key: cord-0710466-w3umjaho
authors: Posch, Wilfried; Vosper, Jonathan; Noureen, Asma; Zaderer, Viktoria; Witting, Christina; Bertacchi, Giulia; Gstir, Ronald; Filipek, Przemyslaw A.; Bonn, Günther K.; Huber, Lukas A.; Bellmann-Weiler, Rosa; Lass-Flörl, Cornelia; Wilflingseder, Doris
title: C5aR inhibition of non-immune cells suppresses inflammation and maintains epithelial integrity in SARS-CoV-2-infected primary human airway epithelia
date: 2021-04-20
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2021.03.038
sha: 422e2606ffd2c1423ffaf5df7a2d931f7b83a2ca
doc_id: 710466
cord_uid: w3umjaho

Background Excessive inflammation triggered by a hitherto undescribed mechanism is a hallmark of severe SARS-CoV-2 infections and is associated with enhanced pathogenicity and mortality. Objective Complement hyper activation promotes lung injury and was observed in patients suffering from MERS-CoV, SARS-CoV-1 and SARS-CoV-2 infections. Therefore, we investigated the very first interactions of primary human airway epithelial cells upon exposure to SARS-CoV-2 in terms of complement C3-mediated effects. Methods For this, we used highly differentiated primary human 3D tissue models infected with SARS-CoV-2 patient isolates. Upon infection, viral load, viral infectivity, intracellular complement activation, inflammatory mechanisms and tissue destruction were analyzed by real-time RT-PCR, high content screening, plaque assays, luminex analyses and TEER measurements. Results Here we show that primary normal human bronchial and small airway epithelial cells respond to SARS-CoV-2 infection by an inflated local C3 mobilization. SARS-CoV-2 infection resulted in exaggerated intracellular complement activation and destruction of the epithelial integrity in monolayer cultures of primary human airway cells and highly differentiated, pseudostratified, mucus-producing, ciliated respiratory tissue models. SARS-CoV-2-infected 3D cultures secreted significantly higher levels of C3a and the pro-inflammatory cytokines IL-6, MCP-1, IL-1α and RANTES. Conclusion Crucially, we illustrate here for the first time, that targeting the anaphylotoxin receptors C3aR and C5aR in non-immune respiratory cells can prevent intrinsic lung inflammation and tissue damage. This opens up the exciting possibility in the treatment of COVID-19.

Early events occurring directly after SARS-CoV-2 transmission to respiratory tissues can 127 influence the outcome in the context of disease severity -in some patients, infection 128 with COVID-19 results in excessive activation of the immune response at 129 epithelial/immune barriers and the generation of a pro-inflammatory milieu (1, 2). The 130 development of a cytokine storm and acute lung injury, causing acute respiratory 131 distress syndrome (ARDS), are potential undesirable consequences of the disease. 132 ARDS accompanied by systemic coagulopathy are critical aspects of morbidity and 133 mortality in 4) . These overshooting immune responses triggered by 134 incoming viruses result in extensive tissue destruction during severe cases, resulting in 135 tissue injury and multi-organ failure (5, 6). Complement may be among the factors 136 responsible for the immune over activation, since complement deposition and high 137 anaphylatoxin serum levels have been reported in patients with severe/critical disease 138 (7). As recently shown in transcriptome analyses of bronchoalveolar lavages of patients, 139

