Complement Membrane Attack Complexes Disrupt Proteostasis to Function as Intracellular Alarmins

Internalized pools of membrane attack complexes (MACs) promote NF-kB and dysregulated tissue inflammation. Here, we show that C9, a MAC-associated protein, promotes loss of proteostasis to become intrinsically immunogenic. Surface-bound C9 is internalized into Rab5 + endosomes whose intraluminal acidification promotes C9 aggregates. A region within the MACPF/CDC domain of C9 stimulates aggrephagy to induce NF-kB, inflammatory genes, and EC activation. This process requires ZFYVE21, a Rab5 effector, which links LC3A/B on aggresome membranes to RNF34-P62 complexes to mediate C9 aggrephagy. C9 aggregates form in human tissues, C9-associated signaling responses occur in three mouse models, and ZFYVE21 stabilizes RNF34 to promote C9 aggrephagy in vivo. Gene-deficient mice lacking ZFYVE21 in ECs showed reduced MAC-induced tissue injury in a skin model of chronic rejection. While classically defined as cytotoxic effectors, MACs may impair proteostasis, forming aggregates that behave as intracellular alarmins.


INTRODUCTION
Membrane attack complexes (MACs) are immune mediators promoting tissue injury 1 that have been therapeutically targeted in ~ 40 in ammatory conditions. 2 MACs share structural and functional properties with perforin and e ciently promote osmolysis of anucleated red blood cells 3 and xenogeneic pathogens. 4,5[8] Complement proteins inclusive of MACs show diagnostic, 9 prognostic, 10 and therapeutic value [11][12][13] in chronic antibody-mediated rejection (CABMR).5][16] A similar process occurs in connective tissue disorders, 17 autoimmune vasculitis, 18 and viral infection including SARS-CoV-2, 19,20 suggesting a generalizable form of immunogenicity linked to MACs that is separable from their cytolytic effects.
A single MAC pore contains ~ 20-25 C' proteins with a molecular weight of ~ 1,600 kD.This de novo protein burden on ECs is anticipated to signi cantly challenge proteostasis, particularly during chronic in ammatory states like CABMR where MACs may become persistently assembled and internalized.C9 comprises the majority of MACs with 13-18 molecules per MAC; and ~ 7-8% of C9 consists of intrinsically disordered regions (IDRs, https://mobidb.bio.unipd.it,http://biocomp.chem.uw.edu.pl/A3D2),suggesting susceptibility to aggregate formation.Alarmins are endogenous proteins that are basally non-immunogenic but become pro-in ammatory after losing their homeostatic compartmentalization 28 or after becoming pathologically modi ed as is the case for amyloidogenic proteins. 29Based on the spatially restricted responses of MACs and the aggregate-prone features of its majority component, C9, we tested the hypothesis that intracellular C9 impairs proteostasis in ECs, generating aggregates that operationally behave as alarmins.

RESULTS
Protein Aggregates and EC Activation in CABMR Biopsies.We initially surveyed CABMR biopsies, excluding patients with confounding conditions characterized by protein aggregates including primary amyloidosis and post-transplant lymphoproliferative disorder (PTLD, Supplementary Table 1). 30In immune-EM, we observed that certain adluminal cells contained lamentous inclusions enriched for C9 which appeared to be contained within intracellular vesicles (Fig. 1a).We did not detect lamentous inclusions in glomerular ECs or in control tissues from transplant patients undergoing routine surveillance biopsies.
Following treatment, PRA generates alloAb, anaphylatoxins, and MACs.To identify culprit mediator(s) causing thio avin uorescence, we performed sera fractionation and recombination studies.We separated PRA sera into its IgG + and IgG-fractions and found that while these fractions showed only minimal effects individually, combining the IgG-and IgG + fractions signi cantly potentiated thio avin uorescence (Fig. 2g).This ruled out IgG or contaminant(s) as a principal cause for increased thio avin staining and suggested a role for C' activity.
