Neutrophils and extracellular traps in crystal-associated diseases

Deposits of crystals within the body, or exposure to external crystalline material, can cause diverse metabolic and inflammatory acute as well as chronic medical disorders.Neutrophils are crucial in the immune response to these pathogenic crystals, from contributing to necroinflammation and driving further tissue damage through the release of proinflammatory mediators and neutrophil extracellular trap (NET) formation to limiting inflammation.The mechanisms of how neutrophils respond to these pathogenic crystals, specifically their mode of cell death (e.g., NETosis, necrosis, necroptosis, and/or pyroptosis), depends on the crystal type, site of crystal deposition, and amount of crystals, all of which influence the intensity of inflammation.Targeting NET components and certain forms of neutrophil death as well as crystallization are novel treatment targets that may improve outcomes in crystal-associated disorders. Crystalline material can cause a multitude of acute and chronic inflammatory diseases, such as gouty arthritis, silicosis, kidney disease, and atherosclerosis. Crystals of various types are thought to cause similar inflammatory responses, including the release of proinflammatory mediators and formation of neutrophil extracellular traps (NETs), processes that further promote necroinflammation and tissue damage. It has become apparent that the intensity of inflammation and the related mechanisms of NET formation and neutrophil death in crystal-associated diseases can vary depending on the crystal type, amount, and site of deposition. This review details new mechanistic insights into crystal biology, highlights the differential effects of various crystals on neutrophils and extracellular trap (ET) formation, and discusses treatment strategies and potential future approaches for crystal-associated disorders. Crystalline material can cause a multitude of acute and chronic inflammatory diseases, such as gouty arthritis, silicosis, kidney disease, and atherosclerosis. Crystals of various types are thought to cause similar inflammatory responses, including the release of proinflammatory mediators and formation of neutrophil extracellular traps (NETs), processes that further promote necroinflammation and tissue damage. It has become apparent that the intensity of inflammation and the related mechanisms of NET formation and neutrophil death in crystal-associated diseases can vary depending on the crystal type, amount, and site of deposition. This review details new mechanistic insights into crystal biology, highlights the differential effects of various crystals on neutrophils and extracellular trap (ET) formation, and discusses treatment strategies and potential future approaches for crystal-associated disorders. Crystals can either form through aberrant crystallization of intrinsic organic material inside the human body or enter the body (usually via the lung) as extrinsic crystals and microparticles. Intrinsic crystals from inorganic minerals are physiologically important and tolerated by the immune system to provide structural stiffness and durability. However, abnormal crystallization and deposition of organic material (Box 1) cause diverse medical disorders that can manifest as either acute or chronic organ injuries, including gouty arthritis (see Glossary), gallstones, Alzheimer's disease, and atherosclerosis. Extrinsic crystals (Box 1), such as occupational dust, can lead to pneumoconiosis, silicosis, and cancer [1.Mulay S.R. Anders H.J. Crystallopathies.N. Engl. J. Med. 2016; 375e29PubMed Google Scholar].Box 1Crystal formation and types of crystalsCrystallizationIn general, crystallization inside the human body occurs through reduced solubility (leading to supersaturation), nucleation, and crystal growth [100.Chhana A. et al.Factors influencing the crystallization of monosodium urate: a systematic literature review.BMC Musculoskelet. Disord. 