the complement system was among the most significantly upregulated intracellular 140 pathways following SARS-CoV-2 infection (8). In addition, the transcriptomes of primary 141 normal human bronchial epithelial (NHBE) cells infected in vitro with SARS-CoV-2 142 revealed an enriched complement signature (8). Very recently, Ramlall et al. identified in 143 addition to type I IFN and IL-6-dependent inflammatory responses, a robust engagement 144 of complement and coagulation pathways following SARS-CoV-2 infection (9). Here, we 145 further analyzed local complement activation at the protein level in a highly differentiated 146 primary human 3D respiratory model upon in vitro infection using a SARS-CoV-2-clinical 147 specimen (10, 11) . In such cultures, complete destruction of epithelial integrity was 148 observed upon viral infection and was associated with exacerbated C3 production, MAC 149 formation as well as detachment of cells and release of virus into the supernatants. Virus 150 infection was not only detected in outermost layers but virus particles were seen to 151 penetrate deeply into the pseudostratified epithelium as well, with a concomitant C3 152 activation or probably also deposition on dead or damaged cells being observed in these 153 layers. Besides C3 activation, significantly higher levels of C3a desArg, tissue damage 154 and pro-inflammatory cytokines, such as IL-6, MCP-1, IL-1α and RANTES, were 155 detected in SARS-CoV-2-infected tissue models. While C3aR blocking alleviated viral 156 infection and reduced the inflammatory response in airways, C5aR antagonism 157 J o u r n a l P r e -p r o o f completely restored tissue integrity, significantly down modulated local complement 158 production and associated therewith returned C3a and pro-inflammatory levels back to 159 normal. C5aR antagonism was also shown to correlate with low viral loads. Our results 160 point towards a novel therapeutic intervention strategy whereby complement inhibitors 161 are administered to high risk patients during an early stage of disease progression in 162 order to prevent an excessive inflammatory response and its associated pathological 163 liquid interface (ALI) as described (10, 11). Briefly, cells were cultured in a T75 flask for 177 2-4 days until they reached 80% confluence. The cells were trypsinized and seeded 178 onto GrowDexT (UPM)-coated 0.33 cm 2 porous (0.4 µm) polyester membrane inserts 179 with a seeding density of 1 × 10 5 cells per Transwell (Costar, Corning, New York, NY, 180 USA). The cells were grown to near confluence in submerged culture for 2-3 days in 181 specific epithelial cell growth medium according to the manufacturer´s instructions. 182

Cultures were maintained in a humidified atmosphere with 5% CO2 at 37 °C and then 183 transferred to air-liquid interphase (ALI) culture. The epithelium was expanded and 184 differentiated using airway media from Stemcell The number of days in development 185 was designated relative to initiation of ALI culture, corresponding to day 0. In addition, 186 for real-time RT-PCR analyses of UI and SARS-CoV-2-infected 3D tissues and 187

supernatants, apical-out lung organoids were generated. Briefly, cryopreserved lung 188 organoids derived from human normal lung tissue (left superior lobe) were acquired from 189 J o u r n a l P r e -p r o o f Hubrecht Organoid Technology (HUB), Netherlands (HUB-07-A2-051). These organoids 190

were cultured according to the previously described protocol (12, 13). As the Cov2 receptor, ACE2 is predominantly expressed on the apical side of the airway 192 epithelium (14), apical out lung organoids were generated as recently described to 193 expose the apical side of the lung organoids to facilitate viral infection. Apical out 194 organoids were further differentiated into multi-ciliated cells by using differentiation 195 medium as previously reported by Zhou et al. (15). 196 Vero/TMPRSS2: VeroE6/TMPRSS2 is an engineered VeroE6 cell line expressing high 197 levels of TMPRSS2 and highly susceptible to SARS-CoV-2 infection and is used to 198 expand SARS-CoV-2 viruses from repositories as well as patient isolates. The cell line 199 was obtained via the CFAR (NIBSC) and is described in Matsuyama et al.(16) . Technologies, Santa Clara, CA, USA) were used. To investigate TCC formation the anti-227

C5b-9 (aE11 clone, Abcam, Cambridge, UK) antibody recognizing a neoepitope 228 exposed on polymerized C9 when incorporated in the human TCC was used. 229

Neoantigens were shown to be expressed in both MAC and fluid phase (SC5-9) 230 complex (17). Intracellular SARS-CoV-2 was detected using Alexa594-labeled SARS-231

CoV-2 antibodies against S1 and N (both Sino Biological, Beijing, China). The Alexa594-232 labeling kit was purchased from Abcam, Cambridge, UK. Cells and 3D cultures were 233 thoroughly washed following the staining procedure using the permaebilization buffer, 234 sterile-filtered D-PBS and finally mounted in Mowiol. To study these complex models 235 using primary cell cultured in 3D and to generate detailed phenotypic fingerprints for 236 deeper biological insights in a high throughput manner, the Operetta CLS System 237 

