C-reactive protein (CRP) recognizes uric acid crystals and recruits proteases C1 and MASP1

Gout is caused by crystallization of uric acid in the form of monosodium urate (MSU) crystals, which induce a sterile inflammatory response that is hardly distinguishable from microbe-induced inflammatory responses. It is unclear, if MSU crystals (like microbes) are recognized by specific pattern recognition receptors. To identify possible soluble pattern recognition molecules for MSU crystals, we purified MSU-binding proteins from human body fluids. We identified C-reactive protein (CRP) as a major MSU-binding protein. Binding of CRP was strong enough to specifically deplete CRP from human serum. We found that CRP was required for fixation of complement components C1q, C1r, C1s and MASP1. Thus, we have identified a pattern recognition molecule for MSU crystals that links to the activation of complement. Notably, CRP does not show an even binding to the complete surface of the crystals. It rather binds to edges or distinct faces of the crystals.

eluted with denaturing SDS buffer. The eluted proteins were applied to SDS-PAGE and visualized by coomassie staining (Fig. 1a). The proteins purified from synovial fluid showed a similar pattern to the proteins purified from serum. The proteins purified with MSU crystals, however, showed a mostly distinct pattern from the proteins purified with zymosan. MSU crystals purified mainly two proteins migrating at 250 kDa and 25 kDa. We excised the corresponding bands (indicated by rectangles in Fig. 1a) and identified the proteins within by liquid chromatography-mass spectrometry (LC-MS). The protein with the highest score in the band above 250 kDa was the known MSU crystal-binding protein apolipoprotein B (apoB), while in the 25 kDa band the protein with the highest score was CRP (Fig. S1a). The zymosan purification also showed a prominent band at 25 kDa. LC-MS analysis of the proteins within this band found apoA1 with the highest probability, suggesting that the 25 kDa proteins in both purifications were distinct (data not shown). We measured the concentration of CRP in both synovial fluid and serum and found around 30 µg/ml and 50 µg/ml, respectively. In line with the results from the LC-MS analysis the concentration of CRP in the body fluids was decreased after the incubation with MSU crystals, but not with zymosan ( Fig. S1b), indicating CRP strongly binds to MSU crystals but not to zymosan. Since several other likely MSU-binding proteins were identified in the 25 kDa band besides CRP (apoA1, SAP, Igκ/λ; Fig. S1a), we tested if the prominent band at 25 kDa was indeed CRP. We repeated the purification of MSU-binding proteins with a set of different sera (Fig. 1b): The 25 kDa band (indicated as A in Fig. 1b) did not appear in the purification from low CRP normal human serum (NHS) (lane 1), but from serum from an individual with an acute phase response (APRS, CRP around 100 µg/ml) (lane 2). Addition of purified CRP to the low CRP NHS from lane 1 before purification with MSU crystals (lane 3) or to a solution of 5% bovine serum albumin (BSA) in Hank's Balanced Salt Solution (HBSS) (lane 4) resulted in a comparable band as in lane 2. Depletion of CRP from CRP-containing acute phase reaction serum using the synthetic CRP ligand phosphorylcholine coupled to agarose (PC-agarose) selectively removed the 25 kDa band (lane 5), while reconstituting this depleted serum with purified CRP also reconstituted the 25 kDa band in the MSU purification (lane 6). In the presence of EDTA, which inhibits CRP binding to its ligands 18 , the 25 kDa band did not appear (lane 7). Lastly, when NHS or CRP-containing serum was incubated with the CRP-ligand PC-agarose, the same 25 kDa band is purified only from CRP-containing serum (lanes 8 and 9). Western blot analysis of the samples confirmed that the signal for CRP showed the same pattern as the 25 kDa band (Fig. 1b, bottom panel). Together, this proves that CRP is indeed the major constituent in the prominent 25 kDa band and thus one of the major MSU crystal-binding proteins in CRP-containing body fluids during an acute phase response.