2015; 16: 296Crossref PubMed Google Scholar]. Nucleation begins in the liquid phase, in which small molecules (e.g., minerals) cluster and agglomerate together. After crystals nucleate, they start growing immediately and can eventually form larger crystalline particles, known as aggregates. The crystallization process involves many factors such as pH, ionic strength, body temperature, low solubility, connective tissue factors and proteins, reduced urine volume, or a combination of these factors, which determine crystal type, shape, size, and distribution [101.Durbin S.D. Feher G. Protein crystallization.Annu. Rev. Phys. Chem. 1996; 47: 171-204Crossref PubMed Google Scholar]. Additionally, fibers (probably collagen) in the synovial fluid of patients with gout might also affect MSU crystal formation by templated nucleation [102.Pascual E. et al.Mechanisms of crystal formation in gout – a structural approach.Nat. Rev. Rheumatol. 2015; 11: 725-730Crossref PubMed Google Scholar].Under healthy condition, crystallization is tightly regulated: for example, the formation of life-essential structures such as teeth. At the same time, the body has different strategies in place to avoid harmful crystallization: for example, (i) a strong blood flow can limit crystal nucleation and growth [103.Lemann Jr., J. et al.Urinary oxalate excretion increases with body size and decreases with increasing dietary calcium intake among healthy adults.Kidney Int. 1996; 49: 200-208Abstract Full Text PDF PubMed Google Scholar], (ii) plasma and urine proteins (e.g., apolipoproteins, uromodulin, Tamm–Horsfall protein) and small molecules (e.g., citrate) [104.Worcester E.M. Urinary calcium oxalate crystal growth inhibitors.J. Am. Soc. Nephrol. 1994; 5: S46-S53Crossref PubMed Google Scholar,105.Rasheed H. et al.The relationship of apolipoprotein B and very low density lipoprotein triglyceride with hyperuricemia and gout.Arthritis Res. Ther. 2014; 16: 495Crossref PubMed Scopus (21) Google Scholar], (iii) excretion of minerals via the urinary tract, and (iv) dietary or drug metabolites [106.Borghi L. et al.Comparison of two diets for the prevention of recurrent stones in idiopathic hypercalciuria.N. Engl. J. Med. 2002; 346: 77-84Crossref PubMed Scopus (791) Google Scholar] can reduce supersaturation. However, when crystals aggregate and continue to form in the human body, the overloaded particles will induce necroinflammation, obstruction, fibrosis, and sometimes even organ failure [1.Mulay S.R. Anders H.J. Crystallopathies.N. Engl. J. Med. 2016; 375e29PubMed Google Scholar].Intrinsic pathological crystalsIn the case of an imbalance of proteins, lipids, and mineral precipitation, formation and deposition of endogenous crystals will occur in the body, which applies to various diseases (see Figure 1 in the main text). Many factors contribute to the crystallization process and influence the size, shape, and distribution of crystals. Such pathological crystals can cause not only kidney stones and crystal nephropathy (UA, CaOx crystals) but also gallstones and atherosclerotic plaques, gouty arthritis, neurodegenerative diseases, and the propagation of the malaria parasite within infected patients [1.Mulay S.R. Anders H.J. Crystallopathies.N. Engl. J. Med. 2016; 375e29PubMed Google Scholar].Extrinsic pathological crystalsApart from intrinsic crystals, various extrinsic crystals can enter and deposit in the human body (see Figure 1 in the main text). Airborne environmental pollutants and occupational dusts such as silica and asbestos can be easily inhaled and accumulate in the lung. Other sources of extrinsic crystalline material include cosmetics or implants. In recent years, the exposure of humans to nanoparticles is rising due to their wide applications (e.g., vaccine adjuvants and nanomedicines) [16.Lin M.H. et al.The interplay between nanoparticles and neutrophils.J. Biomed. Nanotechnol. 2018; 14: 66-85Crossref PubMed Scopus (38) Google Scholar]. Several drugs can also precipitate within kidney tubules and cause crystal nephropathy due to their insolubility in the human urine (e.g., sevelamer, acyclovir, methotrexate, indinavir, and sulfadiazine). Crystallization In general, crystallization inside the human body occurs through reduced solubility (leading to supersaturation), nucleation, and crystal growth [100.Chhana A. et al.Factors influencing the crystallization of monosodium urate: a systematic literature review.BMC Musculoskelet. Disord. 