# Cis-aconitate therapy protects against influenza mortality by dual targeting of viral polymerase and ERK/AKT/NF-κB signaling

Adeline Cezard, Déborah Brea-Diakite, Virginie Vasseur, Alan Wacquiez, Loïc Gonzalez, Ronan Le Goffic, Bruno da Costa, Delphine Fouquenet, Séverine Heumel, Arnaud Machelart, et al.

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## To cite this version:

Adeline Cezard, Déborah Brea-Diakite, Virginie Vasseur, Alan Wacquiez, Loïc Gonzalez, et al. Cis-aconitate therapy protects against influenza mortality by dual targeting of viral polymerase and ERK/AKT/NF-κB signaling. 2025. hal-05375838

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Preprint submitted on 21 Nov 2025

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1 *Cis-aconitate therapy protects against influenza mortality by dual targeting of viral polymerase*

2 and ERK/AKT/NF-κB signaling.

3

4 Adeline CEZARD¹,², Déborah BREA-DIAKITE¹,², Virginie VASSEUR¹,², Alan WACQUIEZ¹,², Loic

5 GONZALEZ¹,², Ronan LE GOFFIC³, Bruno DA COSTA³, Delphine FOUQUENET¹,², Severine

6 HEUMEL⁴, Arnaud MACHELART⁴, Eik HOFFMANN⁴, Priscille BRODIN⁴, François TROTTEIN⁴,

7 Cyrille MATHIEU⁵, Lola CANUS⁵, Florentine JACOLIN⁵, Pierre-Olivier VIDALAIN⁵, Laure

8 PERRIN-COCON⁵, Vincent LOTTEAU⁵, Julien BURLAUD-GAILLARD⁶, Dominique TERTIGAS⁷,

9 Michael G. SURETTE⁸, Antoine LEGRAS²,⁹, Damien SIZARET¹⁰, Thomas BARANEK¹,²,

10 Christophe PAGET¹,², Antoine GUILLON¹,²,¹¹ and Mustapha SI-TAHAR¹,²

11

12 1. INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours France

13 2. Université de Tours, Tours, France

14 3. Université Paris-Saclay, INRAE, UVSQ, UMR892 VIM, Jouy-en-Josas, France

15 4. Université de Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 9017 - CIL - Center

16 for Infection and Immunity of Lille, F-59000 Lille, France,

17 5. Centre International de Recherche en Infectiologie (CIRI), Université de Lyon, Inserm, U1111, CNRS,

18 UMR5308, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon, 69007 Lyon, France

19 6. Plate-Forme IBiSA des Microscopies, PPFASB, Université de Tours and CHRU de Tours, France.

20 7. Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

21 8. Department of Medicine, McMaster University, Hamilton, Ontario, Canada

22 9. Service de Chirurgie Thoracique, CHRU de Tours, France.

23 10. Service de Pathologie, CHRU de Tours, France.

24 11. Service de Médecine Intensive -Réanimation, CHRU de Tours, France.

25

26 *Correspondence: Mustapha Si-Tahar, Centre d'Etude des Pathologies Respiratoires (CEPR),*

27 INSERM U1100, Faculté de Médecine - 10, boulevard Tonnellé, 37032 Tours cedex, France. Phone:

28 (00 33 2) 47 36 60 45; Fax: (00 33 2) 47 36 60 49; mustapha.si-tahar@inserm.fr
29 **ABSTRACT**

30 Influenza virus poses a significant global health challenge, causing approximately 500,000 31 deaths annually. Its ability to evade antiviral treatments and vaccine-induced immunity 32 underscores the need for novel therapeutic approaches. Our study identifies cis-aconitate (cis- 33 aco), a mitochondria-derived metabolite, as a potent dual-action agent against influenza, 34 independently of its metabolic derivative, itaconate. Cis-aco impairs viral polymerase activity, 35 suppressing viral mRNA expression and protein synthesis to inhibit replication across a range 36 of influenza subtypes. This antiviral efficacy is confirmed in ex vivo human airway and lung 37 organotypic models. Beyond its antiviral properties, cis-aco exhibits potent anti-inflammatory 38 effects, disrupting key inflammatory cascades and reducing the secretion of inflammatory 39 mediators. In a mouse model of influenza pneumonia, cis-aco mitigates viral replication, 40 inflammation, and immune cell activation, significantly improving survival. Notably, its 41 efficacy persists even when administered at later stages of infection, when 42 oseltamivir/Tamiflu® is no longer effective. These findings position cis-aco as a promising 43 influenza treatment, combining antiviral and anti-inflammatory benefits within a clinically 44 relevant timeframe.

45 **Keywords:** Influenza virus; Therapy, Antiviral; Anti-inflammatory; Pneumonia
46 INTRODUCTION

47 Influenza viruses have long been major causes of morbidity and mortality, with heightened attention
48 since the 1918 pandemic, driving extensive research into therapies [1]. Current approaches, including
49 vaccination and antivirals, often demonstrate limited effectiveness. The short duration of vaccine-
50 induced immunity, combined with the intrinsic antigenic drift of influenza viruses, undermines
51 sustained protection [2, 3]. Skepticism also persists regarding the efficacy of approved anti-influenza
52 drugs, especially when administered later in the course of infection [4–6]. Therefore, developing
53 innovative strategies effective even after infection onset is crucial.

54 The pathophysiology of influenza-related pneumonia stems from the intrinsic viral pathogenicity and
55 the immune response. While a robust immune response is essential for viral clearance, excessive
56 cellular recruitment and the release of cytotoxic molecules can lead to lung hyper-inflammation,
57 resulting in tissue damage, morbidity, and death [7, 8].

58 Interestingly, the recent discovery of metabolic reprogramming of immune cells has opened avenues
59 for innovative therapeutic approaches [9–13]. We and others have demonstrated that hosts develop
60 metabolic countermeasures in response to infection. [9, 14–19]. For instance, using an integrated
61 approach combining metabolomics, *in vitro*, and *in vivo* infection assays, we recently discovered the
62 inhibitory effect of tricarboxylic acid cycle (TCA)-derived succinate on influenza virus infection. This
63 inhibition is primarily associated with succinylation and nuclear retention of the viral nucleoprotein,
64 although additional mechanisms may also contribute [16].

