflux (Hafner-Bratkovic & Pelegrin, 2018;Lima et al., 2013;Petrilli et al., 2007). NIMA-related kinase 7 (NEK7), a serine-threonine kinase, was recently considered to be crucial for NLRP3 inflammasome activation. Upon inflammasome activation, the NEK7-NLRP3 synergy rises, and NEK7 oligomerizes with NLRP3 into a complex that is essential for ASC speck formation and caspase 1 activation. Thus, NEK7 seems to be an important component, in particular to the NLRP3 inflammasome (Schmid-Burgk et al., 2016).
Though most Pattern-recognition receptors have limited specificity for one or few related PAMPs, DAMPs or LAMPs, NLRP3 is exclusive since it is triggered by a wide diversity of unrelated stimuli. NLRP3 is triggered both in pathogen infections and in sterile inflammation. Several different endogenous molecules that are indicative of injury or danger will stimulate the NLRP3 inflammasome, meaning that the NLRP3 inflammasome is able to detect sterile danger signals. These signals include extracellular adenosine triphosphate (ATP), uric acid and hyaluronan that are released by injured cells (Schroder & Tschopp, 2010). The expression of NLRP3 was detected in different cell types such as granulocytes, monocytes, macrophages, B and T lymphocytes, dendritic cells, osteoblasts and epithelial cells, suggesting an important role in the immune response against different threats. Therefore, the majority of studies on NLRP3 inflammasome have been performed in cells of the immune system (Lamkanfi & Kanneganti, 2010).

| INFLAMMASOME DISORDERS AND ITS IMPLICATIONS IN HUMAN DISEASES
Inflammation is a protective response to a noxious stimulus. A defective inflammation leads to continuous infection, and excessive inflammation can originate disease. The inflammasome is one of the most relevant mediators in these processes (Davis et al., 2011).
Consequently, inflammasome activity is highly controlled to avoid excessive cytokine production or cell death, being regulated at two levels, transcriptional (upregulation of inflammasomes components NLRP3, caspase 1 and pro-IL-1β) and post-transcriptional (ubiquitylation, phosphorylation and sumoylation) (Yang et al., 2019). The expression of inflammasome sensors in resting cells is rather low and insufficient to be induced.
Inflammasome alterations have been associated to autoinflammatory, autoimmune and neurodegenerative diseases (multiple sclerosis, Alzheimer's disease (AD), Parkinson's disease), metabolic disorders (atherosclerosis, type 2 diabetes and obesity) and cancer, playing contributing roles in the beginning and development of those pathologies ( Figure 3) (Strowig et al., 2012).
-1111 to overproduction of IL-1 (Dinarello, 2009). The rarity of CAPS and the similarity of symptoms with other diseases delay their accurate diagnosis. IL-1 inhibitory agents, anakinra (a modified IL-1RA that binds and antagonized the IL-1 receptor), canakinumab (an IL-1 neutralizing antibody) and rilonacept (ligand-binding domains of the extracellular portions of IL-1 receptor component) are the main therapeutic approach for CAPS. The blocking of IL-1 leads to a rapid and continual reversal of daily symptoms, diminishing long-term disease consequences (Hoffman, 2009;Lachmann et al., 2009;Sibley et al., 2012). The early diagnosis and treatment for CAPS patients are key to prevent organ damage.  (Yang et al., 2019). However, they have disadvantages since inflammasome activation is important for host defense against a plethora of pathogens, and hence loss of IL-1β can have deleterious effects on immune defense. Over the last years, some blockers of NLRP3 inflammasome pathway have been developed, few of which have been validate in animal models. MCC950 is a compound that specifically inhibits NLRP3 inflammasome activation, but its molecular mechanism has not been fully elucidated (Coll et al., 2015). Cy-09 and OLT1177 can inhibit the ATPase activity of the NACHT domain, which is critical for NLRP3 oligomerization (Jiang et al., 2017;Kuwar et al., 2019). JC-124 blocks the expression of NLRP3, ASC, Caspase 1 and pro-IL-1b, but its molecular mechanism are still under investigation (Marchetti et al., 2015). Due the number of individuals with serious conditions driven by NLRP3, there is a strong motivation for the discovery and clinical development of molecules that selectively antagonize NLRP3. Although these inhibitors have shown therapeutic potential, the food and drug administration and other regulatory agents did not approve any of them. In addition, the NLRP3 structure and activation mechanisms are still poorly understood which has delayed the development of novel therapeutics. Nevertheless, there are pre-clinical evidences F I G U R E 3 NLRP3 inflammasome role in disease. NLRP3 inflammasome has a key function in tissue repair and in host response not only to bacteria, fungi, viruses, and possible parasites, but also to danger-associated molecular patterns (DAMPs) and lifestyle-associated molecular patterns (LAMPs). Anomalous or exacerbated NLRP3 inflammasome activation is linked with the development of many diseases, such as, Alzheimer's Disease (AD), autoinflammatory diseases and cancer. The role of NLRP3 inflammasome in cancer is controversial, since there are evidences of a protective anti-tumorigenic effect as well as a pro-tumorigenic role depending of the cancer type. The correlation of NLRP3 inflammasome with a plethora of diseases hints a substantial interest in the scientific community to determine the effective NLRP3 inflammasome inhibitor [Colour figure can be viewed at wileyonlinelibrary.com] that the pharmacological inhibition of the NLRP3 inflammasome pathway has for example, a neuroprotective role in different disease models, therefore providing convincing arguments to further evaluate the potential of targeting the NLRP3 inflammasome (Jose et al., 2022).