2015; 16: 296Crossref PubMed Google Scholar]. Nucleation begins in the liquid phase, in which small molecules (e.g., minerals) cluster and agglomerate together. After crystals nucleate, they start growing immediately and can eventually form larger crystalline particles, known as aggregates. The crystallization process involves many factors such as pH, ionic strength, body temperature, low solubility, connective tissue factors and proteins, reduced urine volume, or a combination of these factors, which determine crystal type, shape, size, and distribution [101.Durbin S.D. Feher G. Protein crystallization.Annu. Rev. Phys. Chem. 1996; 47: 171-204Crossref PubMed Google Scholar]. Additionally, fibers (probably collagen) in the synovial fluid of patients with gout might also affect MSU crystal formation by templated nucleation [102.Pascual E. et al.Mechanisms of crystal formation in gout – a structural approach.Nat. Rev. Rheumatol. 2015; 11: 725-730Crossref PubMed Google Scholar]. Under healthy condition, crystallization is tightly regulated: for example, the formation of life-essential structures such as teeth. At the same time, the body has different strategies in place to avoid harmful crystallization: for example, (i) a strong blood flow can limit crystal nucleation and growth [103.Lemann Jr., J. et al.Urinary oxalate excretion increases with body size and decreases with increasing dietary calcium intake among healthy adults.Kidney Int. 1996; 49: 200-208Abstract Full Text PDF PubMed Google Scholar], (ii) plasma and urine proteins (e.g., apolipoproteins, uromodulin, Tamm–Horsfall protein) and small molecules (e.g., citrate) [104.Worcester E.M. Urinary calcium oxalate crystal growth inhibitors.J. Am. Soc. Nephrol. 1994; 5: S46-S53Crossref PubMed Google Scholar,105.Rasheed H. et al.The relationship of apolipoprotein B and very low density lipoprotein triglyceride with hyperuricemia and gout.Arthritis Res. Ther. 2014; 16: 495Crossref PubMed Scopus (21) Google Scholar], (iii) excretion of minerals via the urinary tract, and (iv) dietary or drug metabolites [106.Borghi L. et al.Comparison of two diets for the prevention of recurrent stones in idiopathic hypercalciuria.N. Engl. J. Med. 2002; 346: 77-84Crossref PubMed Scopus (791) Google Scholar] can reduce supersaturation. However, when crystals aggregate and continue to form in the human body, the overloaded particles will induce necroinflammation, obstruction, fibrosis, and sometimes even organ failure [1.Mulay S.R. Anders H.J. Crystallopathies.N. Engl. J. Med. 2016; 375e29PubMed Google Scholar]. Intrinsic pathological crystals In the case of an imbalance of proteins, lipids, and mineral precipitation, formation and deposition of endogenous crystals will occur in the body, which applies to various diseases (see Figure 1 in the main text). Many factors contribute to the crystallization process and influence the size, shape, and distribution of crystals. Such pathological crystals can cause not only kidney stones and crystal nephropathy (UA, CaOx crystals) but also gallstones and atherosclerotic plaques, gouty arthritis, neurodegenerative diseases, and the propagation of the malaria parasite within infected patients [1.Mulay S.R. Anders H.J. Crystallopathies.N. Engl. J. Med. 2016; 375e29PubMed Google Scholar]. Extrinsic pathological crystals Apart from intrinsic crystals, various extrinsic crystals can enter and deposit in the human body (see Figure 1 in the main text). Airborne environmental pollutants and occupational dusts such as silica and asbestos can be easily inhaled and accumulate in the lung. Other sources of extrinsic crystalline material include cosmetics or implants. In recent years, the exposure of humans to nanoparticles is rising due to their wide applications (e.g., vaccine adjuvants and nanomedicines) [16.Lin M.H. et al.The interplay between nanoparticles and neutrophils.J. Biomed. Nanotechnol. 