Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.


Introduction
The normal cellular isoform of prion protein, designated PrP C , is a membrane glycoprotein tethered to the outer cell membrane via a glycosylphosphatidylinositol (GPI) anchor moiety and expressed most abundantly in the brain, particularly by neurons, and to a lesser extent in non-neuronal tissues such as heart, lung, and spleen [1][2][3]. Conformational conversion of PrP C into the amyloidogenic isoform, PrP Sc , in the brain is a key pathogenic event in prion diseases, a group of neurodegenerative disorders, which include Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals [1]. Mice devoid of PrP C (Prnp 0/0 ) have been shown to be vulnerable to ischemic injury in the brain, heart and kidney, with enhanced apoptosis in the injured tissues [4][5][6][7][8], suggesting that PrP C could have a cellular protective function in these tissues. It has also been previously shown that lung epithelial cells in Prnp 0/0 mice were highly vulnerable to apoptotic cell death after infection with influenza A viruses (IAVs), suggesting that PrP C could have a protective role for lung epithelial cells [3,9].
IAVs are enveloped, negative sense, single-stranded RNA viruses causing seasonal epidemic outbreaks of the acute upper respiratory disease influenza [10]. Severe influenza infections are often lethal, causing high morbidity and mortality in infected people, particularly in the young and elderly and those with underlying chronic diseases in lung or cardiovascular systems [10]. Currently available main anti-influenza agents are viral protein-targeting agents such as neuraminidase inhibitors. However, emergence of influenza viruses that are resistant to these agents has been reported and may become a serious problem if these drug-resistant IAVs spread in human populations [11][12][13]. It has also been shown that highly pathogenic avian IAVs of the H5N1 and H7N9 subtypes have the potential to infect humans, causing high morbidity and mortality in infected individuals, raising another concern about the pandemic potential of these avian viruses in human populations [14][15][16]. Therefore, identification of new target molecules for anti-influenza agents is urgently awaited.
In this study, we show a novel function of PrP C to induce anti-inflammatory M2 macrophage polarization through activation of Src family kinases (SFKs), and that anti-PrP monoclonal antibodies (mAbs) stimulated the novel activity of PrP C inducing M2 macrophages and thereby protected mice from lethal infection with IAVs. These results suggest that PrP C could be a novel therapeutic target for influenza infection by promoting M2 macrophage polarization.

38-2 anti-PrP mAb provides protection against lethal IAV infection in mice
To examine if PrP C could be stimulated by anti-PrP Abs and provide beneficial effects in IAV infection, various doses of 38-2 anti-PrP IgG mAb, which recognizes residues 41-45 of mouse PrP C [17], was intraperitoneally administered to C57BL/6 wild-type (WT) mice 1 day before intranasal infection with influenza virus strain A/Puerto Rico/8/34 (H1N1) (hereafter referred to as IAV/PR8) with 200 infectious units (IFU). The 38-2 hybridoma was established by fusion of myeloma cells with splenocytes from Prnp 0/0 mice immunized with recombinant mouse PrP [17]. Approximately 90% of control mice, which received vehicle buffer alone, died by 14 days post infection (dpi) (Fig 1A and S1 Fig). However, 38-2 mAb markedly reduced the mortality of infected mice in a dose-dependent manner (Fig 1A and S1 Fig). Weight loss was also mitigated in 38-2 mAb-treated, IAV/PR8-infected mice compared to control mice ( Fig  1A and S1 Fig). These results indicate that targeting of PrP C with 38-2 mAb could confer protection against lethal IAV infection in mice. We then investigated the Fab fragment of 38-2 mAb for its protective activity against lethal IAV infection, by intraperitoneally injecting the 38-2 Fab fragment (1 mg/mouse) into WT mice 1 day before infection with 200 IFU of IAV/ PR8. The 38-2 Fab fragment also significantly reduced the mortality of infected WT mice compared to control Fab fragment (Fig 1B), indicating that the 38-2 mAb-mediated protective activity of PrP C against lethal IAV infection does not depend on cross-linking of PrP C or any Fc-dependent Ab functions.
To confirm that the protective activity of 38-2 mAb against lethal IAV infection is mediated through targeting PrP C , we similarly injected 38-2 mAb (1 mg/mouse) into Prnp 0/0 and WT mice 1 day before infection with 100 IFU of IAV/PR8. Purified unimmunized mouse polyclonal IgG was also administered into WT mice as a control. Since Prnp 0/0 mice had been reported to be highly susceptible to IAV/PR8 infection [3], we used lower doses (100 IFU) of IAV/PR8 in this experiment. 38-2 mAb consistently reduced the mortality and weight loss of infected WT mice (Fig 1C). However, the mortality and weight loss of infected Prnp 0/0 mice was not affected by 38-2 mAb (Fig 1C). These results confirm that targeting of PrP C is essential for 38-2 mAb to elicit its protective activity against lethal IAV infection.

38-2 mAb suppresses IAV-infected lung pathologies
To investigate the effects of 38-2 mAb on IAV-infected lung pathologies, we microscopically examined the lungs of control IgG-and 38-2 mAb-treated WT mice uninfected and at 3 and 5 dpi with IAV/PR8 (200 IFU). No solid lesions with infiltration of inflammatory cells were detected in the parenchymal and bronchiolar areas of uninfected lungs (Fig 2A). However, inflammatory cells had infiltrated these areas of control IgG-and 38-2 mAb-treated, IAV/ PR8-infected lungs, particularly markedly at 5 dpi, with much milder infiltration of inflammatory cells in 38-2 mAb-treated, IAV/PR8-infected lungs than in control lungs at 3 and 5 dpi (Fig 2A). Neither hyaline membrane formation nor hemorrhage was observed in these lungs (Fig 2A). Wet weight of 38-2 mAb-treated, IAV/PR8-infected lungs was significantly lighter than that of control lungs (S2 Fig), suggesting lower exudates in the former lungs than in the latter. Atelectatic lung areas were also smaller in the former lungs than in the latter (Fig 2B).
The NP-positive cells were observed in the airway and parenchymal areas of both IgG-or 38-2 mAb-treated, IAV/PR8-infected lungs at 3 and 5 dpi. However, they were gradually increased in the airway and parenchymal areas of 38-2 mAb-treated lungs, but rapidly in control lungs (Fig 2I and 2J). These results suggest that 38-2 mAb also could affect the susceptibility of lung epithelial cells to IAV/PR8 without altering the cell tropism of IAV/PR8 in the lung. Taken together, these results indicate that 38-2 mAb could suppress IAV infection-induced lung pathologies, including inflammatory cell infiltration, inflammatory cytokine production, epithelial cell damage, and IAV production.