In order to monitor SARS-CoV-2 infection in primary normal human bronchial epithelial 312 (NHBE) cells, monolayers and fully differentiated, pseudostratified epithelia were 313 infected using different clinical isolates derived from SARS-CoV-2 patients (10, 11). The 314 clinical specimens were anonymized before use and the study has been approved by background IC C3 levels (Fig. 1A) . Expression of complement components C3 and C5 332 were analyzed by real-time RT-PCR in UI and SARS-CoV-2-infected HAE cultures (Fig.  333 1B). Consistent with the microscopic data, expression of C3 was significantly higher in 334 SARS-CoV-2-infected cultures, while no differences in C5 were observed (Fig. 1B) . 335

These analyses demonstrate that intracellular C3 is massively induced at protein and 336 gene expression levels in SARS-CoV-2-infected, primary HAE cultures. 337

Next, we characterized the distribution of intracellular C3 in infected HAE monolayer 340 cultures. We found that IC C3 was produced by infected cells and in particular also their 341 neighboring cells ( Fig. 1C ; green). High content image analyses revealed that viral 342 particles (Fig. 1C , red, arrows) were transmitted in C3-containing, activated cells via 343 membrane nanotube formation to neighboring cells (Fig. 1C) , a process also used by 344

HIV-1 to exacerbate infection (21). To quantify nanotubes and C3/SARS-CoV-2-345 containing nanotubes in UI and SARS-CoV-2-infected cells, digital phase contrast (DPC) 346 microscopy on a total number of 5000 cells and concomitant analyses using the 347

Harmony software (Perkin Elmer) were performed (Fig. 1C, bar graphs) . This revealed a 348 significantly higher number of nanotubes and C3/SARS-CoV-2-nanotubes in SARS-349

CoV-2-vs. UI cells (Fig. 1C, bar graphs) . This indicates that SARS-CoV-2 is shuttled via 350 C3-containing nanotubes to neighboring cells. 351

To monitor, if this extensive IC C3 mobilization also occurs within multi-layered human 353 airway epithelia, six-month-old, fully differentiated, ciliated and mucus-producing 354 epithelial tissue models or multi-ciliated apical out lung organoids were infected at a MOI Quantitation of cell numbers in the respiratory tissue models revealed a significant loss 372 of cells in SARS-CoV-2 vs. UI on 2 dpI (Fig. 2D, 1 st chart, table) . 42.3% vs. 2.6% of the 373 analyzed cells were positive for C3 in SARS-CoV-2-infected vs. UI cultures (Fig. 2D, 2 nd  374 chart, table). 27% were positive for SARS-CoV-2 in infected cultures, while 3.3% 375 showed background fluorescence staining in UI (Fig. 2D, 3 rd chart) . About half of the 376 SARS-CoV-2-positive cells (15%) were also stained positive for C3 in infected cultures 377 (Fig. 2D, 4 th chart, table) . A total number of more than 5000 cells was analyzed for each 378 condition. 3D and xyz-analyses of the two signals revealed not only a superficial 379 localization of SARS-CoV-2 and C3, but that the infection and intracellular complement 380 mobilization penetrated deep into the tissue layers as illustrated in Figure 2C (upper 381 right image). In contrast only background signals for SARS-CoV-2 and C3 were 382 apparent in the UI control ( Fig. 2C and 2D) and these epithelia displayed a higher 383 degree of structural integrity (Fig. 2C, lower right image, UI) . 384

Infection of respiratory tissue models with clinical specimens of SARS-CoV-2 and the 387 concomitant considerable IC C3 mobilization resulted in substantial disruption of the 388 epithelial integrity as assessed by measuring TEER (Fig. 3A; left) , release of SARS-389

CoV-2 into the medium (Fig. 3A, middle) , C3a desArg secretion (Fig. 3A, right) and an anti-C5b-9 antibody recognizing a neoepitope exposed on polymerized C9 when 412 incorporated in the human terminal complement complex (TCC) (17). The C5b-9 Ab co-413 localized in many SARS-CoV-2-infected cells (Fig. 3C, 

