key: cord-0028658-r42pm0im
authors: Wessig, Anne Kathrin; Hoffmeister, Leonie; Klingberg, Annika; Alberts, Anika; Pich, Andreas; Brand, Korbinian; Witte, Torsten; Neumann, Konstantin
title: Natural antibodies and CRP drive anaphylatoxin production by urate crystals
date: 2022-03-16
journal: Sci Rep
DOI: 10.1038/s41598-022-08311-z
sha: 3d266a31b47b8448be2dda6adba61be8eecbabb6
doc_id: 28658
cord_uid: r42pm0im

In gout, crystallization of uric acid in the form of monosodium urate (MSU) leads to a painful inflammatory response. MSU crystals induce inflammation by activating the complement system and various immune cell types, and by inducing necrotic cell death. We previously found that the soluble pattern recognition molecule C-reactive protein (CRP) recognizes MSU crystals, while enhancing complement activation. In the absence of CRP, MSU crystals still induced complement activation, suggesting additional CRP-independent mechanisms of complement activation. In the present study, we searched for additional MSU crystal-binding complement activators. We found that all healthy individuals, even unborn children, have MSU crystal-specific immunoglobulin M (IgM) in their blood. This indicates that innate IgM, also known as natural IgM, recognizes these crystals. In serum lacking IgM and CRP, MSU crystals showed negligible complement activation as assessed by the production of the anaphylatoxins C4a, C3a, and C5a (listed in order of production via the classical complement pathway). We show that IgM and CRP both activate the classical complement pathway on MSU crystals. CRP was more efficient at fixating active C1 on the crystals and inducing release of the most inflammatory anaphylatoxin C5a, indicating non-redundant functions of CRP. Notably, while CRP recognizes MSU crystals but not the related calcium pyrophosphate dihydrate (CPPD) crystals, natural IgM bound to both, suggesting common and distinct mechanisms of recognition of individual crystal types by complement activators.