38-2 mAb does not activate SOD1 in IAV-infected lungs
It has been reported that PrP C could provide protection against IAV infection in mice, by reducing reactive oxygen species (ROS) through activation of Cu/Zn-dependent superoxide dismutase (SOD1) in infected lungs [3]. To address if SOD1 activity could be involved in the 38-2 mAb-induced protective activity of PrP C against lethal IAV infection, we investigated whether 38-2 mAb could induce activation of SOD1 in IAV-infected lungs. ROS levels were slightly lower in 38-2 mAb-treated lungs than in control lungs at 3 dpi with IAV/PR8 (200 IFU) (Fig 3A). However, enzymatic activity of SOD1 was similarly elevated in both lungs ( Fig  3B). Protein levels of SOD1 were also unchanged in both lungs (Fig 3C). These results suggest  Eosin-stained lungs from control IgG-and 38-2 mAb-treated mice  uninfected and at 3 and 5 dpi with 200 IFU of IAV/PR8. Bar, 1 mm. Atelectatic area (%) (B) and levels of pro-inflammatory cytokines (C) in the lungs from control IgG-and 38-2 mAb-treated mice uninfected and at 3 and 5 dpi with 200 IFU of IAV/PR8 (n = 3 in each group). Un, uninfected. (D) Western blotting  for the AT1 cell marker podoplanin, the AT2 cell marker SP-C, the Clara cell marker CC10, pro-caspase 3, and the cleaved caspase 3 in lungs from control IgGand 38-2 mAb-treated mice uninfected and at 3 and 5 dpi with 200 IFU of IAV/

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PrP in M2 macrophages and influenza that the SOD1-mediated anti-oxidative activity of PrP C is not involved in the 38-2 mAbinduced protective activity of PrP C against lethal IAV infection.

SFK activation is essential for 38-2 mAb to elicit the protective activity against lethal IAV infection
It has been reported that certain anti-PrP mAbs could activate SFK in neuronal and non-neuronal cells [19][20][21][22]. Therefore, to investigate the mechanism by which 38-2 mAb could provoke protection against lethal IAV infection, we first examined if SFK could be activated by 38-2 mAb in IAV-infected lungs, by performing Western blotting for SFK phosphorylation at tyrosine 416, the activated form of SFK, in the lungs of control IgG-and 38-2 mAb-treated WT mice at 3 dpi with IAV/PR8 (200 IFU). Phosphorylated SFK (Tyr416) was increased in 38-2 mAb-treated, IAV/PR8-infected lungs, compared to control lungs (Fig 4A), indicating that 38-2 mAb could activate SFK in IAV-infected lungs. We then investigated the role of SFK activation in the 38-2 mAb-induced protective activity of PrP C against lethal IAV infection, by administering 38-2 mAb and control IgG together with the SFK inhibitors dasatinib (DS) and PP2 into WT mice 1 day before intranasal infection with 200 IFU of IAV/PR8. DS and PP2 had no effects on the mortality of control IgG-treated, IAV/PR8-infected mice ( Fig 4B, S5 Fig,  S6A Fig). However, both inhibitors abolished the protective activity of 38-2 mAb against lethal IAV infection ( Fig 4B, S5 Fig, S6A Fig). The survival rate of 38-2 mAb-treated, IAV/ PR8-infected mice was markedly decreased by DS (Fig 4B, S5 Fig) and PP2 (S6A Fig). The SFK activation and reduction of viral protein M2 and cleaved caspase 3 fragments in 38-2 mAb-treated, IAV/PR8-infected mice were also inhibited by DS (Fig 4C). DS and PP2 inhibit not only SFKs but also other kinases such as c-Abl and c-Kit. We therefore similarly tested imatinib, which selectively inhibits c-Abl, platelet-derived growth factor receptor, and c-Kit [23]. Imatinib failed to inhibit the protective activity of 38-2 mAb against lethal IAV infection (S6B Fig). These results indicate that 38-2 mAb could activate SFK in IAV-infected lungs, and that the SFK activation is essential for the 38-2 mAb-induced protective activity of PrP C against lethal IAV infection.

38-2 mAb induces M2 macrophage polarization through activation of SFK
To further elucidate the mechanism for the 38-2 mAb-induced protective activity of PrP C against lethal IAV infection, we identified cell-types in which SFK is activated by 38-2 mAb in
To further clarify the role of 38-2 mAb-induced activation of SFK in macrophage polarization, we collected peritoneal macrophages and incubated them with 38-2 mAb. We first confirmed that peritoneal macrophages express PrP C on Western blotting with 38-2 mAb ( Fig  6A). PrP C is a glycoprotein with two glycosylation sites, therefore di-, mono-, and un-glycosylated forms of PrP C are detected as several distinct bands (Fig 6A). 38-2 mAb strongly crossreacted with a non-PrP molecule of around 28 kDa in WT and Prnp 0/0 peritoneal macrophages (Fig 6A). Incubation with 38-2 mAb increased phosphorylated SFK (Tyr416) and MGL1/2 in WT macrophages, but not in Prnp 0/0 macrophages (Fig 6B). The SFK activation and MGL1/2 upregulation in 38-2 mAb-treated macrophages were suppressed by DS (Fig 6B). The M2 cytokines, IL-4 and IL-10, but not the M1 cytokines, IFN-γ and TNF-α, were increasingly released into the medium from 38-2 mAb-treated WT macrophages (Fig 6C). We also directly injected 38-2 mAb and control IgG into the peritoneal cavity of WT mice (1 mg/mouse) and collected their peritoneal macrophages 1 and 3 days after injection. Western blotting showed that WT peritoneal macrophages, but not Prnp 0/0 peritoneal macrophages, had increased phosphorylated SFK (Tyr416) and MGL1/2 after treatment with 38-2 mAb (Fig 6D). The

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PrP in M2 macrophages and influenza

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PrP in M2 macrophages and influenza increase in phosphorylated SFK (Tyr416) and MGL1/2 in 38-2 mAb-treated WT mice was abolished by DS ( Fig 6D). We also treated alveolar macrophages collected from the bronchoalveolar lavage fluids (BALF) of WT mice with 38-2 mAb, and investigated for expression of the M1-and M2-specific genes using real-time RT-PCR. The gene expression of M2-specific molecules, such as MGL1 and IL-10, was increased, but the expression of the M1-specific genes including those for TNF-α and IFN-γ was decreased, in 38-2 mAb-treated alveolar macrophages compared to control IgG-treated alveolar macrophages (S7 Fig). These results suggest that interaction of 38-2 mAb with PrP C on the surface of macrophages could lead to activation of SFK in the macrophages and polarize them to M2 macrophages.

