key: cord-0324256-rkk2fong authors: Santos, Nuno Brito; Vaz da Silva, Zoé Enderlin; Gomes, Catarina; Reis, Celso A.; Amorim, Maria João title: Complement Decay-Accelerating Factor is a modulator of influenza A virus lung immunopathology date: 2021-02-16 journal: bioRxiv DOI: 10.1101/2021.02.16.431406 sha: 87a02c31cf0bf03016ed214ce6af256a2ea99590 doc_id: 324256 cord_uid: rkk2fong Clearance of viral infections, such as SARS-CoV-2 and influenza A virus (IAV), must be fine-tuned to eliminate the pathogen without causing immunopathology. As such, an aggressive initial innate immune response favors the host in contrast to a detrimental prolonged inflammation. The complement pathway bridges innate and adaptive immune system and contributes to the response by directly clearing pathogens or infected cells, as well as recruiting proinflammatory immune cells and regulating inflammation. However, the impact of modulating complement activation in viral infections is still unclear. In this work, we targeted the complement decay-accelerating factor (DAF/CD55), a surface protein that protects cells from non-specific complement attack, and analyzed its role in IAV infections. We found that DAF modulates IAV infection in vivo, via an interplay with the antigenic viral proteins hemagglutinin (HA) and neuraminidase (NA), in a strain specific manner. Our results reveal that, contrary to what could be expected, DAF potentiates complement activation, increasing the recruitment of neutrophils, monocytes and T cells. We also show that viral NA acts on the heavily sialylated DAF and propose that it exacerbates complement activation, leading to lung immunopathology. Remarkably, this mechanism has no impact on viral loads but rather on the host resilience to infection and may have direct implications in zoonotic influenza transmissions. Author summary Exacerbated complement activation and immune deregulation are at the basis of several pathologies induced by respiratory viruses. Here, we report that complement decay-accelerating factor (DAF), which inhibits complement activation in healthy cells, increases disease severity upon Influenza A virus (IAV) infection. Remarkably, DAF interaction with IAV proteins, hemagglutinin (HA) and neuraminidase (NA), resulted in excessive complement activation and recruitment of innate and adaptive immune cells, without affecting viral loads. Furthermore, we observed that viral NA directly cleaves DAF and promotes complement activation, providing a possible link between IAV-DAF interaction and pathology. Therefore, our results unveil a novel pathway that could modulate disease severity, which may help to understand the increased pathogenicity of zoonotic and pandemic IAV infections. 8 31 (X31). X31 is a reassortant strain of PR8 containing segments 4 and 6 from A/Hong 152 Kong/1/68 (HK68) (44) and for clarity purposes, the X31 strain will be mentioned as PR8-153 HK4,6 throughout this work. WT and Daf -/mice were infected with sublethal and lethal 154 doses of PR8 or PR8-HK4,6, and bodyweight loss and mortality assessed for 11 d.p.i.. Taken together, our results suggest a role for DAF in disease outcome. To further 176 dissect the mechanisms behind such role, we focused on infections with PR8-HK4,6 as it 177 is a well-described laboratorial model, with a virulence resembling circulating strains. Protection conferred by DAF depletion could be explained by a decrease in viral 179 burden or by preventing immunopathology (6) . To distinguish between these two Lastly, to assess if protection of Daf -/mice was linked to a decrease in lung damage 196 and immunopathology, a comprehensive and blind histological analysis of lung tissue was 197 performed at day 3 and 6 p.i. (Table S1 ). At 3 d.p.i., Daf -/mice had a histological score of We have shown that Daf -/mice suffer less severe disease than WT mice upon IAV 208 infection by