To test C9 aggregation, we treated HUVECs with the IgG + fraction of PRA combined with C9-de cient reference sera.This permitted IgG binding, anaphylatoxin formation, and oligomerization of MAC proteins to form C5b-8 which has pore-forming capabilities 33 but lacks C9.IgG-induced C' activation with C9-de cient sera minimally increased thio avin uorescence compared to the IgG + fraction alone (Fig. 2h, lane 3 vs lane 4).Addition of C9, which alone showed no effects (lane 1) signi cantly rescued thio avin staining (lane 4 vs lane 5).We separated PRA-treated HUVECs into soluble and insoluble fractions, and detected increased C9 within the SDS-insoluble pellet (Fig. 2i) that, unlike pools of C9 within SDS-soluble supernatants, became resistant to mild proteinase K (PK) digestion, together indicating generation of insoluble C9 (Fig. 2j).
To further verify C9 aggregates, we tandemly expressed elements within the TSP, LDLRA, and MACPF domains of C9 that form b-sheets (www.uniprot.org,prosite.expasy.org/scanprosite/), a conformation frequently adopted in protein aggregates.Each element, ranging from ~ 6-9 kD, was tandemly expressed intracellularly and separated by a exible glycine linker to allow antiparallel b-sheet stacking which occurs upon native assembly of MACs (Fig. 2k).We linked these elements to FLAG as this tag is less prone to aggregation relative to GFP, RFP, and luciferase reporters.
Of elements tested, AA197-270, an element within the MACPF domain containing the majority of predicted IDRs, strongly induced high molecular weight aggregates in HEK293 cells (Fig. 2l).AA197-270 colocalized within large perinuclear punctae, phenocopying distributions of native C9 aggregates in PRAtreated HUVECs (Fig. 2m).In fractionation studies, AA197-270 aggregates exclusively appeared within the insoluble pellet (Fig. 2n).Original and uncropped Western blot lms for Fig. 2 are shown in Supplementary Fig. 3. C9 may form aggregates. Intracellular C9 Form Aggregates Within the Endolysosomal Pathway.We asked whether C9 aggregates formed at the cell surface and/or intracellularly.We previously found that MAC internalization required CME. 23Exploiting this, we pre-treated HUVECs with CME inhibitors, Dynasore and PitStop2, and tested effects on C9 aggregates.Both CME inhibitors increased C9 levels at the cell surface (Fig. 3a, arrows), and this signi cantly reduced thio avin uorescence in PRA-treated HUVECs.Pitstop2 signi cantly decreased insoluble C9:soluble C9 ratios (lane 2 vs lane 4, Fig. 3b), an effect phenocopied by siRNA vs dynamin-2 (DNM2), the EC-speci c dynamin isoform whose membrane scission activity generates endosomes (Fig. 3c).Following MAC assembly, the majority of C9 aggregates form intracellularly.
MACs vigorously assemble under cell-free conditions at pH < 6. 34,35 While C9 is unlikely to encounter such conditions within intravascular or interstitial space, intracellular endolysosomes routinely reach intraluminal pH 4.5-5.5 to regulate vesicular maturation and tra cking. 36Based on this, we tested whether C9 aggregates could form within the endolysosomal system.We resuspended C9 in buffers of varying pH and found that at pH ≤ 6.5, C9 began to form ring-like structures whose aggregation increased in direct proportion to buffer acidity (Fig. 3d) and protein concentration (Fig. 3e).Among MAC components, C9 showed the highest thio avin uorescence (Fig. 3f), altogether suggesting that endolysosomes favor C9 aggregation.
Orthogonally, AA197-270 aggregates upregulated NF-kB marked by NIK (Fig. 5f) in both a dose-and time-dependent manner (Fig. 5g,h).To identify salient genes and/or pathways, we performed RNA seq which showed 2,160 and 2,528 genes that were signi cantly upregulated and downregulated, respectively, by AA197-270 aggregates (Supplementary Fig. 1d).Notably, GSEA uncovered a gene signature for autophagy (Supplementary Fig. 1e) as well as signatures related to allograft rejection, IFNg, and NF-kB responses (Supplementary Fig. 1f).NF-kB and interferon signaling genes were strongly upregulated in AAV197-270 cells (Supplementary Fig. 1g).Based on this, we examined functional intersection between these pathways, speci cally testing IFN-g priming of NF-kB genes, a phenomenon observed in PRA-treated HUVECs 22 as well as macrophages where IFN-g may prime in ammasomes.We pre-treated AA197-270 cells with IFN-g and noted that in ammatory genes previously found to be NF-kBdependent in PRA-treated HUVECs 22 including CCL5 and IL-6 became synergistically or additively potentiated, respectively, by IFN-g (Fig. 5i).Similar to PRA-treated HUVECs, gene-speci c knockdowns of ATG5 signi cantly reduced NF-kB (Fig. 4l) and in ammatory genes in AA197-270 cells (Fig. 5j).Original and uncropped Western blot lms for Fig. 5 are shown in Supplementary Fig. 5. Aggrephagy of C9 promotes NF-κB and EC activation.