PrP in M2 macrophages and influenza
To investigate which types of SFKs could be activated in macrophages treated with 38-2 mAb, we treated peritoneal macrophages with control IgG and 38-2 mAb, and subjected the cell lysates to immunoprecipitation with anti-phosphorylated SFK (Tyr416) Ab and then the precipitates to mass spectrometry. The anti-phosphorylated SFK (Tyr416) Ab most abundantly precipitated Lyn, with the abundance much higher in 38-2 mAb-treated peritoneal macrophages than in control peritoneal macrophages (S8 Fig). To a lesser extent, Hck was also immunoprecipitated with higher abundance in 38-2 mAb-treated peritoneal macrophages than control peritoneal macrophages (S8 Fig). Other SFKs including Fyn, Frk, Yes, Fgr, Lck, and Src were only marginally detectable in the precipitate (S8 Fig). These results indicate that Lyn and to a lesser extent Hck could be predominantly phosphorylated in peritoneal macrophages treated with 38-2 mAb.

38-2 mAb is also therapeutically effective against lethal IAV infection
We also investigated if 38-2 mAb could be therapeutically effective against lethal IAV infection, by intraperitoneally administering 38-2 mAb into WT mice (1 mg/mouse) 3 or 5 dpi with 200 IFU of IVA/PR8. Control IgG was similarly injected into WT mice at 3 dpi. Only 20% of control WT mice survived the infection (Fig 7). However, the 3 dpi-treatment with 38-2 mAb significantly reduced the mortality of infected mice (Fig 7). Weight loss was also reduced in 38-2 mAb-treated mice compared to control mice (Fig 7). The treatment starting even at 5 dpi also tended to be slightly but not significantly effective at reducing the mortality of infected mice (S9 Fig). These results indicate that 38-2 mAb could also be therapeutically effective against lethal IAV infection.

38-2 mAb also protects against lethal infection with other IAV strains
We further investigated if 38-2 mAb could protect against other IAV strains, by intraperitoneally injecting 38-2 mAb (1 mg/mouse) into WT mice 1 day before infection with 600 IFU of A/Aichi/2/68 (H3N2) (referred to as IAV/Aichi) and 3,000 IFU of A/WSN/33 (H1N1) (referred to as IAV/WSN). Control IgG was similarly injected into WT mice. Mortality of the control mice infected with IAV/Aichi and IAV/WSN was about 80% and 50%, respectively (Fig 8A and 8B). However, 38-2 mAb significantly reduced the mortality to about 30% and 5% in mice infected with IAV/Aichi and IAV/WSN, respectively (Fig 8A and 8B). Weight loss was also reduced in infected mice by 38-2 mAb (Fig 8A and 8B). These results indicate that 38-2 mAb could confer protection against lethal infection with various IAV strains.

Other anti-PrP mAbs also evoke protection against IAV infection through a similar mechanism
We also investigated if other anti-PrP mAbs could also evoke protective activity against lethal IAV infection through the same mechanism as 38-2 mAb. 3S9 and 2H9 anti-PrP IgG mAbs recognize residues 141-161 of mouse PrP C and a discontinuous epitope included in residues 151-221, respectively [24,25]. 3S9 and 2H9 mAbs also activated SFK and upregulated MGL1/2 in peritoneal macrophages from WT mice but not from Prnp 0/0 mice (Fig 9A). The SFK activation and MGL1/2 upregulation in 3S9 or 2H9 mAb-treated WT macrophages were abolished by DS (Fig 9A). These results indicate that, similarly to 38-2 mAb, 3S9 and 2H9 mAbs could activate SFK in macrophages and thereby induce M2 macrophage polarization. We then intraperitoneally administered 3S9 and 2H9 mAbs into mice (1 mg/mouse) 1 day before intranasal infection with 100 or 200 IFU of IAV/PR8. Both mAbs significantly reduced the mortality of IAV/PR8-infected mice (Fig 9B). These results indicate that 3S9 and 2H9 mAbs could

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PrP in M2 macrophages and influenza also evoke protective activity against lethal IAV infection by inducing M2 macrophage polarization through activating SFK.

Anti-PrP mAbs specifically recognize PrP C on the cell surface of macrophages
To investigate whether 38-2, 3S9, and 2H9 mAbs could specifically recognize PrP C expressed on the cell surface of macrophages, we first confirmed that all of these mAbs detected PrP C in the brains and lungs of WT mice by Western blotting (Fig 10A, S10A-S10C Fig). Consistent with PrP C being expressed as different glycosylated forms, several distinct bands corresponding to the different glycosylated forms of PrP C were detected with these mAbs in WT brains and lungs, but not in Prnp 0/0 brains (Fig 10A, S10A-S10C Fig). PrP C was detected more easily with 38-2 mAb than with 3S9 and 2H9 mAbs, and the band patterns of PrP C were different with each of the mAbs (Fig 10A, S10A-S10C Fig). This could be because these mAbs recognize different epitopes. These mAbs also weakly cross-reacted with several other molecules except for a molecule of around 28 kDa that was strongly cross-reacted with 38-2 mAb in WT and Prnp 0/0 lungs (Fig 10A, S10A-S10C Fig). A similar cross-reactive band was detected with 38-2 mAb in peritoneal macrophages (Fig 6A). It is thus possible that the cross-reactive band around 28 kDa in WT and Prnp 0/0 lungs might be derived from alveolar macrophages.
We then performed FACS analysis with these mAbs. To avoid confusion that might be caused by expression of endogenous PrP C , we used PrP C -knockout cells, N2aΔPrP cells [26]. The cells were transfected with control vector or pIRES-hrGFP-MoPrP vector, which encodes mouse PrP C together with the transfection marker humanized Renilla green fluorescent protein (hrGFP) under the control of an internal ribosomal entry site (IRES) motif. Western blotting with 38-2, 3S9, and 2H9 mAbs showed several distinct bands of PrP C in pIRES-hrGFP-MoPrP-transfected cells, but not in control vector-transfected cells (Fig 10B). We