2018; 14: 66-85Crossref PubMed Scopus (38) Google Scholar]. Several drugs can also precipitate within kidney tubules and cause crystal nephropathy due to their insolubility in the human urine (e.g., sevelamer, acyclovir, methotrexate, indinavir, and sulfadiazine). Organ injuries and diseases caused by these crystals are commonly associated with inflammation, cell death, fibrosis, thrombosis, and/or obstruction of vessels and ducts. During these processes, neutrophils are crucial. Several forms of crystal-related neutrophil death have been identified, including neutrophil extracellular traps (NETs), apoptosis, NETosis and necroptosis. NETs are important not only for pathogen clearance [2.Poli V. Zanoni I. Neutrophil intrinsic and extrinsic regulation of NETosis in health and disease.Trends Microbiol. 2023; 31: 280-293Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar] during host defense, but are also involved in driving inflammation and further tissue damage (Box 2) [3.Papayannopoulos V. Neutrophil extracellular traps in immunity and disease.Nat. Rev. Immunol. 2018; 18: 134-147Crossref PubMed Scopus (1773) Google Scholar]. Recent evidence indicates that aggregated NETs can even limit inflammation [4.Schauer C. et al.Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines.Nat. Med. 2014; 20: 511-517Crossref PubMed Scopus (693) Google Scholar]. Thus, the identification of NETs has helped us to refine our view of the role of neutrophils in numerous crystal-related diseases (Figure 1). It has become apparent that the type of neutrophil death may vary depending on the crystalline material, the site of crystal deposition, and the amount of crystals in the body [5.Franklin B.S. et al.Crystal formation in inflammation.Annu. Rev. Immunol. 2016; 34: 173-202Crossref PubMed Google Scholar]. This suggests that the immune response and severity of inflammation might differ in the tissue depending on the crystal type. In this review we outline new mechanistic insights into crystal biology and immunity, and address the differential effects of numerous crystals on neutrophils and ET formation as well as on NETs in crystal-related disease manifestations. This will inform our discussion on potential treatment strategies and provide future research opportunities for crystal-associated diseases. Our purpose is to provide a succinct, yet comprehensive, overview of the latest updates in this field.Box 2Mechanisms of ET formation in neutrophilsNeutrophils are crucial for host defense [3.Papayannopoulos V. Neutrophil extracellular traps in immunity and disease.Nat. Rev. Immunol. 2018; 18: 134-147Crossref PubMed Scopus (1773) Google Scholar], but also in non-infectious diseases. Once recruited to the site of tissue injury, neutrophils form NETs and subsequently promote necroinflammation (see Figure 2 and Table 1 in the main text). Recent studies indicate that NETosis can be classified into two forms: suicidal and vital [107.Castanheira F.V.S. Kubes P. Neutrophils and NETs in modulating acute and chronic inflammation.Blood. 2019; 133: 2178-2185Crossref PubMed Scopus (382) Google Scholar]. Suicidal NETosis occurs in a NOX-independent manner through Ca2+ influx and mitochondrial ROS production [108.Yipp B.G. Kubes P. NETosis: how vital is it?.Blood. 2013; 122: 2784-2794Crossref PubMed Scopus (714) Google Scholar]; it involves chromatin decondensation, nuclear swelling, and membrane rupture. By contrast, vital NETosis seems to be oxidant-independent without membrane rupture, where neutrophils release mitochondrial DNA and/or nuclear DNA in vesicles but retain their ability to migrate, phagocytose, and kill pathogens [108.Yipp B.G. Kubes P. NETosis: how vital is it?.Blood. 2013; 122: 2784-2794Crossref PubMed Scopus (714) Google Scholar,109.Yousefi S. et al.Untangling 'NETosis' from NETs.Eur. J. Immunol. 2019; 49: 221-227Crossref PubMed Scopus (0) Google Scholar]. Although it seems that NETs are decorated with a conserved set of proteins (e.g., complement, PAD4, cytokines) and enzymes (e.g., NE, MPO, histones) [110.Ravindran M. et al.Neutrophil extracellular trap formation: physiology, pathology, and pharmacology.Biomolecules. 