65 Building on this finding, our investigation explored a broader range of host metabolites for their ability
66 to regulate influenza virus infection in human lung epithelial cells. Among the TCA intermediates
67 examined, *cis*-aconitate (*cis*-aco) stood out for its potent antiviral and anti-inflammatory properties.
68 We further elucidated the molecular mechanisms underlying *cis*-aco's protective effect, and our *in vivo*
69 experiments confirmed its efficacy in mitigating severe influenza pneumonia. Remarkably, *cis*-aco
70 demonstrated therapeutic benefits even when administered at advanced stages of infection, when
71 conventional treatment with oseltamivir (Tamiflu®, [20]) proved ineffective.
72 **RESULTS**

73 **Cis-aco inhibits both viral replication and production of inflammatory mediators in influenza**
74 **virus-infected lung epithelial cells.**

75 Building on our previous work examining the anti-influenza effects of succinate [16], we selected
76 eight metabolites derived from glycolysis or the TCA cycle for evaluation. Human bronchial epithelial
77 BEAS-2B cells were infected with influenza A virus (IAV; A/Scotland/20/74, H3N2) and treated
78 with 3.4 mM of each metabolite at 4 hours (h) post-infection (p.i.) (Fig. 1a). This concentration
79 was chosen as the highest dose without cytotoxic effects across all tested metabolites (Fig. EV1a-
80 b). Notably, cis-aco treatment at this concentration had no significant impact on cell proliferation,
81 mitochondrial mass, or ROS production (Fig. EV1c-e).

82 To assess the release of neo-virions, we measured neuraminidase (NA) activity in cell supernatants at
83 20 hours p.i.. Treatment with *cis*-aco resulted in a 60-fold reduction in NA activity (p=0.0003), while
84 itaconate treatment led to a smaller, yet significant, 4.8-fold reduction (p=0.028) (Fig. 1b). No
85 significant changes in NA activity were observed with other metabolites. Concomitantly, *cis*-aco
86 treatment also reduced the levels of the inflammatory cytokine interleukin-6 (IL-6) in IAV-infected
87 epithelial cells (p=0.03) (Fig. 1c). We next evaluated the expression of a broader panel of pro-
88 inflammatory cytokines and chemokines, including CCL2/MCP-1, CCL5/RANTES, CXCL1/GROα,
89 CXCL10/IP-10, IL-6, and CXCL8/IL-8. In IAV-infected cells, the levels of these mediators were
90 increased by 4- to 7-fold compared to MOCK conditions (Fig. 1d, pink bars). *Cis*-aco treatment
91 inhibited this inflammatory response by at least 3-fold (Fig. 1d, blue bars). Of note, *trans*-aconitate
92 exhibited similar antiviral and anti-inflammatory activities (Fig. EV2), suggesting that both *cis*-
93 and *trans*-isomers of aconitate possess anti-influenza properties.

94 Both influenza A and B viruses contribute to seasonal flu epidemics, with influenza A encompassing a
95 diverse range of subtypes [21]. To assess the broad-spectrum antiviral potential of *cis*-aco, we
96 evaluated its activity against several influenza strains: influenza A/pandemic/2009 H1N1 (Fig. 1e),
97 influenza A/Puerto Rico/8/1934 H1N1 (Fig. 1f), influenza B Yamagata strain B/Paris/234/2013 (Fig.
98 1g), in comparison with our "reference" strain influenza A/Scotland/20/74 H3N2 (Fig. 1h). Plaque-
forming unit assays revealed a significant decrease in viral particle production in cells treated with cis-aco, irrespective of influenza virus type or subtype (p<0.05; Fig. 3a-c).

The foregoing results were obtained using the immortalized bronchial epithelial BEAS-2B cell line, which can exhibit altered functional and metabolic profiles [22, 23]. To better reflect the lung's physiological conditions, we next tested cis-aco in two dimensional (2D) cultures of primary bronchial epithelial cells. In these cells, cis-aco treatment significantly reduced IAV release in a dose-dependent manner (p<0.0001; Fig. 1i) and inhibited IAV-induced IL-6 production by up to 80% (Fig. 1j). Cis-aco also decreased IAV-induced cell death by approximately 70%, as shown by SYTOX™ labeling (Fig. EV3). While 2D primary bronchial epithelial cultures are closer to in vivo conditions than immortalized cell lines, they still lack the complexity of native tissues. To address this, we developed a 3D culture model using primary airway epithelial cells grown at an air-liquid interface (ALI), forming a differentiated, polarized epithelium with basal, goblet, and ciliated cells (Fig. 2a). This model exhibited a transepithelial electrical resistance of ~400 ohms/cm² and showed beating cilia (not shown). In this more complex system, cis-aco again demonstrated anti-viral and anti-inflammatory effects, confirming its efficacy in a model that more closely mimics the in vivo environment (Fig. 2b-d).

To further model human lung physiopathology, we developed a standardized ex vivo lung organotypic slice culture at the ALI (Fig. 2e). Tissue explants were obtained from patients undergoing surgery for lung cancer (Table 1), predominantly male (6/8), with an average age of 73 [68-78] years and a history of smoking (6/8). Upon tissue infection, IAV localized to restricted areas (Fig. 2f), and infectious particles were detected in culture supernatants (Fig. 2g). Cis-aco treatment reduced viral titers by 95% (Fig. 2g).

121

### Cis-aco confers potent benefits in a murine model of influenza infection.

Before evaluating the potential anti-influenza effects of cis-aco in vivo, we assessed its safety profile using repeated instillations in mice over 15 days. Cis-aco did not significantly affect body weight in either male or female mice compared to controls (Fig. EV4a). No changes in liver function were observed, as measured by serum ALAT levels (Fig. EV4b). Similarly, no alterations
127 in fecal microbiota composition were detected between *cis*-aco-treated and control mice (Fig. EV4c). Flow cytometry analysis of immune cells in BAL fluid (Fig. EV4d) and blood (Fig. EV4e)
128 confirmed that *cis*-aco had no impact on pulmonary or systemic immune responses, regardless of
129 sex.
130

131 By mitigating various aspects of influenza pathogenesis, the preceding *in vitro* and *ex vivo* data
132 (Figs. 1 and 2) suggest that *cis*-aco could prevent lung damage induced by IAV *in vivo*. Therefore,
133 to assess its potential as an anti-influenza drug, we evaluated its effect on the mortality of infected
134 mice, comparing it to the standard of care, oseltamivir (a neuraminidase inhibitor [25]). While all
135 untreated IAV-infected mice succumbed, those treated with 30 mg/kg *cis*-aco or 20 mg/kg
136 oseltamivir within 20 min p.i. reached survival rates of 80% and 90%, respectively (Fig. 3a).
137 Moreover, by 15 days post-treatment, surviving mice had regained their original body weight (Fig.
138 3b). However, delaying oseltamivir administration at 2 days p.i. abolished its efficacy (Fig. 3c-d),
139 whereas *cis*-aco retained its curative effect under the same conditions in both female C57Bl/6 (Fig.
140 3c-d) and male BALB/c mice (Fig. EV5).