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PrP in M2 macrophages and influenza analyzed hrGFP-positive cells for PrP C expression with these mAbs by FACS. Compared to control IgG, these mAbs showed a shifted signal in pIRES-hrGFP-MoPrP-transfected cells, but not in control vector-transfected cells (Fig 10B), indicating that these mAbs could specifically recognize PrP C expressed on the cell surface of N2aΔPrP cells. We also performed FACS analysis of CD11b-positive peritoneal macrophages from WT and Prnp 0/0 mice for PrP C expression with 38-2, 3S9, and 2H9 mAbs. These mAbs also showed a shifted signal in CD11b-positive WT macrophages, but not in CD11b-positive Prnp 0/0 macrophages (Fig 10C), indicating that these mAbs could also specifically recognize endogenous PrP C on the cell surface of peritoneal macrophages.

Discussion
In the present study, we showed that targeting of PrP C with three different anti-PrP mAbs examined, including 38-2, 3S9 and 2H9, conferred protection against lethal infection with

PrP in M2 macrophages and influenza
IAVs in WT mice. These anti-PrP mAbs mitigated inflammation, inflammatory cytokine production, epithelial cell damage and virus production in IAV-infected lungs and eventually reducing the morbidity and mortality of IAV-infected mice. 38-2 mAb failed to protect Prnp 0/ 0 mice from lethal infection with IAV/PR8, confirming that targeting of PrP C with anti-PrP mAbs could elicit the protective activity against IAV infection. We previously showed that, although Prnp 0/0 mice were highly susceptible to IAV infection, they were successfully rescued from lethal infection with the same IFU of IAV/PR8 as used in WT mice, by treatment with butylated hydroxyanisole, a radical oxygen species (ROS) scavenger, and allopurinol, the inhibitor of xanthine oxidase, which is a major ROS-generating enzyme in IAV-infected lungs [3]. These results indicate that the unsuccessful protection of Prnp 0/0 mice by 38-2 mAb could not be due to the higher sensitivity of Prnp 0/0 mice to IAV infection, but due to lack of PrP C in Prnp 0/0 mice. It is thus conceivable that PrP C could be a new therapeutic target for IAV infection.
It has been previously reported that PrP C could regulate SOD1 activity, thereby reducing oxidative stress in IAV-infected lungs and providing protection against IAV infection in mice [3]. However, 38-2 mAb did not increase SOD1 activity in IAV-infected lungs, indicating that the SOD1-mediated anti-oxidative activity of PrP C is not involved in the anti-PrP mAbinduced protective activity of PrP C against IAV infection. We showed that Fab fragments of 38-2 mAb reduced the mortality of IAV-infected mice, similarly to its parental 38-2 mAb. This suggests that the protective activity of the anti-PrP mAbs against IAV infection does not depend on cross-linking of PrP C or the Fc-dependent Ab functions, such as Ab-dependent cell-mediated cytotoxicity, Ab-dependent cellular phagocytosis, and Ab-mediated complement activation leading to complement-dependent cytotoxicity. Consistent with other reports demonstrating that stimulation of PrP C with certain anti-PrP mAbs activated SFK in cultured cells [19][20][21][22], we observed that SFK was activated in 38-2 mAb-treated, IAV-infected lungs. Moreover, DS and PP2, which inhibit SFKs as well as c-Abl and c-Kit kinases, but not the c-Abl and c-Kit kinase-specific inhibitor, imatinib [23], abolished the 38-2 mAb-induced protective activity of PrP C against IAV infection, decreasing the survival rate of 38-2 mAb-treated, IAV-infected mice, suggesting that the anti-PrP mAb-induced SFK activation is essential for the anti-PrP mAb-induced protective activity of PrP C against IAV infection. Administration with DS alone did not affect the mortality of IAV/PR8-infected mice, suggesting that SFK is not critically involved in the pathogenesis of IAV infection. Consistent with this, phosphorylated SFK (Tyr416) was not increased in the lungs of control IgG-treated, IAV/PR8-infected mice.
We found that SFK was predominantly activated in M2 macrophages in 38-2 mAb-treated, IAV-infected lungs, and that DS reduced M2 macrophages and conversely increased M1 macrophages in 38-2 mAb-treated, IAV-infected lungs. We also found that 38-2, 3S9, and 2H9 mAbs activated SFK in peritoneal macrophages from WT mice, but not from Prnp 0/0 mice, and induced M2 polarization in WT macrophages in vitro, and that DS inhibited the anti-PrP mAbs-induced M2 polarization in WT macrophages. We further showed that 38-2 mAb activated SFK in peritoneal macrophages and induced M2 macrophage polarization in vivo. FACS analysis of WT and Prnp 0/0 peritoneal macrophages revealed that 38-2, 3S9, and 2H9 mAbs specifically recognized PrP C on the cell surface of WT peritoneal macrophages. These results suggest that anti-PrP mAbs could activate SFK through interaction with PrP C on the cell surface of macrophages and induce M2 macrophage polarization. Phosphorylated Lyn and to a lesser extent phosphorylated Hck were predominantly precipitated with anti-phosphorylated SFK (Tyr416) Ab in 38-2 mAb-treated peritoneal macrophages, suggesting that these SFKs might be involved in the anti-PrP mAb-induced M2 polarization in macrophages. These results also might be consistent with Lyn and Hck being highly expressed in macrophages PLOS PATHOGENS PrP in M2 macrophages and influenza [27]. IL-4 and IL-10, both of which are known to induce M2 macrophage polarization through the STAT6 pathway [28,29], were abundantly secreted into the culture medium of peritoneal macrophages after incubation with 38-2 mAb. It is thus possible that IL-4 and IL-10 could be involved in the anti-PrP mAbs-induced M2 macrophage polarization.