MSU crystal-specific antibodies in healthy individuals. As described above, we had previously identified MSU crystal-binding proteins by liquid chromatography-coupled mass spectrometry (LC-MS). As a control particle we had used zymosan, a fungal cell wall preparation from S. cerevisiae containing mainly β-glucans. We had incubated both MSU crystals and zymosan in human serum, washed away the unbound proteins, eluted the bound proteins, and subjected them to LC-MS 26 . Among the bound proteins, we had found CRP and the related pentraxin serum amyloid P (SAP) 24 . So far, we were unable to show a role for SAP in complement activation by MSU crystals (data not shown). Thus, we searched for other complement activating proteins within the identified crystal-binding proteins. As antibodies are common activators of complement, we analyzed the fraction of each immunoglobulin isotype bound to MSU crystals and zymosan (Fig. 1a , "Supplementary Dataset"). All isotypes showed some binding to zymosan, likely due to previous immune responses to ubiquitous fungi. IgM was the only isotype showing strong binding to both zymosan and MSU crystals. Consequently, the J-chain, which links IgM and IgA monomers, was also found on both MSU crystals and zymosan. Since these data were based only on two donors, we next purified proteins that bound to MSU from 14 human serum samples obtained from healthy individuals (aged 20-65 years). We performed Western blot analysis to determine the relative amount of bound IgM, IgA, and IgG. Densitometric quantification of the blots showed that, compared to IgA and IgG, a much higher fraction of IgM bound to MSU crystals (Fig. 1b, Supplementary Fig. 1 ). We tested the same for the related CPPD crystals (here, triclinic (t) crystals were used), which showed similar results, albeit the fraction of bound IgM was lower. Binding of IgM to both crystal types was found in every serum sample tested with low variability, indicating that most, if not all individuals have naturally occurring MSU-and t-CPPDspecific IgM antibodies. We confirmed this observation using a flow cytometer. Two independent preparations of MSU crystals preincubated in normal human sera showed a strong signal after staining with a fluorescent anti-IgM antibody, which was comparable to zymosan particles (Fig. 1c) . As a control we used serum samples obtained from patients with common variable immunodeficiency (CVID), which were selected based on very low IgM concentrations (< 0.05 mg/ml; normal range 0.4-2.3 mg/ml 27 ) . IgA was also low in these IgM-deficient sera, while IgG levels were in the normal range. In these IgM/IgA-deficient serum samples very low signals were Figure 1. Natural IgM binds to MSU and CPPD crystals. (a) Serum proteins bound to each indicated particle were analyzed by LC-MS. Fraction of each antibody isotype bound to each indicated particle is shown. Data from 2 individual serum samples is depicted. In one serum sample, IgA2 was not detected; therefore, only one data point is shown. (b) Fraction of immunoglobulin isotypes bound to MSU or t-CPPD crystals from 14 normal human serum samples quantified from Western blot analysis. ND = not detected. Raw data (blot images) is shown in Supplementary Fig. 1 . (c) Individual normal human sera (NHS, including samples from B) or IgM/IgA-deficient serum samples were incubated with the indicated particles, and bound IgM and IgG were detected using anti-IgM PE or anti-IgG PE, respectively. Median fluorescence intensity (MFI) of the particles was normalized by subtracting the MFI of the negative control (FBS). Means of the NHS and IgM/IgA-deficient serum samples were compared by an unpaired t-test. (d) Binding of purified polyclonal (poly.) or monoclonal (mono.) IgM to three distinct preparations of MSU crystals in IgM/IgA-def. serum or HBSS + 10% BSA. (e) Pooled human serum collected either from healthy adults or from human cord blood was incubated with the indicated particles, and bound IgM was detected as in (c). Values for each particle preparation and the mean are shown. (f) MSU (lot 2), silica (SiO 2 ), or calcium carbonate (CaCO 3 ) crystals were incubated in FBS or 4 normal human pool serum samples. Bound human IgM was detected as in (c) and MFI for each serum sample is shown including the negative controls (FBS). Fig. 2b ). Thus, IgM may not only bind directly but also indirectly to the crystals. Finally, to test whether crystal-specific IgM was natural, i.e., it is innate and not produced after encountering a foreign antigen, we compared IgM binding to crystals in pooled serum collected either from adults or from cord blood samples. The binding of IgM to three independent preparations of MSU and two independent preparations of both t-CPPD and monoclinic (m)-CPPD in cord blood serum was nearly as strong as that in adult serum (Fig. 1e) . No zymosan-specific IgM was detectable in cord blood serum. Thus, natural IgM recognizes MSU and CPPD crystals, while zymosan-specific IgM is likely generated in response to foreign antigens. Together, these findings show that most, if not all individuals have MSU-specific IgM in their blood, which is at least partly innate. To test, if IgM binds to all types of crystals or particles, we analyzed binding of IgM in human pool serum to calcium carbonate (CaCO 3 ), silica particles (SiO 2 ), and cholesterol crystals. IgM only weakly bound to these particles as compared to MSU crystals ( Fig. 1f , Supplementary Fig. 2c ), suggesting IgM only recognizes a subset of crystals (as does CRP).

To test the impact of IgM on crystalinduced complement activation, we used the same serum samples obtained from CVID patients with serum IgM concentrations below 0.05 mg/ml. We reconstituted IgM/IgA-deficient serum with polyclonal and monoclonal IgM that we already used in Fig. 1d and analyzed MSU crystal-induced complement activation by measuring the complement activation products C4a, C3a, and C5a. Only polyclonal IgM induced C4a and C3a production in the presence of MSU crystals (Fig. 2) , which is in line with above findings that only polyclonal IgM bound to the crystals in serum (Fig. 1d) . However, at the concentrations used, IgM was not able to induce significant amounts of C5a, while polyclonal IgA did not induce any anaphylatoxin production.

CRP-mediated anaphylatoxin production in the presence and absence of IgM. We started this study to find the complement activator that can compensate for CRP in its absence. To test if CRP has nonredundant roles in complement activation in the presence or absence of the identified second complement activator IgM, we reconstituted normal human pool sera or IgM/IgA-deficient sera with CRP. We analyzed the production of anaphylatoxins (C4a, C3a, and C5a) and the final product of the pathway (sC5b-9) after incubation with MSU or t-CPPD crystals, which are not recognized by CRP, as a control.

In pool sera from healthy individuals, CRP did not significantly enhance C4a and C3a production (the earliest products of the classical complement pathway) but enhanced the production of C5a and sC5b-9 (Fig. 3a) . In the absence of IgM, IgA, and CRP, all complement activation products were hardly present, but their production could be fully reconstituted by the addition of CRP, indicating IgM is indeed the second independent complement activator that we were looking for. The production of the later activation products C5a and sC5b-9 was reconstituted even above values achieved in normal human serum, indicating CRP is especially efficient at inducing later complement activation events. Two of the 11 IgM/IgA-deficient serum samples, however, behaved like normal Fig. 3 ), so either a third IgM-and CRP-independent pathway exists or residual antibodies in the two serum samples have been sufficient to activate the early components of the complement pathways.