141

142 **In vivo** assessment of pathophysiological mechanisms modulated by *cis*-aco in influenza
143 pneumonia.

144 To better understand how cis-aco achieves its protective effect on survival, we next explored its
145 impact on the pathophysiological responses associated with influenza infection (Fig. 4). Mice were
146 intranasally infected with a lethal dose of influenza A/Scotland/20/1974 (H3N2) and treated with
147 cis-aco two days p.i.. Four days p.i., some mice were euthanized to analyze early events in
148 influenza pathophysiology. Cis-aco treatment resulted in a significant reduction in viral load in
149 lung tissues, with a 1-log decrease compared to controls (p<0.0001; Fig. 4a). Additionally, the
150 relative expression of 50 mediators in the BAL fluids - including pro-inflammatory cytokines,
151 interferons, growth factors, and proteases - was reduced in cis-aco-treated animals (Fig. 4b).
152 Among these, the most pronounced reductions were observed in CCL2/MCP-1, C1qR1, G-CSF,
153 CCL17/Tarc, and CCL20/MIP3α, while levels of resistin, myeloperoxidase, pentraxin 2/serum
154 amyloid P, CXCL-10/IP-10, and CXCL9/Mig remained largely unchanged.
155 In a separate series of experiments, mice were sacrificed 8 days p.i. to assess lung injury caused by
156 IAV. Severe pathology was characterized by increased leukocytes infiltration, including
157 neutrophils and NKT cells. While NK cell counts remained stable, their activation, assessed by
158 CD69 marker quantification, was elevated. Similarly, NKT activation was also significantly
159 heightened upon IAV infection (Fig. 4c). Cis-aco treatment consistently mitigated these responses,
160 significantly reducing neutrophil and NKT cell infiltration (p=0.001 and p=0.05, respectively; Fig.
161 4c). Furthermore, NK and NKT cell activation was markedly lower in cis-aco-treated mice
162 compared to infected, untreated controls (p=0.01; Fig. 4c).

163 Histopathological analysis revealed significant reductions in alveolar wall thickening, hyaline
164 membrane formation, epithelial necrosis, and leukocyte infiltration in IAV-infected mice treated
165 with *cis*-aco compared to untreated animals (Fig. 4d). Quantitative assessment confirmed that *cis-*
166 aco significantly alleviated tissue damage caused by IAV infection (p<0.01; Fig. 4e). To further
167 evaluate IAV-triggered inflammation on a broader scale, we measured NF-κB activity in NF-κB-
168 luciferase transgenic mice infected with IAV and treated or not with *cis*-aco (Fig. 4f and 4g). By
169 day 8 p.i., both airway and systemic inflammation were visible (Fig. 4f, central picture). In
170 contrast, NF-κB activation was significantly reduced in *cis*-aco-treated animals (p<0.03; Fig. 4f,
171 right picture, and 4g). Overall, *cis*-aco suppressed critical aspects of influenza pathogenesis,
172 including viral replication, inflammatory signaling, secretion of inflammatory mediators, and immune
173 cell infiltration.

174

175 **Mechanistic insights into the anti-influenza action of cis-aco in inhibiting influenza virus**
176 **replication and inflammatory signaling.**

177 To gain molecular insights into how cis-aco inhibits the production of viral particles, we analyzed its
178 effects on various stages of IAV replication cycle. IAV is an enveloped virus with a genome made up
179 of negative sense, single-stranded RNA and its life cycle comprises the following stages: (i) entry into
180 the host cell; (ii) nuclear import of ribonucleoproteins (vRNP); (iii) transcription of the viral genome;
181 (iv) replication and translation of viral proteins; (v) nuclear export of vRNP; and (vi) assembly and
182 budding at the host cell plasma membrane.
183

184 *Impact of cis-aco on virus budding and viral protein expression.*

185 Transmission and scanning electron microscopy (TEM and SEM) of IAV-infected bronchial epithelial cells revealed a marked reduction in virus budding following *cis*-aco treatment (Fig. 5a). To determine whether this was due to impaired budding or to reduced viral material production, we quantified viral proteins and mRNA in cells, treated or not with *cis*-aco 4 h p.i.. Confocal microscopy (Fig. 5b-c) and western blotting (Fig. 5d-e) revealed significant reductions (~75%, *p*<0.03, Fig. 5e) in NP, NS1, and PA protein expression at 8 h p.i. while qRT-PCR showed a 10-fold decrease in viral M1 mRNA levels at 6 h p.i. (*p*<0.02, Fig. 5f). Given that *cis*-aco was applied at 4 h p.i., it is unlikely that it affects the early stages of viral entry, including cell fusion and nuclear trafficking (completed within 1 h p.i., [26]). These findings suggest that *cis*-aco inhibits IAV genome transcription, leading to reduced viral protein expression and neo-virion production. This hypothesis was confirmed by a minigenome assay in HEK293T cells, where *cis*-aco treatment inhibited luciferase activity by ~75% (*p* < 0.03, Fig. 5g), indicating interference with IAV polymerase activity.

197

198 *Anti-inflammatory effects of cis-aco*

199 We next investigated whether *cis*-aco's ability to decrease inflammation (e.g., IL-6, Figs. 1c-d) was a secondary consequence of its antiviral action or due to intrinsic immunomodulatory properties.
200
201 IAV infection in lung epithelial cells activates intracellular signalling pathways that upregulate the expression of inflammatory cytokines [27–29]. Consistent with this, we observed increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Protein kinase B (PKB; also known as AKT), and the p65 subunit of the nuclear factor-kappa B (NF-κB) at 20 h p.i compared to non-infected cells (MOCK) (Fig. 6a). *Cis*-aco treatment appeared to inhibit the accumulation of these phosphorylated proteins (Fig. 6a-b). To further assess whether *cis*-aco possesses inherent anti-inflammatory properties, we stimulated lung epithelial cells with different inflammatory agonists: (i) Poly(I:C) (PIC), a TLR3 agonist that mimics viral RNA (ii) Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C signaling; and (iii) TNFα, a major inflammatory cytokine.
205
206
207
208
209
As expected, all agonists induced IL-6 secretion (Fig. 6c). Interestingly, *cis*-aco treatment inhibited
211 IL-6 release in a dose-dependent manner (2.3-fold decrease after TNFα stimulation, p<0.04, and
212 >4-fold decrease after PIC or PMA stimulation, p<0.01; Fig. 6c). These findings highlight the
213 intrinsic anti-inflammatory properties of cis-aco, independently of its antiviral activity.