Macrophages are phagocytic cells that play an important role in infectious diseases by triggering innate immunity and regulating various stages of inflammation [28,29]. Based on their phenotype and function, macrophages can be grouped into two subclasses: pro-inflammatory M1 and anti-inflammatory M2 macrophages [28,29]. In the early stages of infection, macrophages are polarized to a M1 phenotype. They produce inflammatory cytokines including IL-1, IL-6, and TNF-α, trigger innate immunity, and eventually protect the host from pathogen colonization. In contrast, M2 macrophages are generated in the late stages of infection, terminating the inflammation and repairing damaged tissues, by releasing anti-inflammatory cytokines such as tumor growth factor-β (TGF-β) and IL-10 and removing damaged cells [28,29]. Several reports indicate that M2 macrophages could be therapeutically beneficial for IAV infection. Mice intranasally administered with exogenous M2 macrophages or transgenically expressing granulocyte-macrophage colony-stimulating factor in lung epithelial cells to increase M2 macrophages were shown to display better clinical scores than control mice after infection with IAV [30,31]. It is thus likely that the anti-PrP mAb-induced M2 macrophage generation through SFK activation is the underlying mechanism for the anti-PrP mAbinduced protective activity of PrP C against IAV infection.
Neuronal loss was reported in mice stereotaxically injected into the hippocampus with D13 and P anti-PrP mAbs, both of which recognize epitopes within residues 95-150 of PrP C [32]. Sonati et al. also reported that anti-PrP mAbs, which recognize the C-terminal globular domain of PrP C , induced degeneration of cerebellar granule cells in cerebellar organotypic cultured slices and in mice by causing oxidative stress, which was mainly produced by NADPH oxidase 2 [33]. These results have raised a safety concern for therapeutic application of PrP Ctargeting agents, including anti-PrP mAbs. However, other investigators reported no neuronal loss in the hippocampus of mice injected with not only D13 and P anti-PrP mAbs but also two other different anti-PrP mAbs [34]. We also observed that treatment with 38-2 mAb suppressed the caspase 3 activity in IAV-infected lungs, indicating that 38-2 mAb is not toxic to cells. Further studies are necessary to clarify whether PrP C -targeting agents including anti-PrP mAbs are safe for therapeutic application.
38-2 and 3S9 mAbs recognize residues 41-45 and 141-161 of PrP C , respectively, and 2H9 mAb reacts with a discontinuous epitope included in residues 151-221 of PrP C [24,25]. Other anti-PrP mAbs, including SAF32, SAF61, and 3F4, which recognize residues 86-103, 142-160, and 109-112 of PrP C , respectively, were also shown to induce SFK activation in cultured cells [19][20][21][22]. It is thus likely that various sites, not just a single site, in PrP C could induce SFK activation after targeting with anti-PrP mAbs. The mechanism by which the binding of anti-PrP mAbs to extracellular GPI-anchored PrP C leads to activation of intracellular SFK still remains challenging. It has been reported that the anti-PrP mAbs-mediated activation of SFK in neuronal cells is dependent on caveolin-1 [19]. However, caveolin-1 is an integrated plasma membrane protein forming the cytoplasmic ridges of caveola, with both the N-and C-termini facing the cytoplasm [35]. It is therefore unlikely that caveolin-1 could directly transduce the signal from the GPI-anchored extracellular PrP C to the intracellular SFK after stimulation with anti-PrP mAbs. It has been shown that a small proportion of PrP C molecules on the plasma membrane could adopt transmembrane forms, with either the N-or C-terminus being exposed to the cytoplasm [36,37], suggesting that the transmembrane forms of PrP C could directly interact with and activate SFK in the cytoplasm after stimulation with anti-PrP mAbs. However, 38-2 mAb reacts with the N-terminal residues of PrP C and 3S9 and 2H9 mAbs recognize the C-terminal residues, indicating that the transmembrane forms of PrP C are not responsible for the anti-PrP mAb-induced activation of SFK. Another possibility is that an as yet unidentified transmembrane molecule(s) could form a link between PrP C and SFK. Anticlathrin Ab was reported to partially reduce the anti-PrP mAb-induced activation of SFK [19], suggesting that clathrin might be a candidate molecule connecting PrP C and SFK. Other candidates have been also suggested, including epidermal growth factor receptor and neural cell adhesion molecule [20,38]. It has been shown that Alzheimer amyloid-β oligomer could bind to PrP C around the central residues 97-100 and activate SFK in neurons [39], suggesting that these residues might be involved in the anti-PrP mAb-induced activation of SFK. The polybasic region and the so-called octapeptide region in the N-terminal domain of PrP C have been suggested to be important to induce intracellular signaling from PrP C [33,40]. However, it is unknown whether or not these regions could be involved in the anti-PrP mAb-induced activation of SFK. Identification of a molecule(s) connecting PrP C and SFK and mediating the anti-PrP mAb-induced activation of SFK and the site involved in the anti-PrP mAb-induced activation of SFK would be helpful for further understanding of not only the mechanism for the anti-PrP mAb-induced M2 macrophage polarization, but also the role of the anti-PrP mAbinduced SFK activation in the normal function of PrP C in neuronal cells.
In short, we showed that targeting of PrP C with anti-PrP mAbs could induce M2 macrophage polarization though activation of SFK and thereby confer protection against lethal influenza infection in mice. These results revealed a novel function of PrP C in M2 macrophage polarization, and suggest that the anti-PrP mAb-induced M2 macrophage-polarizing activity of PrP C could be a novel therapeutic target for influenza infection. Many lines of evidence are emerging that induction of M2 macrophages might be beneficial in other non-infectious chronic inflammatory diseases, including type 2 diabetes, atherosclerosis, and obesity [28,29,41]. Therefore, it might be interesting to investigate if treatment with anti-PrP mAbs could be therapeutically beneficial in these diseases. However, careful attention should be paid to the potential detrimental effects that might be caused by the forced polarization into M2 macrophages by PrP C -targeting agents including anti-PrP mAbs.