CoV-2), while in UI cultures only background signals were obtained using these Abs 415 ( Fig. 3C and 3D , UI). BF images (Fig. 3C, left) , staining of acetylated tubulin recognizing 416 ciliary structures (Fig. 3C, right, pink) and C5b-9-single positive cells (Fig. 3C, right,  417 green arrows) additionally illustrated rounding-up of cells and loss of structured cilia in 418 infected cultures. All experiments were independently repeated at least three times and 419 quantitation was performed in four different fields on at least 2000 cells (Fig. 3D) . These 420 data revealed that local complement activation in pseudostratified HAE cells correlated 421 with the disruption of the epithelial integrity following SARS-CoV-2 infection. 422

Next, we used our highly differentiated HAE model to investigate possible treatment C5aR1 were only expressed on 3D models (Fig. E5) . After verifying the presence of the 436 various anaphylatoxin receptors in our system, cells were infected for 2 days and then 437 stained for SARS-CoV-2 S1/N (red), intracellular C3 (green) and nuclei (blue) for 438 microscopic analyses (Fig. 4A) . As demostrated before, SARS-CoV-2-infected HAE 439 tissue models showed a high level of infection ( Fig. 4A ; SARS-CoV-2, red) and 440 intracellular C3 induction ( Fig. 4A ; SARS-CoV-2, green) as well as disruption of the 441 epithelium compared to the uninfected cultures ( Fig. 4A; UI) . Basolateral pre-incubation 442 of epithelia with C3aR antagonist resulted in reduction of viral loads as well as of C3-443 positive cells and preserved a more intact morphology of the human airway epithelium 444 ( Fig. 4A ; C3aR-Inh/SARS-CoV-2). In addition to imaging analyses, basolateral 445 supernatants of pre-treated and infected HAE cultures were harvested on 2 dpI to 446 perform a plaque assay using TMPRSS2-expressing VeroE6 (VeroE6/TMPRSS2) cells 447

for determining viral infectivity from these cultures (Fig. 4B) . Further, the virus 448 concentration was analyzed by quantitative real-time RT-PCR (Fig.4C) . Both, plaque 449 assay as well as real-time RT-PCR, revealed a slight decrease in PFUs or SARS-CoV-2 450 copy numbers after treatment with C3aR antagonist (C3aR Inh/SARS-CoV-2), but 451 compared to non-treated, infected cells (SARS-CoV-2) this reduction was statistically not 452 significant ( Fig.4B and 4C) .The most convincing results in terms of antagonizing SARS-453

CoV-2-mediated intracellular C3 production were achieved using a C5aR antagonist 454 2). Here, we illustrated that SARS-CoV-2 induced excessive local complement activation 469 can be decreased by antagonizing C3aR and is completely reversed by blocking C5aR. 470

To determine if C3aR/C5aR antagonism is potentially an effective post exposure 472 preventative mechanism, we infected differentiated HAE tissue models with SARS-CoV-473 2 clinical isolates and added the C3aR and C5aR antagonists following a 6h delay. Cells 474 were again analyzed after 2 days. As depicted in Figure 5A , SARS-CoV-2 efficiently 475 infected the culture models and caused tissue disruption ( Fig. 5A ; SARS-CoV-2), while 476 uninfected control cells displayed an intact tissue morphology as illustrated by a uniform 477 phalloidin staining without disruptions (Fig 5A; UI) . Addition of C3aR inhibitor ( Fig. 5A ; 478

C3aR Inh/SARS-CoV-2) led to decreased viral load but the reduction in local C3 479 production was not as obvious as that observed upon pre-administration of antagonist. (1 dpi) and all cells were detached in SARS-CoV-2 and surprisingly also in C3aR 487

antagonist-/SARS-CoV-2-exposed cells on 2 dpi. In contrast, antagonizing C5aR 488 protected from the cells from CPE after SARS-CoV-2 infection and they looked similar to 489 uninfected controls (Fig. 5B) and C5aR antagonist-treated control cells (not shown). 490