Intriguingly, there was a second band at around 35 kDa in the coomassie-stained gel (indicated as B in Fig. 1b), which strongly correlated with the CRP band, but was absent when CRP was purified with MSU from a BSA solution (lane 4). Thus, it may be a post-translationally modified version of CRP or a serum protein recruited by CRP. We excised the band and LC-MS identified the protein C1qB with a higher score than CRP (data not shown). Western blot analysis using a C1qB antibody showed the same pattern as the 35 kDa band in the coomassie-stained gel (Fig. 1b, middle panel). While the Western blot analysis using the CRP antibody showed some additional bands, which may represent adducts of CRP with other proteins, none of these were in the range of the 35 kDa band (Fig. S1c). This indicates that CRP recruits C1q to the surface of MSU crystals.
To confirm that CRP binds to MSU crystals we incubated either self-made (lot 2) or commercial (com.) MSU crystals with serum containing 10.4 µg/ml CRP and stained the crystals with CRP antibody. Using a flow cytometer, we found strong binding of this antibody to both crystal preparations compared to isotype control (Fig. 1c, top panel). To test the specificity of the CRP antibody, we depleted CRP from this serum. This reduced binding of the CRP antibody nearly to isotype levels and reconstituting the depleted serum to 10 µg/ml purified CRP recovered binding of the CRP antibody (Fig. 1c, lower panel). Results from two independent MSU crystal preparations are shown in Fig. S1d.
We already showed that CRP binds to MSU crystals in a solution not containing serum proteins other than BSA (Fig. 1b). To compare the binding of CRP in the presence and absence of serum proteins, we added purified CRP to low CRP serum or a 10% BSA solution in HBSS and incubated these solutions with MSU crystals. Bound CRP was detected using a flow cytometer. Both at 10 and at 40 µg/ml, CRP showed weaker binding in serum than in BSA solution (Fig. 1d), indicating that CRP directly binds to the crystals and may even compete with other serum proteins more than it cooperates.
To test the specificity of the CRP binding, we incubated four different preparations of MSU (one was used untreated and sonicated (s)), two preparations of triclinic CPPD (t-CPPD) and two preparations of S. cerevisiae (zymosan and heat-inactivated yeasts) with human serum supplemented with 30 µg/ml CRP (either purified or recombinant) and analyzed CRP binding as above. As shown in Fig. 1e, both purified and recombinant CRP bound strongly to all MSU crystal preparations. CRP bound only weakly, but significantly, to both preparations of t-CPPD, but not to zymosan and S. cerevisiae. MSU crystals can be used to purify CRP. The immobilized CRP ligand phosphorylcholine (PC-agarose) can be used to both deplete and purify CRP from human body fluids 19 . To test, if MSU crystals may also have the ability to specifically deplete CRP from human body fluids, we incubated different human sera or a BSA solution in HBSS either with zymosan, MSU crystals, or PC-agarose. After 45 min CRP and total protein was analyzed in the supernatant. Compared to zymosan, which does not bind CRP, both MSU and PC-agarose strongly reduced the concentration of CRP but not of total protein in all solutions (Fig. 2a). MSU crystals reduced the concentration of CRP significantly more than 50%, which may be considered depletion. MSU crystals did not reduce the concentrations of known MSU-binding proteins IgM and C3, or albumin as compared with zymosan and PC-agarose (Fig. 2b). Adding EDTA to serum or the BSA solution in HBSS blocked the depletion of CRP by both MSU-crystals and PC-agarose (Fig. 2c). Together, this shows that MSU crystals are able to specifically deplete CRP from human serum in a Ca 2+ -dependent manner. This suggests that MSU may be used to purify CRP from serum. To test this, we incubated serum containing 20 µg/ml CRP with