Ethics statement
The Ethics Committee of Animal Care and Experimentation of Tokushima University approved this study (approval number T30-100 and T2019-81). Mice and chicken embryos were cared for in accordance with The Guiding Principle for Animal Care and Experimentation of Tokushima University and with Japanese Law for Animal Welfare and Care. 10-day old chicken embryos were purchased from Ishii Co., Ltd., Tokushima, Japan. Animals C57BL/6 mice were purchased from Japan SLC Inc. (Shizuoka, Japan). Prnp 0/0 mice (kindly provided by Dr. Stanley B. Prusiner) used in this study had been obtained elsewhere by at least more than 9 time-backcrosses to C57BL/6 with Prnp 0/0 mice originally carrying a mixed background of C57BL/6×129Sv×FVB mice [42,43]. Mice were housed under specific pathogen-free conditions in cages of 5-6 animals with water and food ad libitum. Cages were provided with standard softwood bedding. Mice were kept on a standard 12:12 light:dark cycle. The mouse experiments carried out in this study were reviewed by the Ethics Committees of Animal Care and Experimentation of Tokushima University every year.

Virus preparation
IAV/PR8, IAV/Aichi, and IAV/WSN were grown in the allantoic sac of 11-day-old chicken embryos (Ishii Co., Ltd.) at 36˚C for 48 hours. Eggs were chilled at 4˚C for at least 4 hours prior to harvesting the allantoic fluids. Cellular debris was removed by centrifugation at 2,380×g at 4˚C for 30 min. The clarified supernatant was layered over a 20% sucrose cushion and centrifuged at 25,000×g at 4˚C for 120 min. The virus pellet was suspended in phosphatebuffered saline (PBS), and stored in multiple aliquots at -80˚C until use.

Intranasal infection of IAVs
After anesthesia with intramuscular injection of ketamine (Daiichi-Sankyo Propharma Co., LTD., Tokyo, Japan), male mice aged 5 weeks were intranasally inoculated with IAVs in a total volume of 20 μL (10 μL in each nasal cavity), and monitored for survival, weight loss, and clinical signs of illness for 14 days. The IAV stock aliquots were thawed and diluted in saline before use. To remove tissues, mice were anesthetized with ketamine (Daiichi-Sankyo Propharma Co., LTD.) and subjected to perfusion through the right ventricle with PBS.

Virus titer determination
Virus titers were expressed as IFU/mL, which was determined using Madin-Darby canine kidney (MDCK) cells (kindly provided by Dr. Takaaki Nakaya, Kyoto Prefectural University of Medicine) as follows. MDCK monolayer cells were inoculated with a 10-fold serial dilution of each sample of interest for 14 hours at 37˚C. The cells were then fixed with 4% paraformaldehyde (PFA), permeabilized with 0.3% Triton-X100 in PBS, and immunostained with anti-NP monoclonal antibodies (GeneTex). Signals were visualized using horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibodies (GE Healthcare) and TrueBlue Peroxidase Substrate (KPL, Gaithersburg, MD). IFU/mL was defined as the number of the cells positive for the anti-NP signals in 1 mL of each sample.

Antibody treatments
Mice were intraperitoneally injected with the indicated amounts of respective antibodies at indicated times. Control mouse polyclonal IgG was purchased from Sigma-Aldrich (St Louis, MO). 38-2 [17], 3S9 [24,25], and 2H9 hybridoma cells [24,25] were inoculated into the peritoneal cavity of BALB/cSlc-nu/nu mice (Japan SLC Inc.) pretreated with pristane (Sigma). Abs in their ascites were then purified using a MAbTrap Kit (GE Healthcare) according to the manufacturers' instructions.

Purification of 38-2 Fab
38-2 Fab and control mouse IgG Fab fragments were prepared using a Mouse IgG 1 Fab and F(ab') 2 Preparation Kit (Thermo Scientific Inc., Waltham, MA). In brief, 38-2 mAb and control mouse IgG (Sigma) were respectively subjected to proteolysis with immobilized ficin. The digested Fc portion and undigested IgG fragment were removed by passing over a column of immobilized protein A 3 times and the resulting elutes containing the Fab fragments was collected. The Fab concentration was determined at 280 nm using a Nanodrop 1000 spectrophotometer (Thermo Scientific Inc.).

Oral administration with DS, PP2 and Imatinib
DS was purchased from Santa Cruz Biotechnology, PP2 from Tocris bioscience (Bristol UK), and Imatinib mesylate from Selleck Chemicals (Houston, TX). DS and PP2 were dissolved in dimethyl sulfoxide (DMSO) and stored at -20˚C. Imatinib mesylate was dissolved in water and stored at -20˚C until use. DS was orally administered at 10 mg/kg/day or 20 mg/kg/day, and PP2 was administered at 5 mg/kg/day using a sonde needle daily from -1 to 4 dpi. Imatinib mesylate was orally administered at 200 mg/kg/day twice a day from -1 to 14 dpi.

Western blotting
Total proteins were denatured by boiling for 5 min in Laemmli's sample buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto Immobilon-PVDF membranes (Millipore, Bedford, MA), and membranes were blocked for 2 hours with 5% non-fat dry milk-containing TBST (0.1% Tween-20, 100 mM NaCl, 10 mM Tris-HCl, ph 7.6). Primary antibodies were incubated with the membrane overnight at 4˚C. Signals were visualized using horseradish peroxidase-conjugated anti-mouse, anti-rabbit, antigoat, and anti-rat IgG antibodies (GE Healthcare), and detected using a chemiluminescence image analyzer LAS-4000 mini (Fujifilm Co., Tokyo, Japan). Signal intensities were measured using ImageJ 64 software (NIH, Bethesda, MD). The average and standard deviation (SD) of the signals were calculated using Microsoft EXCEL software, version 16.37.

Pathological examinations
Lung tissues were fixed with 4% PFA, dehydrated, embedded in paraffin, and cut into 5 μmthick tissue sections. The sections were deparaffinized and stained with hematoxylin-eosin (H-E).