214

215 In vivo confirmation of the anti-inflammatory activity of cis-aco.

216 To examine the anti-inflammatory effects of *cis*-aco *in vivo*, we used a murine model of acute lung
217 injury induced by bacterial lipopolysaccharide (LPS; [30]). To this end, NF-kB-luciferase
218 transgenic mice were treated or not with *cis*-aco 15 min post-LPS challenge, and NF-kB activity
219 was measured as a proxy of inflammation (Fig. 6d-e). At 24 h post-stimulation, lung inflammation
220 induced by LPS (Fig. 6d, center picture) was significantly reduced in *cis*-aco-treated animals
221 (~75%, p<0.03; Fig. 6d, right picture and Fig. 6e). Consistent with this observation, TNFα levels in
222 the BAL fluids of LPS-challenged mice were also significantly lower in *cis*-aco-treated animals
223 (70% reduction, p<0.008, Fig. 6f). These *in vivo* experiments thus suggest that *cis*-aco can inhibit
224 inflammatory signaling, resulting in decreased production of pro-inflammatory cytokines.

225

226 The anti-influenza properties of *cis*-aco are independent of itaconate

227 *Cis*-aco is a TCA intermediate that can be converted into itaconate by the mitochondrial enzyme
228 *cis*-aco decarboxylase (CAD), also known as ACOD1 or Irg1 (Fig. 7a) [31]. To verify whether the
229 anti-influenza effect of *cis*-aco is dependent on this conversion, we used a silencing approach
230 targeting the CAD gene. This specific siRNA transfection effectively suppressed CAD expression
231 in bronchial epithelial BEAS-2B cells compared to a control scramble siRNA (Fig. 7b.
232 Interestingly, even under CAD silencing conditions, *cis*-aco retained its antiviral and anti-
233 inflammatory properties, as determined through quantification of IAV infectious particles,
234 neuraminidase activity, and IL-6 release (Fig. 7c-e). These findings suggest that *cis*-aco inhibits
235 IAV infection in lung epithelial cells independently of its conversion into itaconate by CAD. These
236 *in vitro* findings were further supported by *in vivo* experiments, where CAD-deficient mice infected
237 intranasally with IAV and treated with *cis*-aco displayed survival rates similar to wild-type mice (Fig.
238 7f) [32].
241 The preceding data demonstrate that cis-aco prevents influenza-induced lung damage by reducing
242 viral infection and inflammation. To assess its therapeutic potential in a clinically relevant context,
243 we considered the typical delay between symptom onset and treatment in human influenza cases
244 [33]. This delay is critical for evaluating the efficacy of anti-influenza therapies in mice in “real-
245 world” conditions. To complement our experimental study, we examined a prospective clinical trial
246 [34] involving patients with community-acquired pneumonia (CAP) caused by influenza A and B
247 viruses. Among the 153 CAP patients, 37% had viral pneumonia, 24% had bacterial pneumonia
248 and 20% had co-infections. IAV was the predominant pathogen, found in 33% of cases. Detailed
249 characteristics of these CAP patients, particularly those with IAV infection, are provided in Table
250 2. The median [IQR] symptom-to-hospitalization time was 3 [2-7] days for all CAP cases and 4 [3-
251 6] days for those attributed to influenza (A or B) infection (Fig. 8a).

252 To simulate this clinically relevant delay, cis-aco treatment was initiated in mice on day 4 p.i., by
253 which time IAV infection had already progressed to pneumonia, with substantial production of
254 inflammatory mediators (Fig. 4b). To more closely mirror clinical conditions, a second dose of cis-
255 aco was administered on day 5 p.i.. In line with previous clinical observations [35, 36] and
256 reinforcing our data from Fig. 3, delayed oseltamivir treatment did not prevent IAV-induced
257 mortality (Fig. 8b). In contrast, cis-aco treatment on days 4 and 5 improved survival, increasing the
258 survival rate from 0% to approximately 50% (Fig. 8b). These findings highlight the potent curative
259 effects of cis-aco against IAV infection within a clinically relevant timeframe.
260 **DISCUSSION**

261 Influenza pathophysiology is multifactorial, involving direct viral cytopathic effects and
262 dysregulation of the host immune response, which together contribute to lung damage. Currently, the
263 treatment of influenza remains an unmet medical need, as the effectiveness of antiviral therapies is
264 limited, especially when administered late in the course of infection. Moreover, strategies that reduce
265 inflammation without controlling viral replication have been associated with increased mortality [37].
266 In this study, we show that cis-aco treatment addresses both key aspects of influenza pathophysiology.
267 Cis-aco effectively inhibits major human influenza strains by impairing viral RNA and protein
268 expression, while simultaneously reducing influenza-induced inflammatory signaling. Guided by a
269 clinical perspective, we demonstrated the protective anti-influenza effects of cis-aco in human lung
270 tissue explants and in infected mice treated within a clinically relevant timeframe, surpassing the
271 efficacy window of the standard of care, oseltamivir/Tamiflu.

272 By screening a range of related metabolites using an in vitro model of IAV infection in human
273 bronchial epithelial cells, the primary target cells for IAV [38–40], cis-aco emerged as the most
274 promising molecule. It is synthesized in the TCA cycle from citric acid through the action of the
275 mitochondrial enzyme aconitate hydratase (also named aconitase) and can be converted by the enzyme
276 cis-aconitate decarboxylase (CAD; also called ACOD1 or Irg1) into itaconate. This latter metabolite
277 is central in linking the innate immune response to cell metabolism, including during IAV infection
278 [31, 41, 42]. However, the anti-influenza effects of cis-aco were not due to its conversion into
279 itaconate, as antiviral and anti-inflammatory activities persisted despite efficient silencing of CAD
280 using siRNA. These in vitro findings were further confirmed in vivo, with CAD-deficient mice
281 infected intranasally with IAV and treated with cis-aco displaying survival rates comparable to wild-
282 type mice. Collectively, these data demonstrate that the anti-influenza properties of cis-aco are
283 independent of its conversion to itaconate.

284 To further elucidate the mechanism of action of cis-aco, we investigated its effects on various
285 stages of the IAV life cycle. Our results show that cis-aco impairs viral polymerase activity, leading to
286 decreased levels of viral RNA and proteins and preventing the formation of new viral particles. These
287 antiviral effects were confirmed against both influenza A and B viruses, which is particularly
288 noteworthy given that current neuraminidase inhibitors are less effective against influenza B viruses
289 compared to influenza A [5, 43]. However, further research is required to fully elucidate the
290 mechanism of cis-aco, given its likely complex effects on both viral replication and inflammatory cell
291 signaling.