three distinct MSU preparations, washed , acute phase reaction serum (APRS; CRP around 100 µg/ ml) or HBSS (always containing Ca 2+ ) with or without depletion of CRP or addition of purified CRP to 100 µg/ml were incubated with MSU crystals or phosphorylcholine-agarose (PC-agarose) for 45 min at 37 °C. Bound proteins were eluted and subjected to SDS-PAGE and visualized by coomassie. In addition, the same samples were analyzed by Western blot analysis using CRP antibody (lower panel) or C1qB antibody (middle panel). (c) Human serum (CRP = 10.4 µg/ml) was left untreated or CRP was depleted with PC-agarose or was depleted and then reconstituted with 10 µg/ml purified CRP. All three sera were incubated with two preparations of MSU crystals (lot 1 and a commercial preparation (com.)). CRP was stained with CRP antibody and anti-rabbit-PE and analyzed using a flow cytometer. (d) Three different preparations of MSU crystals (lot 1, lot 2 and a commercial preparation) were incubated with either pool serum (NHS) (CRP < 0.3 µg/ml) or 10% BSA in HBSS, both with purified CRP added to the indicated concentrations. Binding of CRP to the crystals was analyzed as in c. Median fluorescent intensity (MFI) is shown. (e) Four different preparations of MSU crystals (one commercial (com) and three self-made (untreated or sonicated (s)), two preparations of t-CPPD (commercial (com.) and self-made (sm)) and two preparations of S. Cerevisiae (zymosan and heat-inactivated yeast) were incubated with NHS (CRP < 0.3 µg/ml) with either 30 µg/ml purified CRP or 30 µg/ml recombinant (rec.) CRP added. Binding of CRP to the crystals and fungal particles was analyzed as in c. MFI of staining with CRP antibodies was divided by MFI of isotype controls. Uncropped images of gels and Western blot are shown in Fig. S3. with 10 µg/ml purified CRP added, a single donor serum with 10.4 µg/ml CRP (Serum10.4) and HBSS 10%BSA with 10 µg/ml purified CRP added, were incubated with 3 mg zymosan, 5 mg MSU (lot 1) or 35 µl PC-agarose for 45 min at 37 °C. Samples were centrifuged and the supernatants were analyzed for CRP and total protein concentration. Using a one-sample t-test, the p-value of MSU samples compared to 50% of CRP concentration of the corresponding zymosan sample was calculated. For the difference of total protein in zymosan or MSU treated samples a paired t-test was used. (b) The concentration of IgM, C3c and albumin was analyzed in samples from a by turbidimetry. (c) For the indicated CRP-containing solutions the experiment was repeated in the presence of 5 mM EDTA and the supernatants were analyzed for CRP and total protein concentration. (d) 200 µl of pool serum containing 20 µg/ml purified CRP was incubated with nothing, three distinct preparations of MSU (5 mg each) or 35 µl PC-agarose for 45 min at 37 °C, washed 1x with HBSS for 5 min and then eluted with 5 mM EDTA in HBSS. Supernatants of each step were analyzed for CRP and albumin concentration by the crystals once with Ca 2+ -containing buffer, and then eluted Ca 2+ -dependent proteins with EDTA. For each step the concentration of CRP and albumin was measured in the supernatant (Fig. 2d). CRP was strongly reduced in serum after incubation with MSU and low in the wash buffer. Nearly half of the starting concentration was recovered in the EDTA-elution. The concentration of albumin was strongly reduced in both wash buffer and in the EDTA-elution compared to input serum, indicating MSU crystals can separate CRP from other serum proteins. PC-agarose was more efficient than MSU crystals in purifying CRP, which may be due to the higher specific surface area of the porous agarose. However, when extensively washed, MSU and PC-agarose purify CRP to a similar purity (Fig. 2e). Together, this shows that the binding of CRP to MSU strongly resembles the binding of CRP to its immobilized ligand phosphorylcholine, arguing that MSU crystals could also act as a genuine ligand.