ZFYVE21 is Required for C9 Aggrephagy.While Rab5 activity was required to form C9 aggregates (Fig. 3c), prior studies have linked Rab5 activity to aggregate degradation, i.e., aggrephagy. 37ZFYVE21 is a conserved Rab5-associated protein implicated in cell motility, 41 and we previously found that ZFYVE21 mediated NF-kB activity. 24,26The functions of ZFYVE21 have not been connected to macroautophagy, and in this context we tested roles for ZFYVE21 in mediating C9 aggrephagy.
We identi ed upregulated proteins in our proteomic datasets containing RING and HECT domains, domains known to show E3 ubiquitin ligase activity.We cross-referenced this list against proteins showing z-scores ≥ 2 in a prior genome-wide siRNA screen for NF-kB. 23Among proteins identi ed, we focused on RNF34.RNF34 is an E3 ubiquitin ligase containing a FYVE domain allowing colocalization to Rab5 + endosomes and a RING domain enabling ubiquitinylation of immune-related substrates. 44,45F34 has not been connected to macroautophagy but was previously found to directly bind ZFYVE21 in a process that enhanced its stability in HUVECs. 26 thus considered whether RNF34 interacted with ZFYVE21 within aggresomes.RNF34 pulled down with ZFYVE21 following PRA treatment, and this interaction persisted to 2-4 hr, timepoints when aggresome formation occurred (Fig. 7a).At these times, RNF34 was found to heavily colocalize with ZFYVE21 + Thio avin + aggresomes (Fig. 7b).RNF34 siRNA reduced C9 ubiquitinylation while potentiating C9 (Fig. 7c,d), and this resulted in attenuated NF-kB (Fig. 7c,d) and decreased EC-mediated T cell activation in EC:T cell cocultures (Fig. 7e).Based on this, we tested RNF34 ubiquitinylation of C9 in cell-free ubiquitinylation studies (Fig. 7f).Among E2 conjugating enzymes tested, we selected UBCH5a due to its strong activity.We found that UBCH5a allowed RNF34 to directly K48 ubiquitinylate C9 in a manner requiring its RING domain which is known to contain E3 ubiquitin ligase activity (Fig. 7g).We subsequently co-transfected CQ-treated HUVECs with RNF34 siRNA in the presence of either Ub-WT or Ub-DN constructs and found that RNF34 siRNA, like Ub-DN, blocked C9 ubiquitinylation (Fig. 7h), indicating that RNF34 ubiquitinylates C9.Ubiquitinylated cargo require binding to proteins containing ubiquitin binding domains (UBDs). 46C9 and ZFYVE21 lack annotated UBDs and did not bind K48 Ub chains in the absence of E2 conjugating enzymes (Supplementary Fig. 2h).In studies above, we tested P62, a UBD-containing protein, which, in contrast to C9 and ZYVE21 showed robust binding to K48 Ub chains (Supplementary Fig. 2h).In ZFYVE21-FLAG ECs, P62 pulled down with ZFYVE21 (Fig. 7i) and heavily colocalized with ZFYVE21 + punctae concurrently showing thio avin staining (Fig. 7j).In pulse-chase studies, P62 siRNA potentiated C9 while inhibiting LC3-II (Fig. 7k).P62 (SQSTM1) siRNA strongly inhibited PRA-induced NF-kB luciferase activity (Fig. 5a), and strongly decreased phosphoP65 in PRA-treated HUVECs (Fig. 7k).Original and uncropped Western blot lms for Fig. 7 are shown in Supplementary Fig. 7. Collectively, our data support a function for ZFYVE21 as an adaptor, bridging LC3 + aggresomes to RNF34-P62 complexes to mediate C9 aggrephagy.