292 Severe viral pneumonia is closely associated with lung hyperinflammation, which can lead to
293 acute respiratory distress syndrome (ARDS), as well as significant morbidity and mortality due to
294 respiratory failure. While antiviral therapy and supportive measures, such as mechanical ventilation,
295 are standard treatments, the use of immune-modulatory agents remains controversial [37]. Proponents
296 argue that anti-inflammatory treatments could reduce lung inflammation whereas opponents caution
297 that such treatments might interfere with immune responses, potentially delaying viral clearance and
298 increasing mortality risk. This debate was particularly evident during the A/H1N1 2009-2010
299 influenza pandemic, when anti-inflammatory therapies like steroids were linked to higher mortality
300 rates [44-46]. As a result, anti-inflammatory therapies are not recommended for influenza-related
301 pneumonia, and the disease was excluded from the largest randomized clinical trial assessing steroids
302 in severe community-acquired pneumonia (i.e. the CAPE COD study) [47].

303 Given this context, cis-aco's dual action as both an antiviral and anti-inflammatory agent holds
304 considerable therapeutic potential. By simultaneously reducing hyperinflammation and enhancing
305 viral clearance, cis-aco could offer a comprehensive treatment strategy. It exerts its anti-inflammatory
306 effects by inhibiting key pro-inflammatory pathways, including ERK, AKT, and NF-κB, which are
307 also crucial for viral replication and often hijacked by IAV [48-55]. For instance, IAV activation of
308 the ERK pathway facilitates the nuclear export of vRNPs [51-54]. By modulating these cellular
309 signaling factors, cis-aco may impair IAV replication more potently than existing antiviral drugs that
310 primarily target viral components [56-58]. Furthermore, because cis-aco acts on cellular pathways
311 rather than solely targeting viral factors, it has the potential to limit the risk of drug resistance by
312 reducing selective pressure on the virus itself [59].

313 To confirm the protective mechanisms of cis-aco in a more representative and challenging
314 context, we conducted in vivo experiments using mice infected with a lethal dose of IAV [16, 60]. Cis-
aco mitigated all key aspects of influenza pathology, including reducing viral replication, controlling excessive inflammatory cytokine production, decreasing immune cells recruitment and activation, and minimizing tissue lesions.

Beyond its efficacy against infectious diseases, our study also emphasizes the broader therapeutic potential of cis-aco. Its potent anti-inflammatory properties position it as a promising candidate for managing non-infectious pulmonary inflammatory conditions, such as asthma or fibrosis. Local administration of cis-aco locally via inhalation could enhance its therapeutic effects while reducing potential systemic side effects [66].

Preclinical studies are essential for advancing potential treatments but traditional models - such as mouse and other animal models or in vitro cell cultures - often fail to accurately predict efficacy in patients. To improve clinical translation, we used a multimodal strategy that included: (i) human lung samples from individuals at high risk for influenza (e.g. elderly), (ii) a dosing schedule mimicking clinical treatment (instead of prophylactic or simultaneous treatment, as typically used), and (iii) comparison with an FDA-approved drug. We demonstrated cis-aco antiviral effects not only in human bronchial epithelial cell lines but also in advanced ex vivo lung culture models such as primary epithelial cells and human organotypic lung cultures. The latter preserves the complex lung tissue architecture and cellular diversity, providing a more relevant evaluation context than conventional cell cultures [61]. Next, we compared cis-aco with oseltamivir, the most commonly recommended anti-influenza drug [20]. While oseltamivir is effective at early infection stages, its limited therapeutic window [62, 63] and delayed administration in many patients restricts its efficacy. For example, only 20% of patients receive oseltamivir within 2 days of symptom onset, while viral pneumonia hospitalization typically occurs 4-5 days post-symptom onset [36, 64]). These findings highlight the importance of evaluating anti-influenza treatments at day 4 post-infection to better reflect clinical realities.

Interestingly, in our IAV-infected mouse model, oseltamivir's therapeutic effect declines markedly when administered at 2 or 4 days p.i., aligning with observations in critically ill influenza patients [36]. In contrast, cis-aco demonstrated a significant survival benefit, with ~70% survival when administered at day 2 p.i. and ~50% survival at day 4. These results are promising as similar efficacy
343 is rarely achieved with FDA-approved influenza drugs under similar experimental conditions [65].

344 The superior efficacy of *cis*-aco across a larger treatment window underscores its potential as a major advancement in influenza therapy, allowing greater flexibility and effectiveness.

346 Our current findings significantly advance the identification and understanding of host-derived metabolites with antiviral activity, building on our previous studies on succinate[16]. Succinate mediates its anti-influenza effects by inducing the succinylation of a single amino acid (K87) on the IAV nucleoprotein, thereby impairing the trafficking of viral ribonucleoprotein complexes and disrupting the replication cycle. However, succinate has several limitations compared to *cis*-aco: it requires concentrations five times higher to achieve optimal anti-IAV inhibition, it is ineffective against influenza B viruses and it does not modulate the inflammatory signaling pathways associated with infection [16]. In contrast, *cis*-aco offers the advantage of dual antiviral and anti-inflammatory actions, positioning it as a more potent endogenous molecule for controlling influenza infection. Thus, *cis*-aco, succinate and other metabolites like fumarate [67] and itaconate [68] are increasingly recognized as endogenous mediators with remarkable antimicrobial activities. This discovery points to an ancient, conserved mechanism among primitive organisms, likely evolved to balance cellular proliferation and defense through a unique molecular system. This hypothesis aligns with theories on the origins of life, where metabolites were not only crucial for growth and development but also served as protective agents against environmental challenges [69]. In a broader context, our findings further support the concept that metabokines are part of a wider family of host defence mediators - including cytokines, chemokines, bioactive lipids, and reactive oxygen species - that collectively orchestrate responses to external threats [70, 71].

364 In conclusion, our study demonstrates that *cis*-aco, a host-derived metabolite, exhibits potent anti-influenza properties with significant translational potential due to its natural origin and anticipated low toxicity. As illustrated in Fig. 9, *cis*-aco shows: (i) antiviral activity through inhibition of the IAV polymerase, (ii) strong anti-inflammatory and anti-cell death effects, (iii) broad-spectrum action against both influenza A and B viruses, and (iv) protective efficacy surpassing that of the reference drug oseltamivir. Altogether, our results pave the way for the development of *cis*-aco-based therapies
370 for influenza virus infections Further studies are warranted to investigate *cis*-aco pharmacokinetics and

371 explore its potential in combination with existing antivirals.
372 METHODS

373 **Viruses.** The influenza strains used in this study were initially provided by partner laboratories and
374 subsequently amplified in M. Si-Tahar's laboratory. Specifically, the mouse-adapted influenza
375 A/Scotland/20/74 (H3N2) strain was kindly provided by Prof. Sylvie van der Werf's team at the
376 Pasteur Institute, Paris, France; the influenza A/PR/8/34 (H1N1) strain by Dr. Georg Kochs at
377 Freiburg University, Germany; the pandemic H1N1 strain by Dr. François Trottein at the Center for
378 Infection and Immunity of Lille; and the influenza B/Paris/234/2013 (Yamagata lineage) strain was
379 obtained through the European Virus Archive Global (EVAg)

