In an extension of previous studies we compared the serum proteins bound to MSU crystals to those 472 bound to the microbial pattern zymosan. We confirmed binding of apolipoproteins (19) and CRP to 473 MSU crystals, but not to zymosan (25). Unexpectedly, we identified the transmembrane receptors 474 MARCO, LDLR, and CD14 as proteins binding to MSU only or both MSU and zymosan,
475 respectively. CD14 is long known to exist in a membrane-bound and a soluble form (27) and we also 476 found it in the input serum. The fact that we newly identified MARCO and LDLR on MSU crystals 477 might be due to the ever increasing sensitivity of LC-MS methodology and the potential possibilities 478 for their release, i.e., cleavage from the cell surface or secretion in a soluble form.
479
For MARCO, a class A scavenger receptor (SR), a second isoform has been described before: it is 480 missing the amino acids (aa) 1–78 (the cytoplasmic domain, the transmembrane anchor protein, and 481 17 aa of the extracellular domain), hence, possibly leading to a soluble form of the protein (28).
482 MARCO is primarily expressed on alveolar macrophages in the lung and thereby important for 483 clearance of exogenous material after inhalation. It has been shown to play a critical role in 484 development of silicosis after inhalation of silica particles (23). Our finding that MARCO binds to 485 various naked, endogenous crystals like MSU, t-CPPD, cholesterol, and calcium oxalate dihydrate in 486 addition to exogenous, inhaled particles (e.g., silica) shows that it might act as a more general
487 receptor for many crystalline structures. It remains to be seen, if immune cells like macrophages at 488 sites of crystal-induced inflammation outside of the lung express MARCO and whether it plays a 489 non-redundant role as in the lung. MARCOs ability to recognize so many distinct crystalline surfaces 490 might be due to the formation of homo- or hetero-oligomers as described for the scavenger receptor 491 SR-B1 (29,30). A multimerization in a crystal-like flat surface leading to high avidity and increased 492 binding to various crystals based on the flat surface may be conceivable.
493
The circulating ectodomain of transmembrane LDLR has first been discovered as an interferon-494 induced antiviral protein (31,32) and has recently been described as a possible novel marker for 495 inflammation (33). We show that recombinant LDLR binds to most opsonized crystals and that this 496 binding correlates with the binding of LDL to the same ones: both LDL and LDLR bind to all 497 crystalline structures tested, but not cholesterol crystals. We also observed binding of LDLR to some 498 unopsonized crystals, so we cannot exclude that it may also act as a receptor for pure particles.
499
ion depe
evious studies we compared the serum proteins bound to e compared the serum proteins bound to he microbial pattern zymosan. We confirmed binding of amicrobial pattern zymosan. We confirmed binding o
ystals, but not to zymosan (25).
ystals, but not to zymosan (25).Unexpectedly, wenexpectedly, w , LDLR, and CD14 as proteins bindingLDLR, and CD14 as proteins bin
ly. CD14 is long known to . CD14 is long known to the input serum. he input serum
e to th e to t
Identification of novel crystal-binding proteins
13 Binding of LDL and LDLR to crystals was conserved between human and mouse. However, we did 500 not see major alterations in phagocytosis or IL-1E production in response to crystals by mouse 501 immune cells deficient for LDLR. Since we expect that most of the crystal surface is covered with 502 LDL in the presence of serum, it is conceivable that either the cells have other recognition
503 mechanisms for coated LDL or different opsonizing proteins like complement. Other receptors could 504 potentially compensate the loss of LDLR: members of the LDLR family, e.g., LDLR-related proteins 505 (LRP) that are expressed in a number of different tissues with a wide range of different ligands, could 506 be able to bind LDL and induce endocytosis of crystals. Alternatively, a recent study suggests that 507 direct interaction of a solid structure with the cell membrane can lead to receptor-independent 508 phagocytosis (34). It would be interesting to see, if LDL or other crystal-coating proteins block this 509 direct interaction with the membrane.
510
LDL, the major cholesterol-carrying lipoprotein of plasma, transports lipids from the liver to 511 peripheral tissues, where it is mainly taken up by binding to the membrane-bound LDLR. LDL is 512 believed to be the main culprit in the development of atherosclerosis. However, it is unclear if 513 elevated LDL is sufficient to trigger plaque formation or if it only binds to the damaged artery wall 514 (35). Potentially, LDL may actually act as a patch to cover damaged surfaces and only the prolonged 515 and excessive plaque formation leads to a detrimental outcome of an originally beneficial
516 mechanism. Previous (19,20) as well as our data demonstrate that LDL shows remarkably strong 517 binding to various crystalline structures. LDL may even be depleted from serum by addition of MSU 518 crystals (data not shown). Such strong and specific interactions would support the notion that LDL 519 acts as a patch on damaged or unnatural surfaces. We further show that LDL opsonization inhibits the 520 production of ROS induced by various crystalline structures in human neutrophils. Probably, LDL-521 coating blocks the interaction of crystals with immune receptors like Mac-1 (CD11b/CD18), Fc-522 receptors, or the membrane itself (36).