2019; 9: 365Crossref PubMed Scopus (162) Google Scholar], the composition of NETs varies depending on the type and concentration of crystals and the time of exposure. Not only the composition but also post-translational modifications of proteins are heterogeneous in NETs [111.Petretto A. et al.Neutrophil extracellular traps (NET) induced by different stimuli: a comparative proteomic analysis.PLoS One. 2019; 14e0218946Crossref Scopus (138) Google Scholar]. Whether different NET compositions can exhibit different biological functions, and whether crystals can induce vital NETs, are currently unknown. Similarly, it is unclear how neutrophils exactly decide between phagocytosis and NET formation when combating crystals. Studies suggest that neutrophils selectively release NETs in response to larger crystals, while smaller particles are phagocytosed [112.Pieterse E. et al.Blood-borne phagocytes internalize urate microaggregates and prevent intravascular NETosis by urate crystals.Sci. Rep. 2016; 6: 38229Crossref PubMed Scopus (30) Google Scholar].NETs are proinflammatory and cytotoxic. Under conditions where NETs are inappropriately released or not rapidly degraded and cleared, they can be pathogenic and cause tissue inflammation and damage (e.g., in Alzheimer's disease, chronic obstructive pulmonary disease, and cystic fibrosis) [113.Conceicao-Silva F. et al.The immune system throws its traps: cells and their extracellular traps in disease and protection.Cells. 2021; 10: 1891Crossref PubMed Scopus (28) Google Scholar]. In non-crystalline autoimmune or autoinflammatory diseases such as rheumatoid arthritis, NETs can even act as autoantigens. In turn, immune complexes containing NET autoantigens – such as double-stranded DNA, histones, and MPO coupled with autoantibodies – can further promote innate and adaptive immune responses [114.Wigerblad G. Kaplan M.J. Neutrophil extracellular traps in systemic autoimmune and autoinflammatory diseases.Nat. Rev. Immunol. 2023; 23: 274-288Crossref PubMed Scopus (89) Google Scholar], suggesting that NETs, if not removed from tissues or the circulation, might exacerbate inflammation and tissue damage. Thus, the immune system has certain mechanisms in place to prevent or diminish inadvertent immunological responses, including clearance of apoptotic neutrophils [115.Savill J.S. et al.Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages.J. Clin. Invest. 1989; 83: 865-875Crossref PubMed Google Scholar,116.Steiger S. Harper J.L. Neutrophil cannibalism triggers transforming growth factor beta1 production and self regulation of neutrophil inflammatory function in monosodium urate monohydrate crystal-induced inflammation in mice.Arthritis Rheum. 2013; 65: 815-823Crossref PubMed Scopus (0) Google Scholar], degradation of NETs by DNase I and complement factor C1q [117.Hakkim A. et al.Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 9813-9818Crossref PubMed Scopus (1139) Google Scholar], as well as removal of NETs [118.Farrera C. Fadeel B. Macrophage clearance of neutrophil extracellular traps is a silent process.J. Immunol. 2013; 191: 2647-2656Crossref PubMed Scopus (314) Google Scholar]. Evidence even suggests now that NETs have anti-inflammatory effects and contribute to the resolution of inflammation. For example, in gouty arthritis, neutrophils when present in high numbers form aggregated NETs in response to MSU crystals and subsequently release proteases that can trap, cleave, and inactivate proinflammatory cytokines [119.Schett G. et al.Why does the gout attack stop? A roadmap for the immune pathogenesis of gout.RMD Open. 2015; 1e000046Crossref Scopus (64) Google Scholar]. Neutrophils are crucial for host defense [3.Papayannopoulos V. Neutrophil extracellular traps in immunity and disease.Nat. Rev. Immunol. 2018; 18: 134-147Crossref PubMed Scopus (1773) Google Scholar], but also in non-infectious diseases. Once recruited to the site of tissue injury, neutrophils form NETs and subsequently promote necroinflammation (see Figure 2 and Table 1 in the main text). Recent studies indicate that NETosis can be classified into two forms: suicidal and vital [107.Castanheira F.V.S. Kubes P. Neutrophils and NETs in modulating acute and chronic inflammation.Blood. 2019; 133: 2178-2185Crossref PubMed Scopus (382) Google Scholar]. Suicidal NETosis occurs in a NOX-independent manner through Ca2+ influx and mitochondrial ROS production [108.Yipp B.G. Kubes P. NETosis: how vital is it?.Blood. 