380

381 **Cell lines culture.** In vitro experiments were performed using human bronchial epithelial BEAS-2B
cells, except for plaque assays which used Madin-Darby Canine Kidney (MDCK) cells, and
382 minigenome assay which used HEK-293T. These cells were cultured in either F-12K Medium (BEAS-
383 2B) or MEM (HEK-293T and MDCK) supplemented with 10% FBS, 100 U/ml penicillin, and 100
384 µg/ml streptomycin. All cells were mycoplasma-free. BEAS-2B cells were infected in medium
385 without FBS for 4 h with IAV at MOI=1 (except for TEM and SEM analysis, for which an MOI=5
386 was applied). Cells were also stimulated in medium with FBS with 2 µg/ml Poly(I:C) or in medium
387 without FBS with 2 µg/ml PMA or with 20ng/ml TNFα. Four hours after the challenge, cells were
388 washed with PBS and incubated for 4 h or 16 h with different concentrations of metabolites (cis-
389 aconitate derivative cis-aconitic acid, trans-aconitic acid, itaconic acid, glucose, pyruvate, oxaloacetic
390 acid, fumarate, isocitric acid) diluted in medium without FBS.
391

392

393 **Studies involving human participants.** Informed consent was obtained from all patients in accordance
394 with the Helsinki Declaration. Tissue and cell collections were declared to the French Ministry of
395 Graduate Study, Research, and Innovation (DC-2008-308, MESRI). Lung lobes were collected
396 immediately following surgical resection at CHRU of Tours. All experiments adhered to The Code of
397 Ethics of the World Medical Association and were approved by the Ethics Committee of the CHRU of
398 Tours. Lung donors for this study varied in age, gender, medical history, and the cause of resection
399 (see Table 1). Prospective data collection presented in Table 2 was conducted in a single center over
400 an 18-month period. The study complied with French law for observational studies and to the
401 STROBE guidelines for observational studies. The study was approved by the ethics committee of the
402 French Intensive Care Society (CE SRLF 13-28), was approved by the "Commission Nationale de
403 l'Informatique et des Libertés" (CNIL) for the treatment of personal health data. We gave written and
404 oral information to patients or next-of-kin. Patients or next-of-kin gave verbal informed consent, as
405 approved by the ethic committee. Eligible patients were adults hospitalized in ICU for CAP.
406 Pneumonia was defined as the presence of an infiltrate on a chest radiograph and one or more of the
407 following symptoms: fever (temperature ≥38.0°C) or hypothermia (temperature <35.0°C),
408 cough with or without sputum production, or dyspnea or altered breath sounds on auscultation.
409 Community-acquired infection was defined as infection occurring within 48 h of admission. Cases of
410 pneumonia due to inhalation or infection with pneumocystis, pregnant women and patients under
411 guardianship were not included. Cases with PaO2/FiO2 ≥ 60 mmHg in ambient air or with the need for
412 oxygen therapy ≤4 L/min or without mechanical ventilation (invasive or non-invasive) were not
413 included.

414

415 **Human primary bronchial epithelial cells (PBEC) culture.** PBEC were isolated from normal
416 bronchial tissues of lung cancer patients undergoing lobectomy at the CHRU of Tours. The cancer-free
417 tissues were washed and incubated for 2 h at 37°C with 0.018% (w/v) proteinase XIV in Ca2+/Mg2+
418 free Hank's Balanced Salt Solution. Epithelial cells were scraped from the luminal surface, washed,
419 and cultured in serum-free keratinocyte medium supplemented with 2.4 ng/ml epidermal growth
420 factor, 25 µg/ml bovine pituitary extract, 1 µM isoproterenol, 100 U/ml penicillin, and 100 µg/ml
421 streptomycin on 6-well plates coated with 30 µg/ml PureCol, 10 µg/ml bovine serum albumin, and 5
422 µg/ml fibronectin. During the first week, 1/500 Primocin was added to the medium. After reaching
423 near-confluence, cells were trypsinized and stored in liquid nitrogen.

424 For mucociliary differentiation, PBEC were cultured in PneumaCult EX medium, with a 3-4 day
425 proliferation step before stimulation with 2 µg/ml Poly(I:C) or 50 nM PMA. The stimulation medium
426 was a 1:1 mixture of BEGM and complete DMEM/F12, supplemented with 100 U/ml penicillin, 100
427 µg/ml streptomycin, 12.5 ml 1M HEPES, and 5 ml GlutaMAX™.

428 For ALI differentiation, PBEC were cultured submerged on semipermeable transwell inserts (0.4 µm
429 pore size) coated with collagen and fibronectin. Once confluent, the apical medium was removed, and
430 cells were cultured at ALI for 3 weeks.

431

432 **Human organotypic lung culture (OLC).** The preparation of OLC was carried out according to a
433 protocol derived from [73], using human lung resections from surgical patients at the CHRU of Tours,
434 collected inHibernate medium containing 1/500 Primocin. Lung explants were sliced using the
435 McIlwain® tissue chopper (Campden Instruments) at 500 µm thickness and placed back in Hibernate
436 medium with 1/500 Primocin for slice dissociation under a dissection microscope before their
437 immediate transfer at ALI. Individualized OLC were placed on semipermeable Millicell® cell culture
438 inserts with PTFE membranes (0.4 µm pore size; Merck) already pre-activated with 1 ml of OLC
439 culture medium. The culture medium is a volume-to-volume mix of BEGM and DMEM medium with
440 100 U/ml penicillin and 100 µg/ml streptomycin. OLC were infected by drop deposition with 2.10⁴
441 pfu of A/Scotland/20/74 (H3N2) virus (IAV). After 2 h, OLC were treated with 3.4 mM of cis-aco. At
442 48h p.i., the OLC subnatants were collected to measure viral titers.

