key: cord-0330300-nb0igepf
authors: Ferreira, André C.; Sacramento, Carolina Q.; Pereira-Dutra, Filipe S.; Fintelman-Rodrigues, Natália; Silva, Priscila P.; Mattos, Mayara; de Freitas, Caroline S.; Marttorelli, Andressa; de Melo, Gabrielle R.; Macedo, Mariana C.; Azevedo-Quintanilha, Isaclaudia G.; Carlos, Aluana S.; Emídio, João Vitor; Garcia, Cristiana C.; Bozza, Patrícia T.; Bozza, Fernando A.; Souza, Thiago M. L.
title: Macrophages undergo necroptosis during severe influenza A infection and contribute to virus-associated cytokine storm
date: 2022-04-20
journal: bioRxiv
DOI: 10.1101/2022.04.20.488871
sha: 3ade5a1dab1d2674cc04454bd86da572c2762176
doc_id: 330300
cord_uid: nb0igepf

Influenza A virus (IAV) causes a major public health concern, because it is one of the leading causes of respiratory tract infections and hospitalization. Severe influenza has been associated with the cytokine storm, and IAV productive infection cell death in airway epithelial cells may contribute to the exacerbation of this proinflammatory event. On the other hand, IAV replication in macrophages is non-permissive and whether this immune cell may contribute to severe influenza physiopathology requires more details. Here, we investigated IAV-induced macrophage death, along with potential therapeutic intervention. We found that IAV or simply its surface glycoprotein hemagglutinin (HA) triggers necroptosis in human and murine macrophages in a Toll-like receptor-4 (TLR4) and TNF-dependent manner. Anti-TNF treatment, with the clinically approved drug etanercept, prevented the engagement of the necroptotic loop and mice mortality. impaired IAV-induced pro-inflammatory cytokine storm and lung injury. Our results implicate macrophage necroptosis with severe influenza in experimental models and potentially repurpose a clinically available therapy. Author Summary Various fates of cell death have an integral role in the influenza A virus (IAV) pathogenesis and lung/respiratory dysfunction. IAV physiopathology is not restricted to airway epithelial cells, where this virus actively replicated. Macrophages should support both viral clearance and priming of adaptative immune response in patients that adequately control influenza. However, during severe IAV infection, macrophages – which are unable to support a permissive viral replication - undergo disruptive cell death and contribute to the exacerbated production of proinflammatory cytokines/chemokines. We characterized this process by showing that IAV or just its surface glycoprotein hemagglutinin (HA) trigger necroptosis, a disruptive and TNF-dependent cells death. Since TNF is a hallmark of pro-inflammatory cell death, we blocked this mediator with a repurposed biomedicine etanercept, which prevented the severe IAV infection in the experimental model. The present work improves the knowledge of influenza pathophysiology by highlighting the importance of macrophage cell death during severe infection.