Hence, we showed that inhibiting C5aR on fully differentiated HAE cells prevented 491 subsequent productive SARS-CoV-2 infection in highly susceptible cells. 492

Since C5aR antagonism appeared to be the most promising strategy for protection 495 against SARS-CoV2 infection due to the down-regulation of local complement activation, 496

we performed more detailed analyses in such cultures in order to account for the 497 absence of infection observed in susceptible indicator cells. We found that TEER values 498 could be restored in SARS-CoV-2-infected HAE cells treated with C5aR inhibitor (Fig.  499 6A). While tissue models were strongly disrupted upon SARS-CoV-2 infection as 500 indicated by a significantly lower TEER value compared to UI ( Fig. 6A ; UI), a highly 501 significant improvement in epithelial integrity was observed in C5aR-treated as well as 502

C5aR and SARS-CoV-2-exposed cultures ( Fig. 6A ; C5aR Inh and C5aR Inh/SARS-CoV-503 2). Also C3-containing nanotube levels were reduced to zero in HAE cultures pre-treated 504 with C5aR antagonist prior to SARS-CoV-2 infection comparable to UI and C5aR Inh/UI 505 and in contrast to SARS-CoV-2-infected cultures (Fig. 6B) . To further characterize, if 506

C5a alone can mediate damage of HAE cultures, cells were treated with 1 µg/ml C5a 507 (C5a) for 24h, but not infected with SARS-CoV-2 (Fig. 6C) . These experiments revealed 508

that TEER values of HAE tissues significantly dropped upon treatment with C5a 509 compared to untreated control cells (Fig. 6B , UI) and microscopic analyses also showed 510 disruption of tissue integrity of these cultures (Fig. E6) . This pointed towards a role of 511 local complement activation in amplifying viral load and worsening tissue injury in 512 COVID-19 patients. 513

Finally, we analyzed the pro-inflammatory signature of HAE tissue cultures during 516 SARS-CoV-2 infection in the presence and absence of C5aR antagonist. As controls, 517 tissue models were left untreated (UI) or treated with C5aR inhibitor only (C5aR Inh/UI) 518 (Fig. 6D) . As assessed by luminex technology and median fluorescence intensities, 519 SARS-CoV-2 induced significantly higher levels of IL-6, MCP-1, IL-1α and RANTES 520 compared to uninfected control cultures ( Fig. 6D ; SARS-CoV-2 vs. UI). IP10 levels 521 fluctuated in various experiments following SARS-CoV-2 infection with an overall modest 522 decrease observed in C5aR Inh-pretreated cultures (Fig. 6D) . Induction of all pro-523 inflammatory cytokines by SARS-CoV-2 infection were reversed by antagonizing C5aR 524 and such pre-treated cultures showed levels similar to uninfected cultures ( Fig. 6D ; 525

C5aR Inh/SARS-Cov-2 and UI, C5aR Inh/UI). Blocking C5aR may therefore be a CD46 and changes in injury markers on HAE cells, such as decreased E-cadherin, and 567 increased cleaved poly-ADP ribose polymerase (28). Further, in patients with idiopathic 568 lung injury, increased soluble C5b-9 levels were analyzed in lung lavage fluid (24). We, 569 too, found a significantly enhanced induction of TCC formation in SARS-CoV-2-infected 570 tissue models compared to UI controls, suggesting cell damage also occurs by formation 571 of MAC membrane channels in infected cells. Additionally, inhibition of C3aR or C5aR in 572 bleomycin-injured mice with fibrotic lungs also led to downregulation of local 573 complement production as well as disease progression (24, 30) . Here we demonstrated, 574 that local complement production and progression of SARS-CoV-2 infection could be 575 arrested in primary human airway epithelia by C5aR antagonist. This was even the case, 576

if the C5aR blocker was administered several hours post SARS-CoV-2 infection. 577

Besides decreasing locally produced complement, blocking C5aR resulted in the 578 restoration of transepithelial electrical resistance, a marker of epithelial integrity. 579