Signaling Responses Related to C9 Aggrephagy Occur In Vivo.We examined the relevance of salient signaling processes in vivo.We initially tested whether C9 aggregates could occur in AV.To do this we employed a humanized model of ischemia reperfusion injury (IRI). 27In this model, human artery segments are subjected to hypoxia in organ culture prior to being implanted as interposition xenografts in descending aortae of SCID/beige immunode cient mice pre-engrafted with human lymphoid cells.This protocol activates complement (C') and forms MACs in adluminal ECs but also the media, a region typically spared from collagen deposition in AV. 9 This allowed us to assess thio avin staining without confounding collagen uorescence which complicated our prior anayses in CABMR biopsies (Fig. 1).Compared to controls, C'-treated grafts developing AV showed increased thio avin staining in medial regions (Fig. 8a), and C'-treated tissues showed increased insoluble C9 (Fig. 8b).Our data showed that C9 aggregates may form during AV.
To further delineate roles for RNF34, we utilized a third approach incorporating collagen-bronectin gels. 24,26HUVECs were embedded in collagen-bronectin gels and implanted subcutaneously into SCID/beige mice, allowing HUVECs to self-assemble into perfused microvessels in vivo. 47Three weeks post-implantation, hosts bearing collagen-bronectin gels and injected with PRA showed perfused Ulex + microvessels co-staining for C9 and VCAM-1 (Supplementary Fig. 2i).PRA-treated gels additionally showed increased punctate thio avin and C9 staining under I.F.(Supplementary Fig. 2i, Fig. 9h).In follow-up studies, HUVECs were transduced with control or RNF34 shRNA prior to gel embedding and subcutaneous implantation.Here, we found that RNF34 shRNA signi cantly potentiated both thio avin staining and C9 MFIs (Fig. 9i), indicating a role of RNF34-mediated C9 aggrephagy in vivo.Original and uncropped Western blot lms for Fig. 8 are shown in Supplementary Fig. 8. ZFYVE21 stabilizes RNF34 to mediate C9 aggrephagy, NF-kB, and EC activation in vivo.
ZFYVE21 in ECs Dictates Alloimmune Tissue Injury.We analyzed effects of ZFYVE21 in a skin allograft model of CABMR. 26In CABMR, ECs become principal targets for MACs. 6,15To de ne roles for ZFYVE21 in ECs, we crossed Cdh5-Cre x ZFYVE21 / mice to generate ZFYVE21 EC −/− mice.Subsequently, male ZFYVE21 EC −/− mice, ZFYVE21 −/− mice, and littermate controls (WT) were injected with anti-H-2 b Ab to form MACs, and twenty-four hours later MAC-treated male skin grafts were placed onto SCID/bg recipients passively receiving female splenocytes.Twenty-one days later, compared to MOPC Ab-treated controls, WT skin grafts treated with anti-H2 b Ab showed increased epidermal thickening (Fig. 10a) and CD45 + immune cell in ltrates (Fig. 10b), both of which became signi cantly reduced in similarly-treated ZFYVE21 EC −/− and ZFYVE21 −/− skin grafts.We noted that the tissue readouts above were reduced in ZFYVE21 EC −/− mice to a degree approximating that of ZFYVE21 −/− mice globally lacking ZFYVE21, indicating a principal role for ZFYVE21 in ECs in mediating alloimmune tissue injury in our disease model.On further phenotyping, we found that ZFYVE21 EC −/− grafts showed decreased cytokines (Fig. 10c) and chemokines (Fig. 10d) vs comparably-treated WT hosts.Finally, to generalize ndings we analyzed public RNA seq data from complement-mediated conditions including CABMR (n = 110), rheumatoid arthritis (n = 23), and lupus nephritis (n = 21).These analyses showed signi cant correlations between genes marking EC activation and aggrephagy (Supplementary Fig. 2j) as well as moderate-high correlations between aggrephagy genes with ZFYVE21 (left, Supplementary Fig. 2k) and RNF34 (right).ZFYVE21 in ECs modulates MAC-induced tissue injury in vivo.