Abstract 27 Influenza A virus (IAV) causes a major public health concern, because it is one of the 28 leading causes of respiratory tract infections and hospitalization. Severe influenza has been 29 associated with the cytokine storm, and IAV productive infection cell death in airway epithelial 30 cells may contribute to the exacerbation of this proinflammatory event. On the other hand, IAV 31 replication in macrophages is non-permissive and whether this immune cell may contribute to 32 severe influenza physiopathology requires more details. Here, we investigated IAV-induced 33 macrophage death, along with potential therapeutic intervention. We found that IAV or simply its 34 surface glycoprotein hemagglutinin (HA) triggers necroptosis in human and murine macrophages 35 in a Toll-like receptor-4 (TLR4) and TNF-dependent manner. Anti-TNF treatment, with the 36 clinically approved drug etanercept, prevented the engagement of the necroptotic loop and mice 37 mortality. impaired IAV-induced pro-inflammatory cytokine storm and lung injury. Our results 38 implicate macrophage necroptosis with severe influenza in experimental models and potentially 39 repurpose a clinically available therapy. Various fates of cell death have an integral role in the influenza A virus (IAV) pathogenesis 44 and lung/respiratory dysfunction. IAV physiopathology is not restricted to airway epithelial cells, 45 where this virus actively replicated. Macrophages should support both viral clearance and priming 46 of adaptative immune response in patients that adequately control influenza. However, during 47 severe IAV infection, macrophages -which are unable to support a permissive viral replication -48 undergo disruptive cell death and contribute to the exacerbated production of proinflammatory 49 cytokines/chemokines. We characterized this process by showing that IAV or just its surface 50 glycoprotein hemagglutinin (HA) trigger necroptosis, a disruptive and TNF-dependent cells death. 51 Since TNF is a hallmark of pro-inflammatory cell death, we blocked this mediator with a and NA assists their final release [9] . During this process, infected cells may succumb to death, 80 and the pathway to death has been associated to IAV pathogenesis [9,10]. In airway epithelial 81 cells, where IAV performs its replicative cycle completely and generates infectious progeny, 82 infection activates the receptor interaction serine/threonine-protein kinases 1 and 3 (RIPK1 and 83 RIPK3) promoting apoptosis and necroptosis [11] [12] [13] [14] . Whereas apoptosis has been proposed as 84 cell death related to limit IAV-induced tissue damage [15], virus-induced necrotic cell death may 85 exacerbate inflammatory engagement [14, 16] . During necroptosis, cells are triggered to shift from 86 an apoptotic-like cell death to the disruption of the plasma membrane, facilitating the release of 87 immunomodulatory damage associated with molecular patterns (DAMPs), leading to 88 inflammation [17] . The pro-inflammatory cytokine storm is an important hallmark of IAV-induced 89 severe pneumonia, strongly associated with lung/respiratory dysfunction [18] . 90 Moreover, IAV physiopathology is not restricted to airway epithelial cells. Macrophages 91 are also exposed to this virus, although they are unable to harbor a permissive virus replication [19] . 92 In the IAV-infected lung, both resident macrophages by the infected and macrophages derived 93 from blood monocytes, since they migrate towards the respiratory tract and differentiate to 109 For initial assessments of IAV-induced macrophage death, we evaluated the cell surface Because necroptosis is associated with the pro-inflammatory response, macrophages 131 infected with IAV or exposed to HA produced nitric oxide ( Figure 1F ) and IL-6 ( Figure 1G ). 132 Nec-1 pretreatment on these cells completely shifted the macrophage phenotype to prevent the 133 pro-inflammatory phenotype and increasing the content of the regulatory cytokine IL-10 ( Figure   134 1F-I). Together, these data suggest that IAV-induced necroptosis in macrophages may be directly 135 related to the pro-inflammatory cytokine storm observed in severe disease.

136 137 138 Because influenza HA has been shown to engage TLR4 as an early signal of innate immune 139 response in leukocytes [27,28], we hypothesized that this event could also occur in macrophages 140 as an upstream event to trigger necroptosis. Thus, macrophages were pre-treated with an TLR4 141 signaling pathway inhibitor (CLI95) and exposed to IAV or HA. CLI95-treated IAV/HA-exposed 142 macrophages display lower levels of LDH ( Figure 2A ) and annexinV + /PI + cells ( Figure 2B) , 143 compared to untreated control macrophages (ctr; Figure 2 ). 144 Next, pharmacological data were further validated with macrophages from TLR4 knockout 145 mice (TLR4-/-), and compared to macrophages from wild-type (WT) and TLR2 knockout (TLR2- 160 in macrophages. 161 Since IAV/HA induced cell death with the engagement of TLR4 signaling, we next 162 evaluated whether TNF-α, a downstream pro-inflammatory cytokine produced during these events, 163 could be involved in the necroptotic loop. IAV-infected or HA-exposed macrophages were necroptosis and apoptosis have been reported in epithelial airways cells [11] [12] [13] [14] , where virus actively 218

replicates. Here, we further described thatmacrophages, non-permissive cells to IAV replication, 219 exposed to this virus or just its surface glycoprotein HA experience necroptosis, which leads to the 220 imbalance of pro-inflammatory and regulatory modulators associated with cytokine storm and 221 severe influenza in vitro and in vivo. 222 Indeed, necroptosis has been described as a highly inflammatory process [26] , leading to 223 lung dysfunction and the development of severe multiorgan tissue damage during virus 224 infections [34] [35] [36] . Importantly, the inhibition of necroptosis in IAV/HA-exposed cells shifted 225 macrophage from pro-inflammatory to regulatory phenotype [37] , because IL-6 and nitric oxide 226 levels were reduced and while IL-10 levels were enhanced. 227 We observed that TLR4, which plays a major role in the recognition of PAMPs 