443

444 ***Animal care, handling and study approvals.*** C57Bl/6 female mice (~8 weeks old) were purchased
445 from Centre d'Elevage R. Janvier (Le Genest Saint-Isle, France) and housed under specific-pathogen-
446 free conditions at Tours University animal facility, (France), with *ad libitum* access to food and water.
447 NF-κB luciferase transgenic BALB/C mice were generated by backcrossing NF-κB luciferase
448 transgenic B10.A mice (a kind gift from Prof. Richard Flavell, Howard Hughes Medical Institute) with
449 BALB/C mice to produce transgenic mice with white fur, minimizing light absorption. C57BL/6NJ
450 wild-type mice and C57BL/6NJ-Acod1em1(IMPC)J/J (CAD-/-) mice, deficient in CAD expression,
451 were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All mice were maintained and
452 bred at the Pasteur Institute of Lille, France (agreement B59-350009).
All procedures involving C57Bl/6 mice were conducted in compliance with European animal welfare regulations. Experiments adhered to the ethical standards set by the French government and were approved by our local and national ethics committees (CEEA.19, APAFIS#201604071220401.V2-4885, 2016111512369894 V3 - 7590). Studies using NF-κB transgenic BALB/c mice were approved by the Animal Care and Use Committee at the "Centre de Recherche de Jouy-en-Josas" (COMETHEA) under the relevant institutional authorization (Ministère de l'éducation nationale, de l'enseignement supérieur et de la recherche), authorization number: 2015100910396112v1 (APAFIS#1487). Experiments involving C57BL/6NJ-Acod1em1(IMPC)J/J (CAD-/-) mice deficient in CAD expression were ethically approved by the French Committee on Animal Experimentation and the Ministry of Education and Research (APAFIS#10232-2017061411305485 v6, approved on 14/09/2018).

**Neuraminidase (NA) assay.** The assay measures the release of a 4-methylumbelliferone fluorescent product from the 2'-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (MU-NANA) substrate. 67 µL of cell supernatant was incubated with 33 µL of MU-NANA (50 µM) in black 96-well black micro-plates. Fluorescence was immediately measured in a kinetic assay over 1 h at Ex = 355 nm and Em = 460 nm.

**Protein-array and ELISA.** Protein array and DuoSet ELISA (Human IL-6, and mouse MPO and ALT) were performed according to the manufacturer's instructions (R&D Systems or Clinisciences for ALT ELISA).

**siRNA transfection.** 1.25x10⁵ BEAS-2B cells were seeded in a 12-wells plate the day before transfection with specific siRNA or negative control scramble siRNA. Each siRNA stock was diluted to 50 nM in 100 µL of optiMEM (Gibco) containing RNAiMax reagent (ratio siRNA:RNAiMax of 1:3). After 5 min of incubation at room temperature, 100 µL of each siRNA-mix was added to 900 µL of fresh medium per well. Gene knockdown efficacy was evaluated by RT-qPCR after 48 h (medium replaced after 24 h).
481

482

**RNA isolation and RT-qPCR.** Cells in 6-well plates were lysed with 350 µL RA1 buffer (Macherey-Nagel) and 1/100 diluted β-mercaptoethanol. Total RNA was extracted using the NucleoSpin® RNA

483

kit (Macherey-Nagel), including DNase digestion. RNA concentration was measured with a Nanodrop

484

2000. cDNA synthesis was performed from 500 ng RNA using the High-Capacity cDNA Reverse

485

Transcription Kit, with IAV M1-specific sense primer or random primers. mRNA levels were

486

quantified by RT-qPCR on a LightCycler 480 (Roche) using 10 ng cDNA, 10 µM primers (M1: sense

487

5'-AAG ACC AAT CCT GTC ACC TCT GA-3', antisense 5'-CAA AGC GTC TAC GCT GCA GTC

488

C-3'; CAD: sense 5'-CGT GTT ATT CAG AGG AGC AAG AG-3', antisense 5'-AGC ATA TGT

489

GGG CGG GAG-3'), and 10 µL SYBR® Premix Ex Taq in a 20 µL reaction volume. Reactions were

490

performed in duplicate, and the thermal protocol included initial denaturation at 95°C for 30 s,

491

followed by 40 cycles of denaturation (95°C for 5 s) and annealing/extension (60°C for 20 s). Melting

492

curves were generated to verify reaction specificity.

493

494

495

**IAV titration by plaque-forming units assay.** Titrations in culture media and mouse lungs were

496

performed as previously described [74].

497

498

**Transmission electron microscopy.** Cells were washed with PBS, detached using trypsin, and

499

centrifuged. They were fixed for 24 h in 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M

500

phosphate buffer (pH 7.2). After washing in PBS, cells were post-fixed with 2% osmium tetroxide

501

for 1 h. Samples were dehydrated in graded ethanol and propylene oxide solutions, then

502

impregnated with a 1:1 mixture of propylene oxide/Epon resin and left overnight in pure resin. The

503

samples were embedded in Epon resin and polymerized at 60°C for 48 h. Ultra-thin sections (90

504

nm) were cut using a Leica EM UC7 ultramicrotome, stained with 2% uranyl acetate and 5% lead

505

citrate, and analyzed with a JEOL 1011 transmission electron microscope using Digital Micrograph

506

software.

507
Scanning electron microscopy. Cells were washed with PBS, detached using trypsin, and centrifuged. They were fixed for 24 hours in 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). After washing in PBS, samples were post-fixed with 2% osmium tetroxide for 1 ho. Samples were dehydrated in a graded ethanol series, then dried in hexamethyldisilazane. The dry samples were placed onto carbon disks and coated with 40 Å of platinum using a GATAN PECS 682 apparatus. Observations were made with a Zeiss Ultra Plus FEG-SEM scanning electron microscope.

Confocal fluorescence microscopy. BEAS-2B cells were cultured in 12-well plates on cover slides. After treatments, cells were fixed with 4% formaldehyde for 30 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 30 min. After blocking with PBS containing 1% BSA and 0.1% Tween 20 for 1 h, cells were stained for 2 h at room temperature with anti-NP-FITC (1/30), anti-NS1 (1/200), and anti-PA (1/50) antibodies. Anti-rabbit-AF488 (2 h at room temperature) served as the secondary antibody for NS1, and anti-mouse-AF488 was used for PA.

PBECs were fixed with 4% formaldehyde for 10 min at 4°C, then permeabilized with cold methanol for 10 min at 4°C. After blocking with PBS containing 1% bovine serum albumin and 0.3% Triton X-100 for 10 min, cells were stained for 2 h at room temperature with anti-p63 (1/100), anti-Tubulin (1/100), and anti-Mucin 5AC (1/1000) antibodies. Secondary antibodies used were anti-rabbit-AF546 (for p63), anti-mouse-AF488 (for Mucin 5AC), and anti-mouse-AF647 (for Tubulin). Nuclei were stained with NucBlue reagent for 5 min.