Despite their initial surface distributions, MACs appear to exert the bulk of their immune effects with respect to NF-kB intracellularly.This spatially restricted feature is compatible with known alarmins including certain aggregate-prone proteins like amyloid precursor protein (APP) 48 and prion protein (Prp) 49 which basally localize at the cell surface and become immunogenic upon forming intracellular aggregates.In contrast to these amyloidogenic proteins as well as intracellular C' proteins including C3 [50][51][52][53] and C5, 54,55 C9 is introduced into intracellular space in a cell non-autonomous manner.As C9 has been detected in up to ~ 95-99% of amyloid plaques 56 we surmise that C9 may propagate in ammation by forming de novo protein aggregates secondary to those generated by amyloidogenic proteins.
A recent study identi ed an endocytic pathway, termed aggregation-dependent endocytosis (ADE), allowing internalization of aggregates formed at the cell surface. 57We observed that endocytic blockade did not fully abrogate thio avin staining, indicating that a residual pool of protein aggregates had formed on EC surfaces (Fig. 3a-c), a compartment where alloAbs and MACs initially assemble.While intracellular MACs signi cantly contributed to total levels of protein aggregates, a contribution of ADE remains untested in our current models.

METHODS
PRA Treatment.'High' panel reactive antibody (PRA) sera were obtained as pooled, de-identi ed sera from the tissue typing laboratory at Yale New Haven Hospital.'High' PRA sera were taken from renal transplant candidates showing allo-sensitization of ≥ 80% and negative testing for numerous infectious agents. 21Prior to use, PRA sera was supplemented with human complement (Sigma, #S1764) at a ratio of 1 vial of lyophilized human complement per 25mL PRA sera.PRA sera supplemented as above can be used for 3 weeks, after which complement activity deteriorates.For PRA treatment, HUVEC were pretreated with IFN-g (50ng/mL, Invitrogen) for 48-72h prior to placement in gelatin veronal buffer (GVB, Sigma) at 25% v/v for the indicated times.In pulse-chase studies, HUVECs were pulsed with GVB containing 25% v/v PRA sera for 4h prior to washing and chased using GVB buffer alone for the indicated times.For treatment of human kidneys in organ culture, PRA was added at 1:4 ratio with GVB, for 6 hours prior to analysis.PRA sera were fractionated into IgG-and IgG + fractions as previously described. 21Total IgG concentrations were rst determined in intact sera prior to fractionation by ELISA (Invitrogen).Then, per manufacturer's speci cations using a MAbTrap Kit (GE Healthcare, Piscataway, NJ), 500µL of neat sera were diluted 1:1 in binding buffer and passed through the provided column pre-equilibrated with binding buffer.The column was washed with 5mL binding buffer to collect IgG-fractions, and the IgG + fraction was eluted using 5mL of elution buffer containing 650µL of neutralizing buffer.The 5mL volumes of IgG + and IgG-fractions were then serially concentrated and re-diluted in PBS using ve 30 minute spins at 2100 x g in Amicon Ultra Centrifugal Filter Devices (EMD Millipore).All IgG + fractions were brought to a nal concentration equivalent to the total IgG concentration prior to sera fractionation.All isolated fractions were then used at 1:10 dilution in gelatin veronal buffer.HUVEC Cell Culture Treatments.All protocols were approved by the Yale IRB.