Determination of atelectatic lung areas
The atelectatic lung area was evaluated using Photoshop software (Adobe, San Jose, CA) and ImageJ software (NIH, Bethesda, MD). Briefly, the original RGB color images of H-E stained lung sections were converted to black-on-white images by use of Photoshop software and saved in TIFF format. The binary images in TIFF format were again inverted into a white-onblack image using the ImageJ application. Atelectatic lung area was expressed as the area of white pixels, which represent the lung parenchyma, against total lung area (white and black pixels).

Evaluation of NP-positive cells in the airway and parenchymal areas
Lung slices (n = 3 slices × 3 mice in each group) were stained with anti-NP antibody (Gene-Tex). The NP-positive cells in large airway (larger than 100 μm diameter) was evaluated by scoring 0 to 5 as followed: Score 0, intact epithelial cells in the airway without NP-positive cells; score 1, intact epithelial cells in the airway with <50% NP-positive cells; score 2, intact epithelial cells in the airway with >50% NP-positive cells; score 4, <50% epithelial cell injuries in the airway with NP-positive cells; score 5, >50% epithelial cell injuries in the airway with NP-positive cells. The NP positive cells in the parenchymal areas were counted using Photoshop software (Adobe) and ImageJ particle count command (NIH).

ELISA for cytokines
IL-6, IFN-γ, TNF-α, IL-4, and IL-10 levels in samples were determined using a Quantikine ELISA kit (R&D systems, Minneapolis, MN) according to the respective protocols provided by the manufacturer. In brief, the samples were diluted 1:1 with the assay diluent provided in the kit and added to the ELISA microplate wells. The plates were then left for 2 hours at RT. The wells were washed with the wash buffer 5 times and added with the mouse IFN-γ, TNF-α, IL-4, and IL-10 conjugate for 2 hrs. The wells were then washed with the wash buffer and the substrate reagent was added for 30 min. The reaction was stopped by addition of the stop solution. The optical density of each well was measured at 450 nm in an automated microplate reader (Thermo LabSystems, Waltham, MA).

PLOS PATHOGENS
PrP in M2 macrophages and influenza TUNEL staining TUNEL staining was performed using the in situ cell death detection kit and fluorescein (Roche Diagnostics, Mannheim, Germany) in accordance with the manufacturers' protocol. In brief, deparaffinized tissue sections were treated with 20 μg/mL proteinase K (Wako Pure Chemical Industries) in 10 mM Tris-HCl for 30 min at RT and incubated in the TUNEL reaction mixture for 1 hr at 37˚C in a humidified dark chamber. The sections were washed with PBS for 5 min 3 times and signals were detected using BIOREVO BZ-9000 (Keyence).

RNA extraction and real-time RT-PCR
Total RNA was first extracted from cells and tissues using RNeasy Mini Kit (QIAGEN, Valencia, CA). Homogenates in buffer RLT were transferred to a QIAshredder spin column (QIA-GEN). The flow-through was mixed with 1 volume of 70% ethanol and then transferred to an RNeasy spin column (QIAGEN). Total RNA bound to the membrane was washed with buffer RW1 and then with buffer RPE, and eluted with RNase-free water. Total RNA was used in a reverse transcription reaction using M-MLV Reverse Transcriptase (Promega, Madison, WI) and real-time PCR was performed with FastStart Universal SYBR green PCR Master (Roche Diagnostics) according to the manufacturers' protocols. Optimal PCR conditions were 40 cycles of 3-step cycling (Denaturation at 95˚C for 15 sec, Annealing at 56˚C for 30 sec, Extension at 72˚C for 50 sec) after an initial denaturation step (95˚C for 10 min). For the relative comparison of each gene expression, the data of real-time PCR were analyzed with 2 -ΔCt method. All real-time PCRs were run using the Applied Biosystems 7300 system (Foster City, CA), and the cycle threshold (CT) value for each sample was obtained using ABI 7300 system sequence detection software, version 1.3. Relative quantitative levels for each sample were calculated on the basis of the CT value by standard curve methods and were normalized to endogenous control β-actin expression. The normalized data of each gene were analyzed with 2 -ΔCt method. The experiments were done in triplicate for each sample. Sequences of the primers used for each gene are given in S1 Table. ROS measurement ROS concentration in samples was measured using an OxiSelect Intracellular ROS Assay Kit (Cell Biolabs, San Diego, CA). The assay uses 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA), which is deacetylated to non-fluorescent 2',7'-dichlorodihydrofluorescin and then oxidized by ROS to highly fluorescent 2',7'-dichlorofluorescin (DCF). Each of the samples were mixed with 1×DCFH-DA solution in a 96-well black plate and incubated at 37˚C for 48 hrs. ROS concentration in the samples was measured by determining the fluorescence intensities of DCF at 480 nm using Spectra Max Gemini EM (Molecular devices, Sunnyvale, CA).

Measurement of SOD activity
SOD activity in samples was determined using an OxiSelect Superoxide dismutase activity assay kit (Cell Biolabs). This assay uses a xanthine/XO system to produce superoxide anions, which reduce chromagen to produce a formazan dye, which is colorimetrically detectable at 490 nm. SOD activity in the samples was determined as the inhibition of formazan dye production. Each of the samples was mixed with 1× XO solution in a 96-well black plate and incubated at 37˚C for 60 min and the formazan dye produced was colorimetrically detected at 490 nm using Spectra Max Plus (Molecular devices).

PM preparation
After anesthesia with intramuscular injection of ketamine (Daiichi-Sankyo Propharma Co., LTD.), PM was obtained by inoculating 10 mL of cold PBS into the abdominal cavity by using 26-gauge needles attached to a 20-mL syringe. After a soft massage of the abdomen for 30 seconds, the peritoneal liquid was collected with 18-gauge needles attached to a 20-mL syringe. This procedure was repeated 2 times for each animal. Total peritoneal liquids were centrifuged at 300×g for 10 minutes to collect PM. The collected cells were adjusted to a concentration of 2.5×10 6 cells/mL in Dullbecco's modified Eagle's medium (DMEM, Wako Pure Chemicals) supplemented with 10% FBS and cultured in a 96-well plate for 3 hrs. The cells were then cultured in DMEM medium containing 1mg/mL of control IgG, 38-2, 3S9 or 2H9 mAb together with DS or control DMSO for 3hrs before being subjected to Western blotting, ELISA, realtime RT-PCR, FACS, and immunoprecipitation followed by mass spectrometry.