Furthermore, we observed a normalization of pro-inflammatory cytokine levels back to 580 those of healthy untreated HAE tissue models or cultures incubated with the antagonists 581 alone. In COVID-19-positive individuals, the levels of 10 circulating cytokines (IL-1α, IL-582 1RA, IL-6, IL-10, GM-CSF, IP-10, MCP-1, RANTES, TSLP and TNFα) were reported to 583 being significantly elevated and different expression profiles were associated with 584 different disease severity (31). Inflammation is among the first coordinated lines of 585 defense following tissue damage by either infection or injury, but excessive immune 586 responses convert the protective mechanism into a harmful one as observed in COVID-587 19 patients (32). Besides the aforementioned biomarkers for disease severity, the 588 anaphylatoxins C3a and C5a are secreted by airway epithelial cells or C3 in mucus 589 might also serve as valuable indicators for disease severity or progression. As illustrated 590 in our highly differentiated, pseudostratified HAE, extremely high levels of C3a desArg 591 are secreted from the airway epithelium upon interaction with SARS-CoV-2. Both, C3a 592 desArg and C5a, comprise important effector molecules attracting, activating and 593 regulating components of innate and adaptive immunity (33). C5a was demonstrated to 594 induce expression of IL-1β and IL-8 in mononuclear cells and augments the release of 595 IL-6 and TNF-α (34). Further, several studies illustrated that C5a and C5aR may be 596 responsible for inflammation in the lung, i.e. during IgG-mediated inflammation or during 597 ARDS (35, 36), and blocking C5aR attenuated IgG-complex-mediated inflammation. 598

Although we were not able to detect C5a in basolateral supernatants of SARS-CoV-2-599 infected epithelia this observation might be attributable to its previously described 600 intracellular engagement and capacity to activate C5aR1 and induction of inflammasome 601 activity (37). Though this ´complement-metabolism-inflammasome´ axis (rev. in (38)) 602 was first demonstrated in immune cells such as CD4 + T cells, it is also conceivable that 603 such pathway is also activated in the airway epithelium, showing expression of C5aR1 at 604 the protein and the mRNA level (39, 40) . Herein, all anaphylatoxin receptors (C3aR, 605 C5aR1, C5aR2) were shown to be expressed on 3D ALI tissue models. As recently 606 reviewed in Kulkarni et al. (41) , all components of lectin and alternative complement 607 pathways are expressed in or produced by human airway epithelial cells, making it likely 608 that upon SARS-CoV-2 infection, exaceberating or chronic amplification of local 609 synthesis of these complement proteins putatively propagates epithelial injury. 610 Furthermore, we found a significantly higher expression of pro-inflammatory cytokines 611 following stimulation of HAE tissue models with SARS-CoV-2, which is in line with C5a 612 effector functions. Importantly, the up-regulation of pro-inflammatory cytokines IL-6, 613

MCP-1, IL-1α or RANTES was efficiently blocked by C5aR antagonism. Besides C5a, 614

C3a desArg may bind C5aR2 at a site distinct from the C5a binding site (42-44), but this 615 issue was controversially discussed over years (45) as well as the role of C5aR2 as 616 either immune-activating or -dampening (rev.in (46)). Since we found a highly elevated 617 induction of C3a desArg upon SARS-CoV-2 infection and abundant expression of 618

C5aR2, some of the observed effects may be due to interaction of these complement 619 components. More detailed analyses of these interactions during SARS-CoV-2 infection 620 are necessary, but would have exceeded the scope of this study. Yet, these data 621 provide a promising base for future translational studies of modifying C3a desArg or 622

C5a/C5a desArg and C5aR1 or C5aR2 interactions. 623

In summary, we propose that the application of complement inhibitors represents a 624 promising approach in SARS-CoV-2 disease management. Complement inhibitors (e.g. 625 AMY-101, eculizumab, or its variant ravulizumab) are currently in advanced stages of 626 SARS-CoV-2 clinical trials and can be administed to the lungs through nebulizers (47-627 49). Beside anaphylatoxin targeting, another yet unexplored option is the targeting of 628 regulators of complement activation such as CD46 or CD55 in the lungs of COVID-19 629 patients in order to reduce inflammation (48, 50). As illustrated herein and also reviewed 630 in Jodele and Koehl, complement may serve as a powerful new therapeutic target for 631 COVID-19 treatment, since severely ill COVID-19 patients with atypical ARDS showed 632 an extensive complement activation both systemically and within the lungs. (7, 8, (51) (52) (53) . 633