To show the specificity of CRP-induced complement activation, we also tested complement activation by t-CPPD crystals (which are not recognized by CRP 24 ) (Fig. 3b ). Both in normal pool sera and in IgM/IgA-deficient sera, CRP did not alter production of any of the complement activation products (Fig. 3b) , confirming the specificity of CRP for MSU crystals. However, a significant reduction in C4a and to a lower extend in C3a levels in the IgM/IgA-deficient serum samples was observed, indicating that IgM also activates the classical complement pathway on t-CPPD crystals, while additional IgM-and CRP-independent mechanisms for complement activation by t-CPPD seem to exist.

To verify that CRP-mediated complement activation is dependent on the presence of MSU crystals, we repeated the experiment with three IgM/IgA-deficient sera and incubated them without crystals, MSU crystals, and cholesterol crystals, which are also recognized by CRP (Fig. 4) . CRP did not induce anaphylatoxin production in the absence of crystals, and MSU crystal-induced anaphylatoxin production was largely dependent on CRP in these sera. In line with previous reports 25 , CRP induced C4a generation by cholesterol crystals, while some C3a and C5a production was seen in the absence of CRP, likely activated by redundant complement activators 6,29,30 . Activation of C1s by IgM and CRP. In our previous study, we showed that CRP was required for fixation of active complement factors C1s (the catalytic subunit of C1) on the surface of MSU crystals even in IgM-sufficient serum 24 . Since IgM is known to activate the classical pathway via C1, we analyzed C1s, C3, and C5 fixation on MSU crystals in the absence of IgM (samples from Fig. 3a) . One representative Western blot image of a normal serum and two IgM/IgA-deficient sera is shown in Fig. 5a (the remaining Western blots used for quantification are shown in Supplementary Fig. 4 ). In line with our previous data 24 and the ability of CRP to induce the release of C5a, the addition of CRP induced fixation of cleaved versions of C1s, C3, and C5 on MSU crystals (in both normal and IgM/IgA-deficient sera). C1s is activated by cleavage and one of the cleavage products is observed at 55 kDa (denoted as C1s*). Upon activation, we always observed another band of C1s, which has an apparent molecular weight higher than full length C1s (denoted C1s + X). As it is stable in reducing SDS-PAGE, it is likely a fusion with another protein via a peptide bond. In the absence of IgM/IgA, we did not see reduced binding of the activated C1s. When we quantified the intensity of the band of activated C1s* observed on blots of four normal and six IgM/IgA-deficient serum samples, the difference was not statistically significant (Fig. 5b, left panel) . We also quantified the C1s + X activation product: no statistically significant difference between IgM-sufficient and IgM-deficient serum was found (Fig. 5b, right panel) . However, when we evaluated active C1s* released into the supernatant and not fixated on crystals, we found strongly increased activated C1s* in the presence of IgM (Fig. 5c, left panel) . Similarly, the larger activation product (C1s + X) was strongly increased in presence of IgM (Fig. 5c, right panel) . This was not due to different levels of C1s in the sera, as the normal full length C1s was even reduced in the presence of IgM (Fig. 5c, middle panel) . Similarly, when we added polyclonal IgM to two IgM/IgA-deficient sera, activated C1s was again detectable after incubation with MSU crystals (Supplementary Fig. 6 ). Together, this suggests that IgM activates C1s on the surface of MSU crystals but cannot fixate it as efficiently as CRP. Accordingly, fixation of C3 and C5 was largely independent of IgM (Fig. 5a, Supplementary Fig. 5 ). www.nature.com/scientificreports/ In summary, we have identified natural IgM as a CRP-independent complement activator for MSU crystals, while we show that CRP is more efficient at fixating complement factors on the crystals and inducing later complement activation products C5a and sC5b-9.