523
One puzzling finding that we cannot explain is the fact that LDL strongly inhibits cholesterol crystal-524 induced ROS production while we were unable to show any binding of LDL to cholesterol crystals.
525 Maybe this interaction is too weak or transient to be observed with the methods used, or cholesterol 526 crystals induce ROS by a distinct pathway that does not rely on the interaction with the crystal 527 surface, e.g., release of soluble cholesterol that interacts with the cells.
528
Furthermore, our findings indicate that LDL reduces IL-1Eproduction of PBMCs in response to 529 some crystals. However, this alteration correlates only partly with the reduced ROS production. For 530 rather small MSU crystals (lot2) and silica crystals, we see a strong reduction in ROS but not in 531 IL-1Eproduction. In some experiments, we even saw increased IL-1Eproduction. It appears 532 reasonable to speculate that the determining factor for this phenomenon might be the size of the 533 particles as this specific lot of MSU crystals and the silica crystals are very small. Thus, LDL may 534 inhibit IL-1Eproduction by crystals that are too large to be phagocytosed. Smaller crystals that may 535 be phagocytosed with or without the need for LDL binding receptors may activate other pathways. It 536 is possible that LDL inhibits the activation of the inflammasome by direct membrane interaction 537 which does not require phagocytosis (37), but LDL may be unable to inhibit phagosomal
538 destabilization after phagocytosis, which also activates the inflammasome (38). Alternatively, other 539 activating receptors may only be active in the endocytic compartment. CD36 recognizes oxidized 540 LDL and activates TLR signaling only after endocytosis (39). Since the LDL used in our experiment 541 could get oxidized during the incubation period, small crystals could deliver the oxidized LDL to 542 CD36 in the endocytic compartment while large crystals could not. It remains to be seen, if the 543 observed effects of LDL coating on IL-1Eproduction are actually caused by priming, i.e., pro-IL-1E 544 production or inflammasome activation.
545
In review
Howeve only binds to the to cover damaged surfaces ancover damaged surfaces a detrimental outcome of an originally bal outcome of an original as well as our data demonstrate that LDL shows redemonstrate that LDL show rystalline structures. LDL may even be depleted from seres. LDL may even be depleted from ser ata not shown). Such strong and specific interactions woa not shown). Such strong and specific interactions w
patch on damaged or unnatural surfaces.
patch on damaged or unnatural surfaces. We furWe fu n of ROS induced by various crystalliof ROS induced by various cryst
ocks the interaction of crysks the interaction of cry or the membranethe membran
This is a provisional file, not the final typeset article 14
Together, our unbiased LC-MS approach has identified one receptor that directly recognizes disease-546 associated crystals (MARCO) and with LDLR another receptor that binds to all crystals tested, 547 except cholesterol, when they are opsonized with LDL. We further show that LDL not only binds to 548 MSU crystals but all crystals tested, apart from cholesterol crystals or microbes like fungi. This LDL 549 binding shows strong inhibition of ROS production but variable effects on IL-1E production
550 depending on crystal sizes.
551
There is a large range of crystal-associated pathologies which makes it important to investigate if 552 there is a shared molecular basis between the recognition of different crystals. We think these data 553 are a step toward answering this question and hopefully they will spur future research into the 554 functional role of these three proteins in crystallopathies.
555
5 Conflict of Interest 556
The authors declare that the research was conducted in the absence of any commercial or financial 557
relationships that could be construed as a potential conflict of interest. 558
6 Author Contributions 559
KN supervised the research, AA and KN planned experiments, AA, AK, LH, and AW performed 560 experiments, AP performed LC-MS analysis. AA and KN wrote the manuscript. All authors analyzed 561 and interpreted data, contributed to manuscript revision, read, and approved the final version.
562
7 Funding 563
This work was funded by a grant from Deutsche Forschungsgemeinschaft (DFG) to K.N. (grant 564 number: NE 2206/1-1).
565
8 Acknowledgments 566
We thank Sebastian Burbano de Lara for kindly donating HepG2 cells, Sabine Meier for her
567 expertise on crystal characterization and phagocytosis, Tamara Tuchel for help with mouse breeding, 568 and Mareike Diekmann and René Huber for advice and helpful discussions. We are very grateful to 569 the Research Core Unit for Laser Microscopy at Hannover Medical School for their support with 570 confocal microscopy of crystals and particles, and to the Research Core Unit for Cell Sorting at 571 Hannover Medical School for single cell sorting of HepG2 cells.
572
ch, AA and KN planned experiments, AA, AK, LH, andlanned experiments, AA, AK, LH P performed LC-MS analysis. AA and KN wrote the manMS analysis. AA and KN wrote the man
rpreted data, contributed to manuscript revision, read, preted data, contributed to manuscript revision, rea ndinging
was funded byas funded b E 22
E 2
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