CRP recruits C1 and MASP to the surface of MSU crystals. In Fig. 1 we already showed that CRP enhances binding of C1qB to MSU crystals in human serum. To globally assess how CRP alters the opsonization of MSU we performed an exploratory proteomics experiment. We incubated low CRP serum and plasma of a single donor with MSU in the presence or absence of added purified CRP. Unbound proteins were washed away and bound proteins were eluted with denaturing SDS buffer and subjected to SDS-PAGE (Fig. 3a). CRP addition led to the appearance of several additional protein bands in both serum and plasma (lane 2 and 4). To identify any proteins that were enriched or depleted on the surface of the crystals in the presence of CRP, we subjected all proteins purified from plasma in the absence of CRP (lane 3) and in the presence of CRP (lane 4) to LC-MS analysis (whole lanes). Peptides were identified and quantified using MaxQuant software. A list of the 30 proteins with the highest intensity identified from lane 4 is shown in Table S1a. Of these 30 proteins, 6 proteins were increased at least 5-fold by addition of CRP (Thrombin, C1qA, C1qB, C1qC, C1r and C1s). We set out to validate these 6 proteins and 3 proteins with a high intensity that showed only a weak increase (C3, Fibrinogen, and SAP). To this end, we repeated the experiment with plasma from three distinct donors, purified both MSU crystal-and PC-agarose-binding proteins, and applied them to Western blot analysis (Fig. 3b). All five components of the C1 complex showed strongly enhanced binding to both MSU crystals and PC-agarose in the presence of CRP. For MSU crystals, binding of the catalytic subunits C1r and C1s were mainly increased in their proteolytically cleaved/active form. An enhanced binding of thrombin in the presence of CRP could not be verified, but specific binding of thrombin (in the form of prothrombin) to MSU crystals could be shown. The sample used for the LC-MS analysis experiment (Fig. 3a) did show increased thrombin binding, suggesting this discrepancy is not due to an inaccurate quantification by LC-MS but rather experimental or donor variance (data not shown).
For C3 we found an increase in C3 and a C3 degradation product (the lower band at around 39 kDa constitutes the C-terminal C3 degradation product C3c α2, according to the epitope of the used antibody) on MSU crystals in the presence of CRP, while C3 already strongly bound to PC-agarose even in the absence of CRP (Fig. 3b). In line with LC-MS results addition of CRP did not change binding of fibrinogen (γ-chain) and SAP to MSU crystals (Fig. 3b). Among the proteins identified by mass spectrometry with a low intensity, a few other proteins showed a strong increase (>10-fold) in the presence of CRP (Table S1b). From these we could verify enhanced binding of MASP1 (Fig. 3b), while MBL2 showed an inconclusive band pattern and coagulation factor VII (F7) did not show an increase (data not shown).
We repeated the experiment twice using different sera instead of plasma with similar results showing enhanced recruitment of all C1 complex subunits, MASP1 and C3 ( Fig. S2; uncropped in Fig. S6, 7).
To confirm this is not due to the specific MSU crystal preparation used, we also confirmed the increased recruitment of C1 and MASP1 using five distinct preparations of MSU crystals (Fig. 3c). Note that for C1r, C1s, MASP1 and C3 we did observe protein bands that appeared to have a molecular weight greater than the full-length form of the proteins after binding to MSU crystals (e.g. Fig. 3a,b), as we saw for CRP (Fig. S1c). The reason for this is unclear, but may be due to formation of intermolecular amide bonds by C3 or transglutaminases.
Since the effect of CRP on C3 fixation was weak as compared to C1, we quantified C3 fixation after the addition of 0, 30 or 100 µg/ml CRP. We found significantly more C3 and a degradation product (C3c α2) on the surface of MSU crystals when 30 µg/ml CRP was added, while no further increase could be observed at 100 µg/ml (Fig. 3d). Our LC-MS analysis did not show enhanced fixation of complement factors downstream of C3 in the presence of CRP. However, when we tested the amount of the terminal complement complex (SC5b-9) on MSU crystals opsonized in the absence or presence of CRP, we found a small but significant increase in the presence of CRP (Fig. 3e).