Inflammation during a gout flare is hardly distinguishable from inflammation induced by microbes. Thus, it is likely driven by the same pattern recognition molecules/receptors as microbe-induced inflammation. What remains unclear so far is if these receptors specifically recognize the crystals. The NLRP3 inflammasome is a main driver of crystal-induced inflammation, but it does not seem to interact with crystals but rather senses damage induced by the crystals to plasma membrane or lysosome 31, 32 . Uptake of crystals not necessarily requires specific receptors either, as the crystals directly interact with the plasma membrane 33, 34 . However, several transmembrane receptors have been shown to recognize MSU crystals rather specifically and/or to be involved in MSU crystalinduced inflammation 26, [35] [36] [37] [38] [39] . Additionally, we found a remarkably specific binding of CRP to MSU crystals but not the related CPPD crystals, suggesting soluble pattern recognition molecules recognize crystal surfaces. In this study, we identify natural IgM as a second soluble molecule for MSU crystal recognition by the complement system. Most proteins that show strong binding to MSU crystals have in common that they have multiple binding sites: CRP and IgM are homopentamers, MARCO is a homotrimer, and Clec12A oligomerizes 40 . Multiple interactions of low affinity may lead to a high avidity interaction of the whole molecule with the crystal surface, which is probably necessary since a classical key-lock recognition mechanism seems unlikely for the various crystal surfaces. In line with this notion, the CRP related pentraxin SAP and ficolin 2, both pentamers bind to cholesterol 25, 30 , and we found both also on the surface of MSU crystals 24 .

Natural IgM is believed to be produced by B1 B-cells independent of any foreign antigens, presumably in response to self-antigens 41 . It has been shown not only to bind various microbes but also to play a role in complement activation by necrotic cells and the heparin:PF4 complex, which is targeted in heparin-induced thrombocytopenia [42] [43] [44] . Other "naturally occurring" antibodies arise from the poly-reactivity or cross-reactivity of IgM produced in infancy in response to ubiquitous microbes, as has been established for antibodies against ABO blood type antigens 45 . We found similar binding of IgM to MSU crystals incubated in cord blood or adult serum, suggesting that the MSU-specific IgM in adult serum is natural. As we cannot differentiate between the origins of the MSU crystal-binding IgM in adult serum, we also cannot exclude the possibility that IgM raised against foreign antigens contributes to the pool of crystal-binding IgM in adult serum. Still, natural IgM recognizes MSU crystals and unlike CRP also binds to CPPD crystals.

As expected, we found that both IgM and CRP activate the classical complement pathway on the surface of MSU crystals. Notably, IgM was less efficient at fixating complement factors on the surface of the crystals. This www.nature.com/scientificreports/ could be due to a weaker or unstable binding of IgM to the crystals or partly indirect binding. While we showed that IgM is in principle capable of binding the crystals directly (Fig. 1d) , it may still bind indirectly in the presence of crystal-coating serum components. We tried to block IgM-binding by coating the crystals with serum or purified proteins, but the results were variable and donor specific. Further research is thus needed to clarify the binding mechanism of IgM to crystals. IgM has been shown before to weakly bind to cholesterol crystals in normal human serum. Intriguingly, IgM bound more strongly in the absence of C1q 30 . It will be interesting to see, if absence of C1q also enhances binding of IgM to MSU and CPPD crystals. CRP binds directly to the crystals and is especially efficient at inducing the late complement activation products C5a and sC5b-9. Both C5a and C5b-9 have been shown to be involved in MSU-induced inflammation in animal models. C5a acts as a chemoattractant and can induce expression of inflammatory genes like proIL-1β 16, 17 , and C5b-9 may activate the NLRP3 inflammasome in bystander cells 18, [46] [47] [48] . Thus, it is likely that high CRP levels during an acute phase reaction increase the likelihood of the initiation of a gout attack.

Before our findings, CRP had already been shown to recognize cholesterol crystals to activate the complement system, and to co-localize with C5b-9 complexes in human atherosclerotic plaques 25 . Thus, also on those crystals, CRP drives the complement system to the very end. This is in contrast to other ligands of CRP, where CRP additionally recruits the inhibitory complement factor H (CFH) to stop complement activation beyond C3 49 . This does not appear to happen on MSU and cholesterol crystals for yet unknown reasons.