| THE ACTIVATION OF THE INFLAMMASOME BY BIOMATERIALS
Tissue engineering and regenerative medicine are focused in the development of therapies to regenerate or replace injured, diseased, or defective cells, tissues, or organs to restore or establish function and structure (Daar & Greenwood, 2007). Many of the developed approaches are biomaterial-based which makes the understanding of innate and adaptive immune responses critical for the success of their application (Sefton et al., 2008). Great advances in this area have been achieved by James Anderson that has presented us a pathologist perspective on the foreign body reaction (FBR) to biomaterials (Table 1) in several manuscripts that are still today a reference for researchers in this field (Anderson, 1988(Anderson, , 2001Anderson et al., 2008). More recently, Franz et al. (2011) presented a thorough review on the immune responses to biomaterials and were one of the first authors to address the impor- The process of implantation of a biomaterial causes injury to tissues that will release DAMPs and may lead to the activation of the inflammasome pathway. Tissue resident macrophages will be one of the first immune cells to respond to injury. When activated by DAMPs, these tissue resident macrophages will release chemokines and cytokines that will prime the recruitment of polimorphonuclear leukocytes and monocytes to the injured site further activating the inflammatory response (Raziyeva et al., 2021). In Table 2 we summarize some general concepts of the inflammatory response related to the host response to biomaterials.
Inflammasome stimulation by biomaterials is being investigated mainly with bioengineered nanomaterials (Christo et al., 2016;Silva et al., 2017). It has been reported as a component of the inflammatory response to several biomaterials such as gold nanoshells (Nguyen et al., 2012), silver nanoparticles (Yang et al., 2012) and chitin/chitosan (Bueter et al., 2011) but these studies are based mainly in the assessment of the production of IL-1β. There are however more detailed studies available in the literature that address the biological effects of different biomaterials, mainly nanomaterials, on immune cells and on the NLRP3 inflammasome activation that we summarize in the following paragraphs and review on Table 3. On the following section of this manuscript we explore some applications on the targeting of the inflammasome pathway in the context of tissue engineering and regenerative medicine.
Based on the available literature it can be concluded that there is a clear tendency for the activation of the inflammasome pathway by nano-and micro-particles. Reisetter et al. (2011) have performed in vitro studies with macrophages exposed to carbon black nanoparticles that triggered inflammasome activation evaluated by the activation of caspase-1 and ensuing IL-1β production.   (Anderson, 2001;Anderson et al., 2008;Christo et al., 2015;Franz et al., 2011)

Biomaterial implantation
The implantation of a biomaterial or biomedical device causes injury in tissues or organs leading to the onset of an inflammatory response.
Protein Adsorption Blood proteins will adsorb to the surface of the biomaterial leading to the activation of the coagulation and complement system and to the activation of platelets. and platelet-derived growth factor (PDGF) that recruits and activates fibroblasts and endothelial cells. Activated fibroblasts will synthesize collagen leading to the formation of a fibrous capsule and consequently biomaterial encapsulation.

General concepts of an inflammatory response
Acute inflammation Initial phase of the inflammatory response of relatively short duration (minutes to days). Mainly characterized by the exudation of plasma proteins and fluid and by the emigration of leukocytes, mainly polymorphonuclear leukocytes (PMNs), from blood vessels to the injured site.