Reagents 250 We used different pharmacological inhibitors throughout this study ( to different concentrations of this protein or lipopolysaccharide (LPS; Sigma Aldrich). We 261 observed that 10 ng/mL of HA was the minimal dose to induce TNF ( Figure S1 ) and 10 ng/mL of 262 LPS was used as positive control ( Figure S1 ). were identified by incubating the membrane with IRDye® LICOR secondary antibodies in TBST, 404 followed by fluorescence imaging detection using the Odyssey® system (CLx Imaging System). 405 Protein bands were quantified by densitometric image analysis using the ImageJ software. 

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of BMDM cultures after 24h of stimulus. * P < 0.05 in comparison to the respective non-infected 596 groups (MOCK) and # P < 0.05 versus respective Wt infected/stimulated group. Graphs are 597 representative of three independent experiments

Macrophage cultures were 600 pre-treated with 1 ng/mL anti-TNF-α antibody and then infected with IAV (MOI of 0.25) or 601 exposed to 10 ng/mL of viral HA for 24 h. Cell death analysis was performed by flow cytometry 602 analysis of annexinV/PI positive cells (A-B). Assessment of cell viability through the measurement 603 of LDH release in the supernatant of BMDM (C). (D) The expression of p-RIPK1 were detected 604 in BMDM by Western blotting. β-actin levels were used for control of protein loading. (E) Graphs 605 of bands densitometry

Levels of nitrite were measured by Griess method in supernatant of 607 BMDM cultures after 24h of stimulus. The levels of (H) IL-6 and (K) IL-10 were measured by 608 ELISA assay in supernatant of BMDM cultures after 24h of stimulus

05 versus respective untreated infected/stimulated group.. (I) Model of A/HA-triggered 611

IAV/HA-triggered necroptosis by a loop of events involving TLR4, TNF-α, RIPK1, 612 leading to exacerbation of inflammation

Etanercept reduced weigh loss improved survival and during IAV lethal infection 615 in mice. C57Bl/6 mice were inoculated intranasally with 10 3 PFU of IAV

Data are show as percentage of survival and weight. Graphs are representative of three 619 independent experiments. * P < 0

Etanercept reduced necroptosis in vivo. C57Bl/6 mice were inoculated intranasally 622 with 10 3 PFU of IAV with and without daily treatment with Etanercep (ETN, 2.5 mg/kg, i.p.)

Mono: 626 mononuclear leukocytes, PMN: polymorphonuclear leukocytes. (C-D) Mononuclear cells death 627 was evaluated by annexinV/PI labelling. (E) Lung damage was evaluated by measuring LDH 628 release in the bronchoalveolar lavage (BAL) (F-I) The expression of p-RIPK1, MLKL, and 629 cleaved Caspase-8 were detected in the lung tissue homogenates by Western blotting. β-actin 630 levels were used for control of protein loading) . (G-I) Graphs of bands densitometry obtained 631 after loading normalization and expressed as fold change over Mock control. Data are expressed 632 as means ± SEM; One-way ANOVA

At days 3 and 5 post-infection (DPI), animals were euthanized and the 639 bronchoalveolar lavage (BAL) was collected for quantification of the of total protein (A) and 640 inflammatory mediators. The level of CXCL1/KC (B)

ANOVA with Dunnett's post-hoc test. * P < 0.05 in comparison to Mock and # P < 0.05 in 643 comparison to IAV

 418 We would like to thank Dra. 429 The authors declare no conflicts of interest. 430