decreasing tissue damage. Next, we aimed at dissecting the mechanism. DAF 209 being an RCA, we first focused on determining the role of the complement pathway. For The complement pathway is a cascade of reactions that will release cytokines for 228 recruitment and activation of the immune system, and culminating in the formation of a 229 cytolytic pore (C5b-9). Our results showed that knocking out Cd59, inhibitor of C5b-9, does 230 not impact disease outcome in the context of IAV infection (Fig. 2- as heat-inactivated serum did not increase cell death , and confirms that Daf -/-238 mice protection is not dependent on complement cytolytic attack. Given that Daf -/mice have lower complement activation but that protection does Cytokines are also key players in the recruitment and activation of the immune Taken together, these results suggest that lower complement activation leads to a 268 reduced immune response and recruitment of innate immune cells, such as neutrophils 269 and monocytes. This will allow a reduction in tissue damage, ameliorating disease 270 outcome. Interestingly, and counter-intuitively, the decrease in complement activation is a 271 consequence of the absence of a major complement regulator, DAF. We observed that lack of DAF protected mice from infection with PR8-HK4,6, but To better understand the role of HA-DAF interaction in disease outcome, we 308 analyzed complement and immune cell recruitment in the lungs of PR8-HK6 infected mice. Interestingly C3 -/and C3 -/-/ Daf -/mice had similar bodyweight loss when infected with NA is an widely studied sialidase with described roles in mucus penetration, cell egress 393 and recently even in viral entry (50). Remarkably, NA has also been reported to cleave 394 sialic acid residues from exogenous proteins inside the cell (51). As DAF is a highly 395 sialylated protein, we hypothesized that the interaction between DAF and NA resided in Protein glycosylation type and levels may greatly vary between organisms (54). As 408 previous results were obtained using human cell lines, we wanted to confirm that infection The affinity of the IAV HA and NA for respective sialic acid conformation is one of the host 461 species restriction factors (60), avian strains preferring α2,3-linked sialic acids, whereas 462 human strains are able to cleave 2,6-linked sialic acids. In accordance with that, 463 transfection of HEK293T cells with avian-adapted NAs did not impact DAF MW (Fig. 9 -K). Remarkably, transfection with NAs from a H7N9 isolated from a human patient In fact, we found that HA-DAF interplay impacts recruitment of CD4 + and CD8 + T The link we identified via NA, DAF and complement establishes a viral mediated 545 mechanism for maintaining inflammation via increasing the recruitment of immune cells. The model that we propose and that is depicted in Figure 10 has not been reported before. DAF cleavage provides a possible link between DAF-NA interaction and in vivo 574 pathology. Given that our study shows that sialic acids cleaved by DAF are α2,6-linked to 575 O-glycans, this mechanism may have implications in host species jumps, as for example, 576 IAV adapted to birds exhibit preference for α2,3-linked sialic acids. Interestingly, we 577 present evidence that NAs derived from two avian-adapted strains, H5N6 and H7N9, were 578 able to cleave human DAF (Fig. 9-J) . As H7N9 and H5N6 outbreaks provoked severe Unspecific staining was minimized with Fc blocking (rat anti-mouse CD16/CD32, IGC 638 antibody facility, clone 2.4G2). Cells were incubated with primary antibodies (Table S2) Table S2 . Primer sequences are indicated in Table S3 . Early local immune defences in the respiratory 763 tract Innate immunity to influenza virus infection Let's talk about sex in the context of COVID-19 Impact of sex 769 and gender on COVID-19 outcomes in Europe COVID-19 and Sex Differences: Mechanisms