Alveolar macrophage preparation
Lungs were lavaged twice by injection with 2.0 ml of sterile saline using a syringe and the lavaged fluids were harvested by gentle aspiration. The fluids were then centrifuged at 300×g for 5 minutes to collect cells. The cells were adjusted to a concentration of 2.5×10 5 cells/mL in DMEM supplemented with 10% FBS and cultured in a 96-well plate for 3hrs. The cells were then treated with 1mg/mL of control IgG or 38-2 mAb in DMEM supplemented with 10% FBS for 3hrs before being subjected to real-time RT-PCR.

Construction of pIRES-hrGFP-MoPrP
The DNA fragment encoding full-length mouse PrP C was amplified by PCR with a sense primer (5'-tcggatcccgtcatcatggcgaac-3'; the underlined sequence, a BamH I site; the bold sequence, a start codon) and an antisense primer (5'-ccctcgaggctcatcccacgatcaggaag-3'; the underlined sequence, a Xho I site; the bold sequence, a stop codon) using pcDNA3.1/MoPrP plasmid encoding mouse PrP C [44] as a template. After confirmation of the DNA sequences, each DNA fragment was digested by BanH I and Xho I and introduced into a pIRES-hrGFP-2a vector (Stratagene, La Jolla, CA).

Flow cytometry
N2aΔPrP cells [26] were plated onto 6-well plates on the day before transfection. pIRES-hrGFP-MoPrP vector and control pIRES-hrGFP-2a vector (Stratagene) were transfected into N2aΔPrP cells using Lipofectamone 2000 (Invitrogen) according to the manufacturers' instructions. The cells were harvested 72 hrs after transfection using PBS containing 20 mM EDTA and washed with FACS buffer (5% FBS in PBS). PM cells collected from the peritoneal cavity were washed with PBS, incubated with ACK lysing buffer (150 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM EDTA) for 5 min to lyse red blood cells. The PM cells were then washed again with PBS and incubated with FACS buffer containing Fc-blocker (Biolegend, San Diego, CA; Cat. No. 101302) for 30 min on ice. Transfected N2aΔPrP and PM cells were incubated with 4 μg/mL of control IgG (Sigma) or 38-2, 3S9, and 2H9 anti-PrP mAbs in the FACS buffer for 1 hr on ice. For PM cells, we also used PE anti-mouse/human CD11b antibody (Biolegend). The cells were washed with the FACS buffer 3 times to remove unbound antibodies, incubated with 1:800 diluted Alexa Fluor 647 anti-mouse antibody (Invitrogen), washed with PBS 3 times, suspended in PBS containing 10 mM EDTA and 0.2% bovine serum albumin, and analyzed using a CytoFLEX Flow Cytometer with CytExpert software, version 2.3 (Beckman

Mass spectrometry analysis
PM were treated for 3 hrs in DMEM medium containing 1 mg/mL of control IgG or 38-2 mAb as described above, followed by two washes with ice-cold PBS. Cells were lysed in RIPA lysis buffer (1% NP-40, 0.25% Sodium deoxycholate, 0.05% SDS, 150 mM NaCl, 20 mM HEPES-NaOH, pH 7.5, 1 mM EGTA, and 1 mM MgCl 2 ) containing Complete protease inhibitors (Merck Millipore, Darmstadt, Germany), PhosSTOP phosphatase inhibitors (Merck Millipore), and 50 units/ml of benzonase endonuclease(Merck Millipore) on ice for 10 min. After centrifugation at 20,000×g for 15 min at 4˚C, the supernatants were incubated with anti-phosphorylated SFK (Tyr416) antibody (Cell Signaling Technology) that was immobilized on SureBeads Protein G magnetic beads (Bio-Rad, Hercules, CA) for 3 hrs at 4˚C with rotation. The beads were washed four times with RIPA buffer and then with 50 mM ammonium bicarbonate twice. Proteins on the beads were digested with 200 ng trypsin/Lys-C mix (Promega) for 16 hrs at 37˚C. The digests were reduced, alkylated, acidified, and desalted using GL-Tip SDS (GL Sciences, Tokyo, Japan). The eluates were evaporated and dissolved in 3% acetonitrile (ACN) and 0.1% trifluoroacetic acid. LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC (Thermo Fisher Scientific) connected to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 75 μm inner diameter ×150 mm C18 reversedphase column (Nikkyo Technos, Tokyo, Japan) with a linear 4-32% ACN gradient for 0-100 min followed by an increase to 80% ACN for 10 min. The mass spectrometer was operated in a data-dependent acquisition mode with a maximum duty cycle of 3 sec. MS1 spectra were measured with a resolution of 120,000 and a mass range from 375 to 1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an isolation window of 1.6 m/z and a normalized collision energy of 30. Dynamic exclusion was set to 15 sec. Raw data were directly analyzed against the SwissProt database restricted to M. musculus using Proteome Discoverer version 2.4 (Thermo Fisher Scientific) with Mascot search engine version 2.5 (Matrix Science, London, UK). The search parameters were as follows: (i) trypsin as an enzyme with up to two missed cleavages; (ii) precursor mass tolerance of 10 ppm; (iii) fragment mass tolerance of 0.6 Da; (iv) carbamidomethylation of cysteine as a fixed modification; and (v) acetylation of the protein Nterminus and oxidation of methionine as variable modifications. Peptides and proteins were filtered at a false-discovery rate (FDR) of 1% using the percolator node and the protein FDR validator node, respectively. Label-free precursor ion quantification was performed using the precursor ions quantifier node, and normalization was performed such that the total sum of abundance values for each sample over all peptides was the same. The abundance for each protein was analyzed using Proteome Discoverer version 2.4 (Thermo Fisher Scientific).

Statistical analysis
Survival rates were analyzed using the log-rank (Mantel-Cox) test (GraphPad Prism software package version 5.04). All other statistical analysis data were determined using a Student's ttest (Microsoft EXCEL software, version 16.37).  Fig. 38-2, 3S9, 2H9 mAbs recognize PrP C on Western blotting. Uncropped, full picture of Western blotting for PrP C with 38-2, 3S9, 2H9 mAbs in the brains (Br) and lungs (Lg) from WT and Prnp 0/0 mice in Fig 10A. (TIF)