Together, this shows that CRP recruits and activates C1 and MASP1 to MSU crystals, which leads to fixation of complement from C3 up to the terminal complement complex, while there seems to be additional CRP-independent mechanisms for activation and fixation of the complement factors from C3 to the terminal complement complex. CRP binds to distinct faces or the edges of MSU crystals. We expected the strong binding of CRP to the crystals would displace a lot of the known MSU-binding proteins. The LC-MS data suggested only a few proteins with reduced binding in the presence of CRP e.g. apoB, fibronectin and lipoprotein(a) ( Table S1a). However, our attempts to verify reduced binding of apoB in the presence of CRP were inconclusive (data not shown). We also expected coating of the crystals with CRP would alter the immune cell responses to the opsonized crystals. turbidimetry. (e) 100 µl of a serum containing 30 µg/ml CRP was incubated with 9 mg MSU or 35 µl PC-agarose for 45 min at 37 °C. MSU/PC-agarose was washed 5x in HBSS, and CRP was eluted by HBSS + 5 mM EDTA. Eluted proteins were applied to SDS-PAGE and proteins were visualized by coomassie staining. Uncropped image of the gel is shown in Fig. S3 (right gel). Each experiment was repeated at least once with similar results. (2020) 10:6391 | https://doi.org/10.1038/s41598-020-63318-8 www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ However, when we tested inflammasome activation in a monocytic cell line (THP-1), we did not see any change in IL1β production in response to MSU crystals in the presence or absence of CRP (Fig. S2c).
A likely explanation for this discrepancy would be that CRP does not bind to the complete crystal surface. We thus incubated the largest MSU crystals (lot 2) that we have generated within this project with CRP-containing serum and analyzed CRP binding by fluorescence microscopy. We indeed found that CRP was mostly located at the edges and sometimes showed a punctured localization (Fig. 4a). To test, if binding of CRP to the edges is dependent on other serum proteins, we incubated the crystals with CRP in buffer containing only BSA. Here, the binding of CRP to the edges was at least as pronounced as in serum, indicating that CRP itself preferentially binds to edges of the crystals (Fig. 4b). The areas bound by CRP may either be edges between crystal faces or narrow crystal faces. Binding to a subset of faces or the edges may explain why CRP only weakly competes with other known MSU-binding proteins. This may not be limited to MSU crystals, as a similar uneven binding of CRP was also observed for CPPD crystals (Fig. 4c). To test, if the fixation of complement induced by CRP also happens at the same sites, we co-stained CRP with C3 on the surface of MSU crystals. On two distinct crystal preparations we found co-localization of C3 at the same edges as CRP, while C3 also bound to other locations on the crystals (Fig. 5a,b). This suggests that CRP recruits C3 to the edges while an additional CRP-independent mechanism of C3 binding to MSU crystals exists.

Discussion
In this study we were searching for MSU binding proteins that could link to the activation of the immune system. The most abundant protein on MSU crystals by a wide margin seems to be apoB, which has been described before to inhibit neutrophil activation by MSU crystals 20,21 . The second most abundant protein was CRP. It is unclear, why this has not been found before. It is likely because most studies on MSU crystal binding proteins were performed before LC-MS was available. To exclude the possibility of CRP-binding to only one potentially compromised MSU crystal preparation, we confirmed that CRP bound with similar strength to up to 6 distinct preparations, two of them from a commercial vendor. We further show binding of endogenous CRP in synovial fluid, plasma and serum from different donors as well as of CRP purified from human body fluids and recombinant CRP produced in a human cell line, indicating no special conditions or co-factors are required for binding of CRP to MSU crystals.
We show that CRP directly binds to MSU crystals with a similar binding strength and Ca 2+ -dependency as its synthetic ligand PC-agarose, which makes MSU crystals a suitable matrix for purification of CRP. We know that PC-agarose remains superior to MSU crystals for purification of CRP, because it has a higher binding capacity and because the CRP-related SAP also binds to MSU crystals. However, the fact that MSU crystals could be used for CRP purification at physiological conditions is a strong argument that MSU could be a genuine CRP ligand.
Our unbiased LC-MS experiment also showed that CRP altered opsonization of MSU crystals in a similar way as other CRP-ligands. We mainly found that CRP recruits active complement component C1 to the surface of MSU crystals. We further show recruitment of the lectin pathway protease MASP1 by CRP. This is likely due to previously described interactions of CRP with lectin pathway pattern recognition molecules like ficolin 22,23 . Together, this suggests that CRP activates both classical and lectin pathway of complement activation on the surface of MSU crystals. In line with this notion we found enhanced C3 and terminal complement complex fixation in the presence of CRP. The effect was smaller than for C1 and MASP1, suggesting that additional CRP-independent complement activating pathways are triggered by MSU-crystals or that CRP also recruits proteins that inhibit terminal complement complex formation as described before 24 . Russel et al. have made the observation that MSU crystals deplete complement from human serum but not from serum from about half of the patients with common variable immunodeficiency (CVID). Addition of CRP restored the ability of the crystals to deplete complement 25 , strongly supporting the notion that CRP is a pattern recognition molecule for one, albeit non-exclusive, pathway of complement activation by MSU crystals. We observed only weak binding of C1 in the absence of CRP to MSU crystals, which may be direct binding of C1 to MSU crystals 26 or mediated by other opsonins (e.g. IgM, IgG 21 , or SAP ( Fig. 3b; Table S1a)). It remains to be seen what other complement activating pathways are responsible for the CRP-independent complement activation and fixation.