Chronic inflammation
Occurs when the inflammatory stimuli persists. Monocytes are recruited to the inflammatory environment and differentiate into macrophages. Macrophages become the predominant inflammatory cell. The constant release of inflammatory mediators leads to permanent activation of macrophages, and the production of chemokines leads to the recruitment of additional inflammatory cells.

Systemic inflammation
Generalized inflammatory response throughout the whole body. Characterized by the inflammatory reactivity of endotheliocytes, plasma and blood cell factors.

Sterile inflammation
Sterile inflammation is an inflammatory response that occurs in the absence of microorganisms. Is associated with the recognition of molecules released from injured cells (DAMPs: Damage-associated molecular patterns). Biomaterials induce sterile inflammation.

PAMPs
Pathogen-associated molecular patterns: Molecular structures produced by microorganisms that are recognized as foreign by the innate immune system.

DAMPs
Damage-associated molecular patterns: Molecules released upon non-physiological cell death, damage, or stress that are indicative of danger and are sensed by the innate immune system and activate immune cells. There are also exogenous DAMPs such as airborne particles.

PRRs
Pattern recognition receptors: Expressed on leukocytes interact with PAMPs and DAMPs leading to leukocyte activation. There are different families of PRRs such as toll-like receptors (TLRs) and NOD-like receptors (NLRs).

Apoptosis
Apoptosis is a process of programmed cell death. This process occurs normally during development and aging but it has also an important role in immune responses.

Pyroptosis
Pyroptosis is an inflammatory type of programmed cell death that is typically elicited by the inflammasome. It is characterized by the permeation of the plasma membrane leading to the subsequent release of intracellular contents. Although significantly fewer, studies using biomaterials too large to be phagocytosed were also performed to evaluate the role of the inflammasome in phagocytosis-independent inflammatory response to biomaterials. Court et al. (2019) documented that collagen 3D scaffolds induced assembly of the NLRP3 inflammasome causing an increased IL-1β secretion observed in human macrophages. We have used 3D chitosan scaffolds (Vasconcelos et al., 2019) and observed that these scaffolds impair NLRP3 inflammasome assembly in macrophages. Remarkably, the results that we obtained are different of other studies reported in the literature that demonstrate that chitosan is an inducer of the NLRP3 inflammasome in nanoscale chitosan products. Taken together, these results suggest that phagocytosis could be important in inflammasome assembly and activation, and suggest a different role of the inflammasome in the host response to nano-and large-scale biomaterials.

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Interestingly, the macrophage response to biomaterials is sizedependent. Macrophages have the ability to phagocyte small fragments and particles (<10 μm). When the particle size is larger (between 10-100 μm) macrophages fuse and form foreign body giant cells (FBGCs) that will engulf and digest the particle. For even larger particles, the digestion occurs via extracellular degradation in a phenomenon described as frustrated phagocytosis (Sheikh et al., 2015). This concept was first described by Henson et al. (Henson, 1971) and has great importance in the host response to biomaterials. It has been demonstrated that FBGCs adherent at the surface of implanted biomaterials present a reduced phagocytic activity but an enhanced degradative capacity through the increased secretion of reactive species (Franz et al., 2011). As far as our knowledge, there are no studies that relate the formation of FBGCs, frustrated phagocytosis and the inflammasome activation which in our opinion could be a breakthrough in this area of research.
In conclusion, there is not yet a clear understanding on how the inflammasome pathway will be triggered by different biomaterials, despite recognizing its key importance. More detailed studies, namely on the importance of the different inflammasome constituents, are required to fully appreciate the importance of the activation of this pathway in the inflammatory response to biomaterials. The modulation of inflammasome activity appears nonetheless as an important strategy to develop effective approaches for successful biomaterial integration that is a central challenge in regenerative medicine research.