2013; 122: 2784-2794Crossref PubMed Scopus (714) Google Scholar]; it involves chromatin decondensation, nuclear swelling, and membrane rupture. By contrast, vital NETosis seems to be oxidant-independent without membrane rupture, where neutrophils release mitochondrial DNA and/or nuclear DNA in vesicles but retain their ability to migrate, phagocytose, and kill pathogens [108.Yipp B.G. Kubes P. NETosis: how vital is it?.Blood. 2013; 122: 2784-2794Crossref PubMed Scopus (714) Google Scholar,109.Yousefi S. et al.Untangling 'NETosis' from NETs.Eur. J. Immunol. 2019; 49: 221-227Crossref PubMed Scopus (0) Google Scholar]. Although it seems that NETs are decorated with a conserved set of proteins (e.g., complement, PAD4, cytokines) and enzymes (e.g., NE, MPO, histones) [110.Ravindran M. et al.Neutrophil extracellular trap formation: physiology, pathology, and pharmacology.Biomolecules. 2019; 9: 365Crossref PubMed Scopus (162) Google Scholar], the composition of NETs varies depending on the type and concentration of crystals and the time of exposure. Not only the composition but also post-translational modifications of proteins are heterogeneous in NETs [111.Petretto A. et al.Neutrophil extracellular traps (NET) induced by different stimuli: a comparative proteomic analysis.PLoS One. 2019; 14e0218946Crossref Scopus (138) Google Scholar]. Whether different NET compositions can exhibit different biological functions, and whether crystals can induce vital NETs, are currently unknown. Similarly, it is unclear how neutrophils exactly decide between phagocytosis and NET formation when combating crystals. Studies suggest that neutrophils selectively release NETs in response to larger crystals, while smaller particles are phagocytosed [112.Pieterse E. et al.Blood-borne phagocytes internalize urate microaggregates and prevent intravascular NETosis by urate crystals.Sci. Rep. 2016; 6: 38229Crossref PubMed Scopus (30) Google Scholar]. NETs are proinflammatory and cytotoxic. Under conditions where NETs are inappropriately released or not rapidly degraded and cleared, they can be pathogenic and cause tissue inflammation and damage (e.g., in Alzheimer's disease, chronic obstructive pulmonary disease, and cystic fibrosis) [113.Conceicao-Silva F. et al.The immune system throws its traps: cells and their extracellular traps in disease and protection.Cells. 2021; 10: 1891Crossref PubMed Scopus (28) Google Scholar]. In non-crystalline autoimmune or autoinflammatory diseases such as rheumatoid arthritis, NETs can even act as autoantigens. In turn, immune complexes containing NET autoantigens – such as double-stranded DNA, histones, and MPO coupled with autoantibodies – can further promote innate and adaptive immune responses [114.Wigerblad G. Kaplan M.J. Neutrophil extracellular traps in systemic autoimmune and autoinflammatory diseases.Nat. Rev. Immunol. 2023; 23: 274-288Crossref PubMed Scopus (89) Google Scholar], suggesting that NETs, if not removed from tissues or the circulation, might exacerbate inflammation and tissue damage. Thus, the immune system has certain mechanisms in place to prevent or diminish inadvertent immunological responses, including clearance of apoptotic neutrophils [115.Savill J.S. et al.Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages.J. Clin. Invest. 1989; 83: 865-875Crossref PubMed Google Scholar,116.Steiger S. Harper J.L. Neutrophil cannibalism triggers transforming growth factor beta1 production and self regulation of neutrophil inflammatory function in monosodium urate monohydrate crystal-induced inflammation in mice.Arthritis Rheum. 2013; 65: 815-823Crossref PubMed Scopus (0) Google Scholar], degradation of NETs by DNase I and complement factor C1q [117.Hakkim A. et al.Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 9813-9818Crossref PubMed Scopus (1139) Google Scholar], as well as removal of NETs [118.Farrera C. Fadeel B. Macrophage clearance of neutrophil extracellular traps is a silent process.J. Immunol. 