and Biomarkers Disease tolerance as a defense strategy The pathology of influenza virus infections High-risk Groups for Influenza Complications Compromised Defenses: Exploitation of Epithelial 787 Responses During Viral-Bacterial Co-Infection of the Respiratory Tract Direct interactions with influenza promote bacterial adherence during respiratory 791 infections Influenza promotes pneumococcal growth during 793 coinfection by providing host sialylated substrates as a nutrient source Extracellular Matrix Proteolysis by MT1-MMP Contributes to Influenza-Related Tissue 797 Damage and Mortality Immunopathology in influenza virus infection: uncoupling the friend from foe The host immune response in respiratory virus 802 infection: balancing virus clearance and immunopathology Systems biological assessment of immunity to mild versus severe COVID-19 infection in 806 humans Impaired 808 type I interferon activity and inflammatory responses in severe COVID-19 patients Multifarious roles of sialic acids in immunity Box 1 as a Biomarker and a Therapeutic Target during Respiratory Virus 1019 Infections. mBio An investigational antiviral drug, DAS181, effectively inhibits replication of zoonotic 1022 influenza A virus subtype H7N9 and protects mice from lethality New insights into upper airway innate immunity The Interaction between Respiratory 1027 Pathogens and Mucus Replication and plaque assay of influenza virus in an 1029 established line of canine kidney cells Rab11-and microtubule-dependent mechanism for cytoplasmic transport of influenza A 1034 virus viral RNA Quantification of complement C5b-9 binding to cells by 1036 flow cytometry Isolation of Mouse Embryo 1038 A DNA transfection 1040 system for generation of influenza A virus from eight plasmids Influenza A virus 1043 PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window Functional neuraminidase inhibitor resistance motifs in avian 1046 influenza A(H5Nx) viruses Escape Adaptive Mutations in the H7N9 Avian Influenza Hemagglutinin Protein Increase 1049 Virus Replication Fitness and Decrease Pandemic Potential Fouchier 1051 RAM. A reverse-genetics system for Influenza A virus using T7 RNA polymerase Efficient generation and growth of influenza virus A/PR/8/34 from eight 1055 cDNA fragments Samples collected at 3 d.p.i. (A, n = 13 and n = 14 for WT and Daf -/-1104 respectively) and 6 d.p.i. (B, n = 18 and n = 19 for WT and Daf -/-respectively). C: 1105 Immunohistochemistry detection of IAV nucleoprotein (NP) in WT or Daf -/-mice 3 d.p.i. with 1106 1000 PFU of PR8HK4,6 (+ healthy; + infected). D: Quantification of infected bronchioli (n = 1107 6 per group). E, F: Histological score of C57BL/6J WT or Daf -/-mice Samples collected at 3 d.p.i. (E, n = 11 and n = 10 for WT Results are expressed as mean±sd. Statistical analysis 1111 detailed in materials and methods 4 -Daf -/-mice have reduced complement activation and recruitment of 1114 innate immune cells C3 -/-or C3 -/-/ Daf -/-mice infected with 500 1116 PFU of A/X-31 (PR8-HK4,6) (Inf n = 10, n = 6 and n = 10 Results are expressed as mean±sd. B: C3a levels 1118 in BALs of C57BL/6J WT (n = 7) or Daf -/-(n = 8) mice 6 d.p.i. with 1000 PFU Results are expressed as mean±sd. C: Cell death of primary lung cells derived 1120 from WT or Daf -/-mice infected or mock-infected with PR8-HK4,6 and treated with serum Results are expressed as mean±sd from 3 replicates from 2 independent experiments Analysis of NK cells (D, n = 6 and n = 3 for WT and Daf -/-respectively), neutrophils 1123 (E, n = 11 and n = 10 for WT and Daf -/-respectively) and monocytes (F, n = 11 and n = 10 1124 for WT and Daf -/-respectively) levels in BALs of WT or Daf -/-mice Results are expressed as mean±sd CD8 + T cells (J) and IFN-γ (K) levels in BALs of WT or (PR8-HK6). (A: Inf n = 16 and n = 18 for WT and Daf -/-respectively). Results are 1136 expressed as mean±sd. C, D: Lung viral titers of C57BL/6J WT or Daf -/-mice infected with 1137 20 PFU of PR8-HK6. Samples collected at 3 d.p.i. (C) and 6 d F: Quantification of 1140 infected bronchioli (n = 5 per group). G, H: Histological score of C57BL/6J WT or Daf -/-1141 mice infected with 20 PFU of PR8HK-6. Samples collected at 3 d.p.i. (E, n = 10 and n = 7 1142 for WT and Daf -/-respectively) and 6 d.p.i. (F, n = 10 and n = 8 for WT and Daf -/-1143 respectively). Results are expressed as mean±sd 6 -Daf -/-mice have reduced complement activation and T cell 1147 recruitment upon PR8-HK6 infection Daf -/-mice infected with 20 PFU 1149 of A/Puerto Rico/8/1934 with segment 6 from A/Hong Kong/1/68 (PR8-HK6) (Inf n = 10 Results are expressed as mean±sd. B: C3a levels in BALs of C57BL/6J WT (n = 10) or = 8) mice 6 d.p.i. with 20 PFU of PR8-HK6. Results are expressed as mean±sd. C Analysis of neutrophils (C) and monocytes (D) levels in BALs of WT Results are expressed as mean±sd = 10) or Daf -/-(n = 8) mice, 6 d.p.i. with 20 PFU PR8-HK6. Results are 1157 expressed as mean±sd. Statistical analysis detailed in materials and methods 7 -DAF interaction with NA modulates immunopathology Bodyweight loss (A) and mortality (B) of C57BL/6J WT or Daf -/-mice infected 1161 with the indicated doses of A/Puerto Rico/8/1934 with segment 4 from A/Hong Kong/1/68 1162 (PR8-HK4). (A: Inf n = 14 and n = 10 Results are 1164 expressed as mean±sd. C, D: Lung viral titers of C57BL/6J WT or Daf -/-mice infected with 1165 100 PFU of PR8-HK4. Samples collected at 3 d.p.i. (C, n = 9 and n = 8 for WT and Daf -/-1166 respectively) and 6 d.p.i. (D, n = 9 per group). Results are expressed as mean±sd. E: 1167 Immunohistochemistry detection of IAV nucleoprotein (NP) in WT or Daf -/-3 d.p.i. with 100 1168 PFU of PR8-HK4 (+ healthy; + infected). F: Quantification of infected bronchioli (n = 5 per 1169 group) Inf n = 16 and n = 18, mock n = 6 and n = 7 for 1171 WT and Daf -/-respectively) and 6 d.p.i. (H, Inf n = 13 and n = 9, mock n = 4 and n = 2 for 1172 WT and Daf -/-respectively). Results are expressed as mean±sd 8 -Daf -/-mice present lower complement activation and neutrophil 1176 recruitment at 3 d.p.i. upon PR8-HK4 infection A: Bodyweight loss of C57BL/6J WT C3 -/-or C3 Results are expressed as mean±sd. B: C3a levels in BALs of C57BL/6J WT 1181 or Daf -/-mice 6 d.p.i. with 100 PFU of PR8-HK4 (n = 9 per group). Results are expressed 1182 as mean±sd. C, D: Analysis of neutrophils (C) and monocytes (D) levels in BALs CD4 + T cells (G) and CD8 + 1185 T cells (H) levels in BALs of WT or Daf -/-mice, 6 d.p.i. with 100 PFU PR8-HK4 (n = 9 per 1186 group). Results are expressed as mean±sd 9 -Influenza A virus neuraminidase cleaves DAF through its sialidase 1190 activity A: Western blot detection of complement decay-accelerating factor (DAF) in A549 1192 cells upon infection with A B: The 1194 proportion of cleaved DAF was measured in each lane as the ratio of low molecular weight 1195 (MW) to total DAF pixel densitometry. C: Western blot detection of DAF in mouse 1196 embryonic fibroblasts (MEFs) derived from C57/BL6 WT or Daf -/-mice upon infection with 1197 PR8 or PR8-HK4,6 at a MOI of 5. D: The proportion of cleaved DAF was measured in 1198 each lane as the ratio of low MW to total DAF pixel densitometry. (B, D: data shown as 1199 mean±sd, from three independent experiments). E: Western blot detection of DAF in 1200 HEK293T cells after transfection with plasmids encoding the eight different PR8 viral 1201 segments. F: Western blot detection of DAF in HEK293T cells transfected with eight 1202 plasmids encoding each of the PR8 segments, including wild-type NA (WT) or the 1203 catalytically-impaired mutant NA-E229A (E229A). G: Western blot detection of DAF S3 -Representative flow cytometry gating strategy Nuno Brito Santos 1,6 , Zoé Enderlin Vaz da Silva 1,6 , Catarina Gomes 2,3 , Celso A.