Crucially, we show here for the first time, that targeting the anaphylotoxin receptors 634 in Vero/TMPRSS2 Cells, were quantified using real-time RT-PCR with SARS-CoV-2 specific primer/probe pairs for the nucleocapsid N1 and N2 regions. A representative PCR quantification from 3 different isolates using the N2 primer/probe set is illustrated on the left with the standard curve shown on the right. Tables below depict log starting quantities and TCID50. Using these isolates, a TCID50 on Vero/TMPRSS2 cells was performed and copy numbers calculated to infect 50% of the tissue cultures. an MOI of 0.1.1. 2 DPI cells were fixed and stained for Höchst (blue), SARS-CoV-2 S1/N (red), acetylated tubulin (green) and mucus producing cells (orange) and then analysed by confocal microscopy. On the left a representative overview of HAE epithelial cells stained with höchst for nuclei (blue), acetylated tubulin (green), mucus-producing cells (orange) and virus (red) is depicted, right the höchst channel was switched off in order to more clearly show viral distribution over the epithelium tissue models. In contrast, all three receptors (C3aR, orange; C5aR1, green; C5aR2, pink) were illustrated by confocal microscopy on 3D tissue models. Experiments were repeated thrice independently. 

COVID-19: Review on latest available drugs and therapies against SARS-CoV-660 2. Coagulation and inflammation cross-talking

A Novel Coronavirus from 662 Patients with Pneumonia in China

Abnormal coagulation parameters are associated with 664 poor prognosis in patients with novel coronavirus pneumonia

Clinical Characteristics of 138 667 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan

Clinical and immunologic 670 features in severe and moderate Coronavirus Disease

Clinical features of patients infected 672 with 2019 novel coronavirus in Wuhan

Tackling COVID-19 infection through complement-targeted 674 immunotherapy

SARS-CoV2 drives 676 JAK1/2-dependent local and systemic complement hyper-activation. Res Sq. 2020

Immune 678 complement and coagulation dysfunction in adverse outcomes of SARS-CoV-2 infection

Upside-Down in Cellulose for Improved Culturing and Imaging of Respiratory Challenges within 682 a Human 3D Model

Fast-track 684 development of an in vitro 3D lung/immune cell model to study Aspergillus infections

Long-term expanding human airway organoids for disease modeling

Challenges in Establishing Pure Lung Cancer Organoids Limit Their Utility for Personalized 690 Medicine

Nasal Epithelial Cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals 693 similar viral kinetics and host transcriptional profiles

Differentiated human airway 695 organoids to assess infectivity of emerging influenza virus

Enhanced 698 isolation of SARS-CoV-2 by TMPRSS2-expressing cells

Monoclonal antibodies recognizing a 701 neoantigen of poly(C9) detect the human terminal complement complex in tissue and plasma

Morphogenesis and cytopathic 704 effect of SARS-CoV-2 infection in human airway epithelial cells

Isolation and characterization of current human coronavirus strains in primary human epithelial 708 cell cultures reveal differences in target cell tropism

SARS-CoV replication and pathogenesis in 710 an in vitro model of the human conducting airway epithelium

Membrane nanotubes physically connect T cells over long distances presenting a novel route for 713 HIV-1 transmission

Syncytia 715 formation by SARS-CoV-2-infected cells

Persistence of viral 717 RNA, pneumocyte syncytia and thrombosis are hallmarks of advanced COVID-19 pathology

720 Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary 721 fibrosis

Association of 723 COVID-19 inflammation with activation of the C5a-C5aR1 axis

Complement and viral pathogenesis

Clinical course and risk factors for 727 mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Crosstalk between TGF-beta 1 and complement activation augments epithelial injury in 731 pulmonary fibrosis