Cholesterol crystals additionally activate the alternative pathway 6, 29 , the lectin pathway via ficolin-2, and recruit C1q via IgM 30 , showing some overlap with MSU crystals. Two early reports also demonstrated alternative pathway activation by MSU crystals 21, 22 . Whether this requires CRP or IgM remains to be seen. Recently, it was shown that several nanoparticles activate the alternative pathway via IgG 50 . This may also happen on MSU crystals, depending on the presence of MSU crystal-binding IgG. We have found very low levels of MSU crystal-binding IgG in most individuals, at least when tested in serum. In line with earlier results 51 , we found that purified IgG binds to MSU crystals in the absence of competing serum proteins (data not shown), similar to the unspecific monoclonal IgM we used in this study (Fig. 1d) . This may become relevant in body fluids low in serum protein (mainly apolipoproteins 26, 52, 53 ) , or under certain experimental conditions. One report showed that addition of IgG could enhance complement activation by MSU crystals 20 . Thus, it remains to be seen under which conditions IgG activates complement on MSU crystals. It is possible that gout patients develop MSU crystal-specific IgG as MSU crystals may act as antigens for an immune response, or that there are individuals with autoantibodies reacting with serum proteins that coat the crystals. At least one serum we used in our study showed IgG binding to MSU crystals. So, it may be worthwhile to screen patients for these antibodies to find out if these antibodies are associated with a medical condition and what they bind to: crystals or coated proteins.

In two out of eleven IgM/IgA-deficient sera in which CRP was depleted, early complement activation was preserved (Fig. 3a) . These may have contained residual IgM, sufficient crystal-binding IgG, or a completely independent complement activator. Thus, there could be a third complement activator for MSU crystals, which we may search for in a future study.

Besides activation of the complement system, binding of CRP or IgM to crystals may alter gout pathophysiology also by other mechanisms. In mice, MSU crystal-binding IgM are required for precipitation of uric acid and its adjuvant properties 54 , while the pentraxin PTX3 has been shown to inhibit crystallization of calcium oxalate 55 . It remains to be seen if CRP or human IgM also alter crystallization of urates, the prerequisite for the development of gout.

Human serum and buffers. We used left-over diagnostic samples of serum obtained from healthy individuals (age range 20-65 years, 9 female and 5 male) and 11 CVID patients who showed IgM and IgA levels below 0.05 g/l (age range 31-55 years, 5 female and 6 male). All serum samples had IgG levels in the reference range, while we did not inquire in which cases this was due to immunoglobulin replacement therapy. Most sera were collected before any SARS-CoV-2 vaccines were available in Germany. Samples were frozen at − 80 °C until use. CVID patient sera were kept at 3-5 °C o/n before freezing to allow completion of diagnostic tests first. Integrity of the complement system in those sera was verified by measuring CH50 before freezing. Only serum samples that were above the mean of the normal range of the test were used.

Three additional normal single donor complement-preserved serum samples (#ICERS10ML), and pooled cord blood serum samples (#IRHUCDS1ML) were obtained from Innovative Research, Inc, Novi, MI. Pooled serum was generated by mixing serum of 3 single donors. All donors gave informed written consent. This study was approved by the Ethics Committee of the Hannover Medical School (Ref. No: 3395-2016) and was performed in accordance with the ethical standards laid out in the 1964 Declaration of Helsinki and its later amendments.

Whenever crystals were in contact with buffer, HBSS containing 1.26 mM Ca 2+ , 0.9 mM Mg 2+ , and 5.5 mM d-glucose (Thermo Fisher Scientific, #14025050) was used. It was saturated with sodium urate to prevent dissolution of the MSU crystals. t-CPPD and m-CPPD were prepared as previously described 56 using sodium pyrophosphate instead of potassium pyrophosphate as the starting material. Lot 1 was prepared in the laboratory of KN, and lot 2 was prepared in the laboratory of Christèle Combes, CIRIMAT, Université de Toulouse, Toulouse INP-ENSIACET, 31030, Toulouse, France.

Silica (Silicon dioxide) particles (− 325 mesh) were purchased from Alfa Aesar (#88316). Calcium carbonate crystals were prepared as described 26 .

Zymosan was purchased from Merck KGaA (#Z4250), washed 3× in 100% ethanol, stored in 100% ethanol at − 20 °C. Before use, zymosan was washed 3-4 times with HBSS.

Analyses of protein binding to crystals. Flow cytometry. Crystals (100 µg) were incubated in 50 µl serum or HBSS + 10% BSA for 30 min at 37 °C with mild agitation. Crystals were harvested by centrifugation at 1000×g for 2 min and washed with HBSS. IgM and IgG were detected using PE anti-human IgM antibody (#314508, BioLegend) and PE anti-human IgG Fc antibody (#410708, BioLegend) (both diluted 1:25 in HBSS + 5% BSA), respectively. Particle fluorescence was analyzed using a FACS Canto II (BD Biosciences) and BD FACSDiva software version 6.1.3 (www. bdbio scien ces. com). Flowing Software version 2.5.1 (Perttu Terho, University of Turku, Finland, https:// biosc ience. fi/ servi ces/ cell-imagi ng/ flowi ng-softw are/) was used for analysis of data obtained.