CRP in its native form is a disc-shaped homopentamer. Upon binding to its ligands on damaged and apoptotic cells, it undergoes a conformational change that allows for binding and activation of C1 and may lead to dissociation to monomeric CRP 18,24,[27][28][29][30] . Our finding that CRP recruits and activates C1 on the surface of MSU crystals suggests that CRP binds MSU crystals via its binding face, which leads to a conformational change that enables C1 binding to the effector face of CRP. Intriguingly, the conformational change in CRP has been postulated and shown to depend on the curvature of membranes 31,32 . We found that CRP recognizes mainly the edges or distinct faces of the triclinic crystals. The edges may provide the required curvature for binding and conformational and was incubated with four distinct preparations of MSU crystals (lots 1-4) for 30 min at 37 °C. Bound proteins were eluted as in a and subjected to Western blot analysis using C3 antibody. Signal for full length C3 (>170 kDa) and its degradation product C3c α2 (39 kDa) were quantified by densitometry and normalized to the intensity of full length C3 in the absence of added CRP. (The corresponding Western blot is shown in Fig. S5). A paired two-tailed t-test was used to compare vehicle (0) with 30 µg/ml purified CRP. (e) Two distinct lots of MSU crystals were incubated in 4 individual human sera with 0 or 40 µg/ml purified CRP added for 30 min at 37 °C, extensively washed and stained with rabbit anti SC5b-9 plus anti rabbit PE. MSU crystals were analyzed using a flow cytometer. Median fluorescence intensity (MFI) of PE / 1000 is shown. A paired two-tailed t-test was used to compare vehicle (0) with 40 µg/ml purified CRP. Experiments from b-e are representative of at least 2 independent experiments. Uncropped images of the gel and Western blots are shown in Fig. S4  www.nature.com/scientificreports www.nature.com/scientificreports/ activation of CRP. This may explain why it appears in confocal microscopy that not all CRP co-localizes with C3: Some CRP binding sites may not provide the required curvature for activation. Alternatively, distinct crystal faces may have distinct surface properties as they expose distinct sides of the urate molecule 33 . This would explain selective binding of CRP to one or more distinct crystal faces.
Similar to the preferential binding of CRP to the edges or specific sides of MSU crystals, we also found irregular binding of CRP to CPPD and it seems that the previously described binding of CRP to cholesterol crystals is also limited to certain areas of the crystals 34 .
Our mass spectrometry experiment validated most of the known MSU-binding proteins like C1, apolipoproteins, fibronectin and fibrinogen 20,21,26 . Many other proteins in the list (Table S1a) are probably also MSU-binding proteins, but we only validated two more notable novel MSU-binding proteins: Thrombin (F2) and SAP (APCS), which both bound independently of CRP. Binding of both pentraxins CRP and SAP has also previously been shown for cholesterol crystals 34 . The third member of the pentraxin family PTX3 has been shown to inhibit calcium oxalate crystallization and nephrocalcinosis 35 , and to contribute to MSU-induced inflammation in mice 36 . Thus, pentraxins seem to have evolved to detect and remove not only dead cells and tissue debris but also diverse crystallized materials. www.nature.com/scientificreports www.nature.com/scientificreports/ Gout patients can exhibit very high serum concentrations of CRP during an acute attack 10 . Whether CRP binding to MSU crystals modulates the inflammation during the attack or whether this is the futile attempt of the immune system trying to remove the crystals remains to be seen. Still, the identification of CRP as a genuine MSU crystal recognition molecule more than 40 years after the conclusion that MSU crystals are the cause of gout 37 points to a more specific interaction of the immune system with crystalline structures than previously anticipated.