HUVECs were isolated as healthy, de-identi ed tissues from the Dept of Obstetrics and Gynecology at Yale New Haven Hospital as previously and plated onto microtiter wells pre-coated with 1% gelatin. 21EK293 cells were commercially obtained (ATCC).HUVEC were pooled from 3 human donors and cultured in complete EBM media (Lonza) containing bullet supplements (Lonza).Where indicated, HUVEC were pre-treated with Dynasore (80µM), Pitstop2 (30µM) for 30min, or ba lomycin (100nM) for 24h in GVB prior to PRA treatment.For studies involving NH 4 Cl, HUVEC were placed in NH 4 Cl (10µM) dissolved in GVB for 24 hrs in GVB prior to PRA treatment.Following indicated treatments, to assess cellular aggregates, thio avin T (SigmaAldrich, #T1892), was added at nal concentration of 5µM to HUVECs for 30min at 37°C prior to uorescence measurements.Thio avin T uorescence was measured at an excitation wavelength of 349nm and emission wavelength of 454nm using ≥ 6 experimental replicates per treatment group (Molecular Devices, SpectraMax iD3).CD4 + CD45RA-HLA-DR-T cells were isolated from human PBMCs following depletion with anti-human CD45RA Ab (eBioscience, clone H100) and HLA-DR Ab (clone LB3.1, gift from Jack Strominger, Harvard University) using anti-human CD4 conjugated magnetic beads according to manufacturer's speci cations (Invitrogen) and co-cultured with allogeneic HUVEC pretreated with IFN-γ for 48 h and then pre-treated for an additional 6 hours with PRA sera, control sera or complement-inactivated PRA sera in round bottom 96-well tissue culture plates (BD Biosciences) at a T cell:EC ratio of 30:1 in a volume of 200µL of RPMI 1640 containing 10% fetal bovine serum with 2% L-glutamine and 1% penicillin/streptomycin. T cell proliferation assayed by labeling T cells with CFSE at 5µM (Invitrogen) and assessed by ow cytometry at seven days.All samples were acquired using a FACSCalibur ow cytometer (Becton Dickinson) and analyzed using FloJo computer software (TreeStar, Inc., Ashland, OR).
HUVECs were fractionated into soluble and insoluble fractions as previously described. 64For proteinase K treatments, soluble and insoluble C9 fractions were incubated with 12.5 µg/ml proteinase K for 30 min at RT prior to Western blot analysis.
siRNA Transfection of HUVECs.HUVEC were pre-treated with IFN-g for 48 hours prior to siRNA transfection.siRNA targeting ZFYVE21, ATG5, ATG16L1, DMN2 and RNF34 or non-targeting siRNA (target sequence UAA CGA CGC GAC GUA A) were purchased as pooled siRNA (Horizon Discovery) and transfected into HUVEC at ~ 50-70% con uency in 24-well plates (BD Falcon).siRNAs were diluted at 20-40nM concentration in Opti-Mem culture media (Gibco) and mixed at equal volume with RNAiMax transfection reagent (Invitrogen) diluted 1:50 in Opti-Mem for 15 minutes at room temperature as per the manufacturer's speci cations.This mixture was then added to HUVEC cultures at 1:6 ratio for 37°C for 6 hours prior to washing and buffer exchange with EGM2.IFN-g was added at 50ng/mL, and cells were then analyzed by Western blot, luciferase assay, RT-PCR, or T cell functional assays 48 hours later (72 hrs after transfection).
Real Time Quantitative Reverse Transcription-Polymerase Chain Reaction (Quantitative RT-PCR.RNA was isolated from treated HUVEC according to the manufacturer's speci cations (Qiagen) and reverse transcribed (Applied Biosystems, Foster City, CA).Respective cDNA was ampli ed in a CFX Realtime System (Biorad, Hercules, CA) at a volume of 20µL containing dilutions of 1:20 Taqman probe (Applied Biosystems), 1:2 Taqman Gene Expression Master Mix (Applied Biosystems), and 1:10 cDNA in ddH-2O.RT-PCR gene probes were purchased from Applied Biosystems [IL6 (#Hs00985639_m1), SELE (#Hs00950401_m1)].For ampli cation, samples were heated to 50°C for 2 minutes for once cycle, 95°C for 10 minutes for one cycle, and then 40 cycles where samples were heated to 95°C for 15 seconds proceeded by 60°C for 1 minute.