| THE POTENTIAL OF THE INFLAMMASOME IN IMMUNOMODULATION
There is growing indication that the immune response has an important influence in the process of tissue repair and regeneration (Eming et al., 2009). Activation of the inflammasome can either lead to the resolution of the inflammatory response and healing, or can be continuous, leading to chronic disease or fibrosis (Artlett, 2013).
Therefore, inflammasomes are considered important regulators of the intensity of the inflammatory response, as well as regulators of the ensuing tissue repair (Ouyang et al., 2013).
As described in the beginning of this manuscript, the assembly and activation of the NRLP3 inflammasome leads to the secretion of robust amounts of IL-1β and IL-18 via caspase-1 activation. Interestingly, caspase-1 activated cells secretome contains important proteins to restore tissue homeostasis, such as fibroblast growth factor (FGF)-2 that has an important role in tissue repair and homeostasis, therefore directly associating inflammation to regeneration ( Baroja-Mazo et al., 2014;Keller et al., 2008). Additionally, both IL-1β and IL-18 act on several innate and adaptive immune cells and regulate their activation, differentiation and migration to local tissues. Non-hematopoietic cells as for example, endothelial and epithelial cells are also targeted to contribute to tissue repair (Palomo et al., 2015). Therefore, understanding the signaling that is elicited by inflammasomes can be used to advance healing, making the inflammasome a rather promising target in the research area of immunomodulatory biomaterials directed tissue repair. An improved understanding of the inflammasome biology can potentially contribute to key pathways that regulate immunity, inflammation and homeostasis. Therefore, effective control of the inflammasome activity has become a potential therapeutic strategy and may become a milestone in bioengineering.
Although research on the targeting of the inflammasome and/or inflammasome constituents is still in the beginning, there are some studies reported in the literature that support the potential of this approach and that are revealing some interesting outcomes in different areas of research that include skin wound healing, bone healing, liver regeneration, pancreatic island transplantation and also in neuronal injuries. -Haus et al. (2015) showed that NLRP3 and caspase-1 null mice submitted to a cutaneous wound excision presented a delay in re-epithelization, granulation tissue formation and angiogenesis, demonstrating the importance of NLRP3 signaling in the early events of wound healing. Ito et al. (2018) further documented that NLRP3 signaling is important for the skin healing process. They observed that NLRP3 and ASC knockout (KO) mice presented an impaired wound repair when comparing to the wild type (WT) animals. They further concluded that the administration of ATP, which is a ligand for NLRP3, leads to the activation of inflammasomes causing the up-regulation of the expression of pro-inflammatory cytokines and accelerates the wound healing. Therefore, identifying molecules that have the ability to modulate the inflammasome assembly and activation, or inflammasome-mediated products, can reduce fibrosis and promote wound healing (Artlett, 2013). Chen et al. (2022) investigated the early inflammatory response to titanium screws in bone, and concluded that the administration of ipriflavone, a natural FBR, allowing full healing of the tissue, in comparison with the conventional treatments (dexamethasone), which also blocks FBR but at the same time impair tissue repair.

Weinheimer
Although so far scarce, reports on the importance of the inflammasome in regenerative processes showed encouraging results, indicating the need to pursue this promising area of research.
The combination of inflammasome modulators with biomaterials will most likely yield encouraging results for regenerative medicine applications.

| CONCLUSIONS
The implantation of a biomaterial causes tissue damage. Damaged cells will release the so-called "danger signals" that can be identified by the inflammasome. Therefore, implanted biomaterials are 1116 -VASCONCELOS ET AL. potential activators of the inflammasome pathway. The role of the inflammasome in the host response to biomaterials as well as the stimulation of inflammasomes by implanted biomaterials needs a more in-depth investigation since it is still poorly understood.
It is important to clarify several different aspects on the biomaterial-inflammasome interactions, such as for example,: (i) What are the differences between nano-or micro-particles and large-scale biomaterials, and is the process of phagocytosis essential in the process of inflammasome assembly and activation?
(ii) How can biomaterial surface characteristics modulate innate immune signaling pathways?
(iii) How biomaterial-adherent cells affect these signaling pathways and is surface chemistry a key aspect?
(iv) How does the inflammasome activation influences the progression of the foreign body response to biomaterials?
(v) What is the importance of the different inflammasome constituents?
(vi) What can we learn using genetically modified mice deficient in these key signaling pathways?
It is in our opinion important to better understand how to control the host response by targeting specific points in these innate immune signaling pathways to improve biomaterials biocompatibility Therefore, further research into how biomaterials influence the inflammasome assembly and activation are of great interest.
It is now commonly recognized that the immune system is of major importance in the coordination of an adequate repair process, and since inflammasomes participate in the innate immune response, it is predictable that they have an important role in tissue repair/ regeneration. The assembly of the inflammasome will not only lead to the production of pro-inflammatory cytokines but also to the regulation of the extracellular levels of proteins involved in the processes of tissue repair and cytoprotection.
A thorough understanding of the inflammasome biology will give us key information on different pathways that regulate immunity, inflammation and homeostasis. It is our belief that a successful modulation of the activity of the inflammasome will become a milestone in bioengineering, and namely in the field of tissue repair and regeneration. There is a growing interest in finding effective approaches that will be able to selectively inhibit the inflammasome pathway.