Methods
Human body fluids. We used leftover diagnostic samples of serum and synovial fluid of a male patient with pseudogout, and a serum of a male individual with an acute phase reaction (CRP ca. 100 µg/ml). Normal human serum and plasma was obtained by drawing venous blood from 4 healthy donors (2 male and 2 female) into blood collection tubes coated with hirudin (Sarstedt, #04.1959.001) or clotting activator (Sarstedt, #02.1388). Both were collected by centrifugation at 3000 xg for 10 min after less than 30 min at 4 °C and frozen at −80 °C. All donors gave informed written consent.
Five additional single donor sera were obtained from Innovative Research (#ICERS10ML). Pool serum was generated by mixing serum of at least 3 single donor sera.  MSU, CPPD, Zymosan and S. cerevisiae. Distinct crystallization protocols were used for each lot of MSU crystals: Lot 1-3: 20 mM uric acid (Merck KGaA, #U0881) was dissolved by boiling in ultrapure water containing 20 mM NaOH. After cooling to 60 °C, the pH was adjusted to a pH of 8 (lot 1), pH 9 (lot 2) or pH 7.5 (lot 3) and the solutions were sterile filtered. Solutions were kept under mild agitation at room temperature until crystals formed for 24-72 h. Crystals were harvested on a sterile filter, washed with ethanol p.a. and dried o/n at 60 °C. Crystals were analyzed by FT-IR spectroscopy and taken up in PBS at 50 mg/ml and stored at 4 °C or −20 °C. Lot 1 and 2 were used both as prepared and sonicated to reduce the size of the crystals. Lot 4: 10 mM uric acid was dissolved by boiling in ultrapure water containing 10 mM NaOH. After cooling to 60 °C, 50 mM NaCl was added and the pH was adjusted to 8.5. Crystals formed without agitation at room temperature, harvested and directly taken up in PBS. Birefringence of all preparations was analyzed by polarization microscopy. To exclude contaminations or bias of our crystals, two additional commercial lots of MSU crystals from InvivoGen were used (#tlrl-msu; lots MSU-40-02 (com.), MSU-41-01 (com. lot 2)).
Triclinic-CPPD was from InvivoGen (t-CPPD com.) (#tlrl-cppd) or was prepared (t-CPPD) as previously published by using sodium pyrophosphate as precursor instead of the potassium one 38 .
Zymosan (Sigma-Aldrich, #Z4250) was resuspended in ethanol p.a. at 30 mg/ml and stored at −20 °C. Before experiments, zymosan was washed 2x in HBSS. Dried S. cerevisiae were from Dr. Oetker GmbH and was rehydrated in RPMI1640 for one hour at room temperature, washed in PBS, resuspended in PBS, was heat-inactivated at 80 °C for 10 min, and stored at −20 °C.
Purification of MSU-, zymosan-or PC-agarose-binding proteins. Throughout this study only HBSS containing 1.26 mM Ca 2+ , 0.9 mM Mg 2+ and 5.5 mM D-glucose (Thermo Fisher Scientific, #14025050) was used which was saturated with sodium urate to prevent dissolution of MSU crystals.
Unless otherwise stated, 100 µl of body fluid was incubated with 8 mg MSU crystals, 5 mg zymosan, or PC-agarose (ca. 20 µl packed beads) for 30 min at 37 °C with agitation (1200 rpm). Particles were washed 6x with sodium urate-saturated HBSS by centrifugation at 2000 xg for 1 min and discarding the supernatant. Bound proteins were either eluted by adding 50 µl of 2x SDS-PAGE sample buffer (+DTT) and heating at 70 °C for 10 min or by incubating with HBSS containing 5 mM EDTA for 5 min at 37 °C.
Images were either acquired using an Olympus IX81 inverted microscope and Cell R software (version 3.2; https://www.olympus-lifescience.com/en/software/) or a Zeiss 980 Airyscan 2 in combination with Zeiss ZEN