2013; 191: 2647-2656Crossref PubMed Scopus (314) Google Scholar]. Evidence even suggests now that NETs have anti-inflammatory effects and contribute to the resolution of inflammation. For example, in gouty arthritis, neutrophils when present in high numbers form aggregated NETs in response to MSU crystals and subsequently release proteases that can trap, cleave, and inactivate proinflammatory cytokines [119.Schett G. et al.Why does the gout attack stop? A roadmap for the immune pathogenesis of gout.RMD Open. 2015; 1e000046Crossref Scopus (64) Google Scholar]. The crystallization process is complex and involves many factors which affect the size, shape, rigidity, stiffness, and electrical surface charge of crystals (Box 1), thus dramatically influencing the outcomes of their interaction with tissues and cells in the human body [5.Franklin B.S. et al.Crystal formation in inflammation.Annu. Rev. Immunol. 2016; 34: 173-202Crossref PubMed Google Scholar]. This includes how immune cells sense and select crystals, what factors influence crystallization and immune cell function, the mode of crystal-related cell death, and the associated inflammatory processes. Cells of the immune system can distinguish and differentially respond to small variations in crystal morphology and size. Immune cells, including macrophages and neutrophils, ingest particles within the nanometer to micrometer size range [1.Mulay S.R. Anders H.J. Crystallopathies.N. Engl. J. Med. 2016; 375e29PubMed Google Scholar]. Crystals can be directly recognized by neutrophils and other immune cells via receptors: for example, the inhibitory C-type lectin receptor Clec12a, CD14 and signal inhibitory receptor on leukocytes-1 (SIRL-1) for monosodium urate (MSU) crystals [6.Fernandes M.J. Naccache P.H. The role of inhibitory receptors in monosodium urate crystal-induced inflammation.Front. Immunol. 2018; 9: 1883Crossref PubMed Scopus (15) Google Scholar,7.Neumann K. et al.Clec12a is an inhibitory receptor for uric acid crystals that regulates inflammation in response to cell death.Immunity. 2014; 40: 389-399Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar], macrophage receptor with collagenous structure (MARCO) and low-density lipoprotein receptor (LDLR) for MSU, calcium pyrophosphate dehydrate (CPPD), and calcium oxalate (CaOx) crystals [8.Alberts A. et al.Binding of macrophage receptor MARCO, LDL, and LDLR to disease-associated crystalline structures.Front. Immunol. 2020; 11596103Crossref PubMed Scopus (9) Google Scholar], and Clec4e (known as Mincle) and MACRO for cholesterol crystals [8.Alberts A. et al.Binding of macrophage receptor MARCO, LDL, and LDLR to disease-associated crystalline structures.Front. Immunol. 2020; 11596103Crossref PubMed Scopus (9) Google Scholar,9.Kiyotake R. et al.Human mincle binds to cholesterol crystals and triggers innate immune responses.J. Biol. Chem. 2015; 290: 25322-25332Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar]. However, further studies are needed to identify receptors for the recognition of other crystalline material by neutrophils. Although receptors are important for phagocytosis of larger crystalline material, other physical characteristics of crystals also play an important role in cell activation without involving receptors, including ion influx and efflux [10.Mulay S.R. et al.Calcium oxalate crystals induce renal inflammation by NLRP3-mediated IL-1beta secretion.J. Clin. Invest. 2013; 123: 236-246Crossref PubMed Scopus (361) Google Scholar]. It is currently unknown whether neutrophils require receptors also for the endocytosis of small crystals, or whether this occurs in an indirect way without receptors. The formation of intrinsic crystals occurs in a multistep process involving many factors such as pH, body temperature, concentration, low solubility, proteins, reduced urine volume, or a combination of these factors, which affect crystal properties and the associated immune response (Box 1) [1.Mulay S.R. Anders H.J. Crystallopathies.N. Engl. J. Med. 2016; 375e29PubMed Google Scholar]. For example, temperatures lower than 37°C promote MSU crystal formation in chemico and increase MSU-crystal-mediated NOD-like receptor protein 3 (NLRP3) inflammasome activation and interleukin (IL)-1β release in macrophages [11.Ahn H. et al.Lower temperatures exacerbate NLRP3 inflammasome activation by promoting monosodium urate crystallization, causing gout.Cells. 2021; 10: 1919Crossref PubMed Scopus (14) Google Scholar], while a higher temperature of 39°C seems to influence the characteristics of MSU crys