Complement Activation Contributes to Severe Acute Respiratory Syndrome Coronavirus 734

Parallel activities and interactions between antimicrobial peptides and 736 complement in host defense at the airway epithelial surface

Plasma IP-10 and MCP-3 levels are 738 highly associated with disease severity and predict the progression of COVID-19

Targeting the "cytokine 741 storm" for therapeutic benefit

Novel insights into the expression pattern of 743 anaphylatoxin receptors in mice and men

Recombinant C5a stimulates transcription rather 745 than translation of interleukin-1 (IL-1) and tumor necrosis factor: translational signal provided by 746 lipopolysaccharide or IL-1 itself

Activation of the complement 751 system in the adult respiratory distress syndrome

T helper 1 753 immunity requires complement-driven NLRP3 inflammasome activity in CD4(+) T cells. 754 Science

Intracellular complement -the complosome -in immune 756 cell regulation

RNA-Seq quantification of the human small airway epithelium transcriptome

Expression 761 of the complement anaphylatoxin C3a and C5a receptors on bronchial epithelial and smooth 762 muscle cells in models of sepsis and asthma

The complement system in the 764 airway epithelium: An overlooked host defense mechanism and therapeutic target?

The 767 chemoattractant receptor-like protein C5L2 binds the C3a des-Arg77/acylation-stimulating 768 protein

C5L2 is a 770 functional receptor for acylation-stimulating protein

C3adesArg/acylation stimulating protein (ASP) is highly bioactive: a critical evaluation of C5L2 773 binding and 3T3-L1 adipocyte activation

The Controversial C5a Receptor C5aR2: Its Role in Health 775 and Disease

The Complement Receptor C5aR2: A 777 Powerful Modulator of Innate and Adaptive Immunity

Role of 780 complement in modulation of TH2 response in prostate cancer cells

The first case of COVID-19 treated with the complement C3 inhibitor AMY-101

Eculizumab 786 treatment in patients with COVID-19: preliminary results from real life ASL Napoli 2 Nord 787 experience

Clinical promise of next-generation complement 789 therapeutics

Highly pathogenic coronavirus N 791 protein aggravates lung injury by MASP-2-mediated complement over-activation

Susceptible Patients with Severe SARS-CoV-2 Infection

5000 cells were counted for their presence of nanotubes (DPC), C3 (green) and 898

SARS-CoV-2 (red) under the indicated conditions. (C) Addition of recombinant C5a 899 significantly reduced the epithelial integrity as analyzed by TEER. Experiments were 900 performed thrice and statistical significance evalulated using unpaired Student´s t test

Supernatants from SARS-CoV-2 infected pseudostratified epithelia subsequently 902 treated with C5aR antagonists were harvested 2 dpI and cytokine profiles determined 903 using Luminex technology. Levels of cytokines are expressed in median fluorescence 904 intensities. Two independent experiments in duplicates were performed. Statistical 905 significance was evalulated using One-way ANOVA

infected tissues plus/minus inhibitors. The experiment was repeated thrice and One-way 866

Inh and C5aR Inh treated SARS-CoV-2 infected pseudostratified epithelia. 2 dpI 868 basolateral supernatants harvested and C3a levels determined using a BD Biosciences 869OptEIA Human C3a ELISA kit. C3a levels in ng/ml were determined for each 870 experimental condition and plotted on a bar graph. were infected by apical addition of SARS-CoV-2 (MOI 0.1-1) and following 6 hours were 891 then supplemented basolaterally with either 1µM C3aR (SB 290157) inhibitor or 1µM 892C5aR (mix of W-54011 and DF2593, 1:1) inhibitor. TEER measurement was performed 893 on d2pI for each experimental condition and the results were plotted on a bar graph. 894Experiments were performed thrice and statistical significance evalulated using One-way 895 ANOVA and Dunnett´s post-test. (B) C3-containing nanotubes were reduced to zero in 896C5aR Inhibitor-treated cultures also upon SARS-CoV-2 infection (C5aR Inh/SARS-CoV-897