For skin graft experiments, skin from 6-12 week-old male C57/Bl6 mice, ZFYVE21-de cient strains, and littermate controls were treated with MOPC Ab or anti-H-2 b Ab as above, dorsal skin segments were harvested 24 hrs after Ab injection, and implanted on the dorsal anks of female SCID/bg hosts (#CBSCBG-F, Taconic) as full-thickness xenografts.Seven days later, mice were injected i.p. with 5x10 6 female C57/Bl6 splenocytes, and skin grafts were harvested three weeks later for analysis.Epidermal thickness was quanti ed in 3-5 hpfs using morphometry, and mononuclear cell in ltration was estimated by 3 blinded observers.Proteomic Analyses.For proteomic analyses, HUVECs from 3 separate donors were grown to con uence in 15 T175 asks prior to being treated with vehicle (gelatin veronal buffer) or PRA 25% v/v for 45 minutes.Subsequently, cells were harvested and GFP pulldowns were performed according to the manufacturer's speci cations using GFP-Trap agarose beads (#gta, ProteinTech).Proteins were eluted from agarose beads and subjected to trypsin digestion and label-free proteomic analysis by mass spectrometry-liquid chromatography (MS-LC) as previously described. 24ltiplex Laser Bead Assay.Polystyrene beads containing uorescent dyes were coated with capture antibody speci c for a given protein analyte.Color-coded beads were then analyzed using a bead analyzer (Bio-Plex 200) containing a dual-laser system where the uorescent dye within each bead is activated, and a second laser excites the uorescent conjugate (streptavidin-PE) that has been bound to the beads during the assay.The amount of conjugate detected by the analyzer is in direct proportion to the amount of the target analyte which can be quanti ed using a standard curve (Eve Technologies).Statistical Methods.Paired analyses were performed using two-tailed Student's t test and multiple comparisons were performed using a one-way or two-way ANOVA followed by Tukey's pairwise comparison test using Origin computer software.p-values < 0.05 were considered statistically signi cant.Standard deviations are reported throughout the text.HUVECs were treated with PRA at various dilutions, and thio avin uorescence was assessed at 2 hours (c).Thio avin uorescence was assessed at various times following treatment with PRA at 1:4 dilution (d).Punctae containing thio avin and C9 (e) but not collagen (f) were increased in PRA-treated HUVECs by I.F. at 2 hours.PRA sera were fractionated into IgG-and IgG+ fractions and added to HUVECs for 2 hours prior to assessing thio avin uorescence (g).IgG+ fractions of PRA sera were added to HUVECs with and without C9-de cient reference sera and C9 protein (5 mg/mL) for 2 hours prior to assessing thio avin uorescence (h).HUVECs were treated with PRA for various times, and soluble and insoluble lysate fractions were analyzed by Western blot (i).Human C9 (0.125 mg/mL) was incubated at room temperature with proteinase K (12.5 μg/ml mg/mL) for 30 minutes prior to Western blot analysis (j).AA197-270 cells (k) showed high molecular weight (l) punctate (m) aggregates that became SDSinsoluble (n).Experiments repeated ≥3 times using different HUVEC donors.Scale Bar =5mm (b), 15mm (e), 50mm (m).*p<0.05 using one-way ANOVA (i) or two-way ANOVA (a,c-d,g-h) with Tukey's post-hoc correction or Student's t-test (f,j).transfected with control or DNM2 siRNA were treated with PRA, and soluble and insoluble lysates were assessed for C9 (c).Human C9 protein (0.125 mg/mL) was resuspended in buffers of varying pH, and thio avin uorescence was assessed over time at 37°C (d).Human C9 protein at concentrations indicated were incubated at 37°C and thio avin uorescence was assessed over time (e).Human complement proteins at 0.125 mg/mL were incubated at times indicated at pH 4.5 at 37°C (f).HUVECs were transduced with Rab5 WT and Rab5 DN constructs and treated with PRA for 2 hours prior to I.F.analysis (g).HUVECs were exposed to vehicle, ba lomycin (100 nM), or NH 4 Cl (10 mM) for 2 hours, PRA was added, and thio avin uorescence was assessed 4 hours later (h).Experiments repeated ≥3 times using different HUVEC donors.Scale Bar = 30mm (a,g,h), 20mm (d).*p<0.05 using one-way ANOVA (h) or two-way (a-c) ANOVA with Tukey's post-hoc correction or Student's t-test (g).including Hsp70 (e), vimentin (f), and thio avin (g).HUVECs were treated as indicated with PRA and/or pre-treated with chloroquine (CQ) for 30 minutes, and I.F. was performed 2 hours later (h).HUVECs were transfected with siRNA as indicated and analyzed in pulse-chase studies (i,j).HUVECs were transfected with AA197-270 (k) and analyzed by I.F.48 hours after transfection.AA197-270 cells were co-transfected with ATG5 siRNA and analyzed by Western blot 72 hours later (l).Experiments repeated ≥3 times using different HUVEC donors.Scale Bar = 30mm (b) and 20mm (d-h,k) *p<0.05 using one-way ANOVA with Tukey's post-hoc correction (a-b,i-j,l) or Student's t-test (e-g).