Fluorescence microscopy was performed as described before 26 and images were acquired using an Olympus IX81 inverted microscope and CellR software (version 3.2; www. olymp us-lifes cience. com/ en/ softw are/). Brightness was adjusted, pseudo-color was inserted in the grayscale image, and scale bar was added using ImageJ (version 1.53e; https:// imagej. nih. gov/ ij/).

Western blot analysis. 1 mg crystals were incubated in 50 µl serum for 30 min at 37 °C and harvested by centrifugation at 1000×g for 2 min (for LC-MS analysis 9 mg crystals were incubated with 100 µl serum).

Supernatants were directly diluted in reducing SDS-PAGE buffer (SDS, glycerol, DTT, Tris pH6.8), and crystals were washed 5 × in HBSS saturated with sodium urate and transferred to a fresh 1.5-ml tube before elution of the bound proteins using 40 µl of 2 × reducing SDS-PAGE buffer at 70 °C for 10 min.

Proteins were separated on precast SDS-PAGE gels (SERVA Electrophoresis GmbH, #43289) and transferred to nitrocellulose membranes (GE Healthcare, #10600003). The membranes were blocked in TBST + 5% BSA and incubated with primary antibody in TBST + 5% BSA o/n at 4 °C. After incubation with HRP-coupled secondary antibodies (Cell Signaling Technology, #7074, #7076 S), the blots were subjected to ECL reaction. Images were acquired using a ChemoStar imager (INTAS Science Imaging Instruments GmbH), and quantification of Western blot signals was performed with ImageJ software (version 1.53e).

The following primary antibodies were used at the indicated dilution: anti-C1S (#14554-1-AP; 1:4000), anti-C3/C3b/C3c (#21337-1-AP; 1:1000), anti-C5 (66634-1-Ig; 1:50,000), anti-IgA (11449-1-AP; 1:2000), anti-IgM (66484-1-Ig; 1:20,000) (all from Proteintech), anti-ApoB (#sc-393636; 1:200; SantaCruz Biotechnology), and anti-IgG (H + L) (peroxidase-coupled; #109-035-003; Jackson ImmunoResearch, 1:10,000).

For coomassie staining, gels were incubated in PageBlue (Thermo Fisher Scientific, #11852174).

Quantification of C3a, C4a, C5a, and sC5b-9. 1.5-ml polypropylene tubes were blocked with 30% heat-inactivated FBS at 4 °C o/n. Crystals (1 mg) were incubated in 50 µl of serum in these blocked tubes for 30 min at 37 °C with mild agitation. The supernatant was harvested and C3a, C4a, and C5a concentration was determined using the BD CBA human anaphylatoxin kit (BD Biosciences, 561418, sample dilution 1:15,000) and sC5b-9 concentration was determined using human TCC ELISA (Hycultec GmbH, HK328-02, sample dilution 1:750) according to the manufacturer's recommendations.

Unless otherwise stated in the figure legends, paired or unpaired Student's two-tailed t-test was performed to compare the means of two groups. All analyses were performed using GraphPad Prism version 9 (GraphPad Software; www. graph pad. com/ scien tific-softw are/ prism/). A p value of < 0.05 was considered statistically significant. Data generated using a flow cytometer represents the median fluorescent intensity of 5-10 k particles measured. 

The data that supports the findings of this study are available in the Supplementary Information of this article or can be provided from the corresponding author upon reasonable request.

Received: 31 October 2021; Accepted: 28 February 2022

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We thank Christèle Combes (CIRIMAT, Toulouse INP, France) for providing an independent preparation of t-CPPD and m-CPPD crystals and Hilke Siedersleben for technical assistance. This work was funded by grants from Deutsche Forschungsgemeinschaft (DFG, Grant number: NE 2206/1-1) and Stiftung für Pathobiochemie und Molekulare Diagnostik (SPMD, Grant number: 6-2021) to KN.

K.N. designed and K.N. and T.W. supervised the research. A.W., L.H., and K.N. planned experiments. A.W., L.H., and A.K. performed experiments. A.P. analyzed LC-MS data. A.W., L.H., and K.N. wrote the manuscript. All authors analyzed or interpreted data, contributed to writing of the manuscript, and approved the submitted version.

Open Access funding enabled and organized by Projekt DEAL.

The authors declare no competing interests.

The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598-022-08311-z.Correspondence and requests for materials should be addressed to K.N. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.