Declarations Figures
Figure 5 C9 Aggrephagy Activates NF-kB and Causes EC Activation.HUVECs were stably transduced an NF-kB luciferase reporter 23 and treated with PRA for 6 hours prior to assessing luciferase activity (a).HUVECs were transfected with siRNA against ATG5 (b) or ATG16L (c) and treated with PRA for 4 hours prior to performing qRT-PCR.HUVECs transfected with siRNA against ATG5 (d) or ATG16L (e) were cocultured for 10 days with alloimmune CD4+CD45RO+ T cells, and T cells were harvested for FACS analysis.HEK293 cells were transfected with AA197-270 and analyzed by Western blot as indicated (f-h).
HEK293 cells were pre-treated with or without IFN-g (50ng/mL) for 48 hrs prior to transfection with AA197-270 and qRT-PCR (i).HEK293 cells were treated with IFN-g and transfected with ATG siRNA prior to qRT-PCR (j).Experiments repeated twice using different HUVEC donors (a), 2 HUVEC donors and 3 PBMC donors (d,e), and 2 separate transfectants (i,j).*p<0.05 using one-way ANOVA with Tukey's posthoc correction (b-e,i-j).HUVECs transfected with control siRNA or RN34 siRNA were analyzed in pulse-chase studies (c,d).HUVECs transfected with siRNA against RNF34 were cocultured with alloimmune CD4+CD45RO+ T cells, and T cells were harvested and analyzed by FACS 10 days later (e).Various E2 ubiquitin ligases were coincubated with ubiquitin, E1 enzymes, RNF34 protein, and C9 protein and tested for in in vitro ubiquitinylation of C9 for 4 hours at 37˚C (f).HUVECs were transfected with Ub-HA and GFP-tagged constructs encoding various regions of RNF34 prior to Ub-HA co-IPs (g).HUVECs were co-transfected with Ub-WT and Ub-DN along with control or RNF34 siRNA, treated with PRA, and analyzed by Western blot following C9 co-IP (h).PRA-treated HUVECs were transduced with ZFYVE21-FLAG prior to FLAG pulldowns and Western blot analysis (i).HUVECs were treated with PRA and analyzed by I.F.(j).HUVECs transfected with control siRNA or P62 siRNA were analyzed in pulse-chase studies (k).Experiments repeated ≥3 times using different HUVEC donors.Scale Bar = 20mm (b) and 10mm (j).*p<0.05 using one-way ANOVA with Tukey's post-hoc correction (a,d,e,k) or Student's t-test (j).n=5 per group).Kidney lysates from WT and ZFYVE21 -/-mice were treated as indicated for 24 hours and, total lysates were analyzed by Western blot (e,f, n=3 per group) or by I.F.(g, n=6-8 per group).HUVECs were embedded in collagen-bronectin gels and implanted subcutaneously into anks of SCID/beige mice.Three weeks later, mice were intravenously injected with 200mL PRA, and gels were harvested and analyzed by I.F.24 hours later (h, n=7 per group).HUVECs were transduced with shRNA as indicated prior to gel embedding and implantation into SCID/beige mice.Three weeks later, mice were intravenously injected with 200mL PRA, and gels were harvested and analyzed by I.F.24 hours later (i, n=6-7 per group).Scale Bar = 100mm (c,h,i) and 50mm (g).*p<0.05 using one-way ANOVA (g) or twoway ANOVA with Tukey's post-hoc correction (c) or Student's t-test (h,i).

Figure 1 Protein
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Figure 2 The
Figure 2