OLC were fixed overnight at 4°C in 4% formaldehyde. Aldehyde groups were quenched with two 10-min incubations in PBS with 0.1% glycine, followed by permeabilization with 0.5% Triton X-100 in PBS for 15 min at room temperature. After 2 h of saturation in PBS with 1% BSA, 0.5% Triton X-100, cells were stained overnight at 4°C with anti-Tubulin (1/500) and anti-NP-FITC (1/50) antibodies. The secondary antibody anti-mouse-AF647 was applied for 2 h at room temperature for Tubulin staining. Nuclei were stained with Hoechst reagent (1/2000) for 10 min. Samples were analyzed and 3D reconstructions were generated using a Leica SP8 confocal microscope with Leica LasX Life Sciences Software.
536
537 **Western-blotting.** Cells in 6-well plates were lysed with 150 µL of RIPA buffer (with protease inhibitors or PhosphoSafe Extraction Reagent). After centrifugation at 12,000 g for 10 min, protein concentration was measured using the Pierce™ BCA Kit. Ten µg of protein were mixed with Laemmli buffer, heated at 100°C for 5 min, and separated on 12% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes and probed with primary antibodies: anti-NP (1/500), anti-NS1 (1/1000), anti-PA (1/1000), anti-(P)ERK1/2 (1/2000), anti-(P)AKT (1/1000), anti-(P)p65 (1/3000), or anti-β-actin (1/5000). HRP-conjugated secondary antibodies were used for detection, followed by ECL. Protein bands were visualized using an automated imaging system and analyzed with FUJI FILM MultiGauge software.

546
547 **Minigenome assay.** The minigenome studies were performed in 24-well plates. Briefly, HEK-293T cells were transfected with together with 50 ng of pRF483-PA-RT, 50 ng of pRF483-PB2-RT, 100 ng of pRF483-NP-RT, 50 ng of pRF483-PB1-RT and 150 ng of reporter plasmid pPoll-WSN-NA-firefly luciferase which contains a firefly luciferase ORF flanked by the noncoding regions of the NA segment under the control of human polymerase I promoter. As a negative control, HEK-293T cells were transfected with the same plasmids, with the exception of the PB1 plasmid. The procedure used the Fugene HD transfection reagent according to the manufacturer's instructions. 20h post - transfection cells were treated with different concentrations of *cis*-aco. 48 h post-transfection, cells were washed twice with PBS and lysed in 100 µl of lysis buffer provided with the Firefly Luciferase Assay System. Firefly luciferase activities were measured on 20 µl of cell extracts, using the Firefly luciferase substrate provided with the above-mentioned kit and a Centro luminometer (Berthold).

558
559 **Incucyte® and cell death assays.** PBEC were seeded in 96-well plates in the presence of 2 µM of the fluorescent dye SYTOX™, which binds to DNA and rapidly penetrates dying cells upon membrane permeabilization, and then infected with IAV. Real-time cell death assays were performed using an IncuCyte® two-color incubator imaging system. The images obtained were analyzed using the software supplied with the IncuCyte imager, which enables precise analysis of the number of
564 SYTOX™-positive cells present in each image. Experiments were carried out using a minimum of two
565 separate wells for each experimental condition and a minimum of four image fields *per* well.

566

567 *Animal infection and fluid collection.* 7-week-old C57Bl/6 mice (female or male) were intranasally
568 challenged with 10 µg LPS or 200 pfu A/Scotland/20/74 (H3N2) IAV, and treated with 0.6 mg *cis*-aco
569 (30 mg/kg) at various time points. Blood was collected on the sacrifice day, centrifuged at 10,000g for
570 10 min for serum analysis or heparinized for analysis on a ProCyte Dx hematocytometer. Airways
571 were lavaged with 4x0.5 ml PBS for BAL collection, and lungs were perfused with 10 ml PBS
572 injected into the heart. The left lung was fixed in 4% paraformaldehyde for histology. Right lungs
573 were digested enzymatically using the gentleMACS dissociator, and after centrifugation, lung
574 suspensions and BAL fluids were stored at -80°C for subsequent inflammatory mediator analysis.
575 Leukocytes were isolated, red blood cells lysed, and leukocytes counted *via* flow cytometry.

576 BALB/c NF-κB transgenic mice were used in Ronan Le Goffic's lab [75]. Mice were challenged with
577 10 µg LPS or infected with 300 PFU A/Scotland/20/74 (H3N2) IAV. At 1 or 8 days p.i., luciferin
578 (0.75 mg/kg) was administered intranasally, and luciferase activity was measured using the IVIS
579 system.

580 CAD-deficient C57Bl/6 mice were used in Priscille Brodin and François Trottein's labs. CAD-
581 deficient and wild-type mice (20 animals *per* group) were infected with 100 PFU A/Scotland/20/74
582 (H3N2) IAV and treated with 30 mg/kg *cis*-aco 20 min p.i. Body weight loss and survival were
583 monitored daily.

584

585 *Genomic DNA Extraction and 16S rRNA Sequencing Analysis.* Genomic DNA was extracted from
586 mouse fecal pellets and 16S rRNA sequencing was performed as previously described [76]. The
587 taxonomy of each amplicon sequence variant (ASV) was assigned based on the SILVA database
588 v1.3.8 [77]. ASVs unclassified at the kingdom or phylum level or ASVs classified as Eukaryota or
589 Mitochondria were excluded. Aitchison distances were measured using the microbiome
590 (http://microbiome.github.io) and phyloseq packages [78] in RStudio v4.1.2.

591
592 **Flow cytometry analysis.** BAL, lungs, or human bronchial epithelial cells were dispensed into 593 round bottomed 96-well plates and centrifuged at 300 g at 4°C for 5 min. Samples were further 594 stained using specific antibodies and appropriate isotype controls (listed in Table 3). For each 595 antibody, one well was seeded for the Fluorescence Minus One Control. Dead cells were excluded 596 using the LIVE/DEAD cell staining kit (Invitrogen). Flow cytometry data were acquired on a 597 MACSQuant® Analyzer (Miltenyi Biotec) and analyses were performed using the VenturiOne 598 software (Applied Cytometry).

599

600 **Histopathology.** Lungs were collected after BAL and airways were washed and placed in 4% 601 paraformaldehyde in PBS. Lung sections of approximately 4 µm thickness were cut and stained with 602 hematoxylin-eosin at the LAPV (Amboise, France). A study pathologist examined the tissue sections 603 using light microscopy on a Leica Diaplan microscope in a blinded experimental protocol. All 604 histopathological findings were graded in a semi-quantitative fashion on a scale of 0 to 4 (0: absent, 1: 605 mild, 2: moderate, 3: severe, 4: extremely severe).

606

607 **Cell proliferation and cytotoxicity assays.** Cells in 96-well plates were washed twice with PBS and 608 incubated for 1 h at 37°C with 100 µL of MTS reagent diluted 1/5 for the cell proliferation test. 609 Optical density was measured at 490 nm. Cells were stained for 15 min at 4°C with Live/Dead 610 (1/1000°), anti-Ki67(1/100°) or MitoTracker(1/5000°), or 5 min at 37°C with 611 Dihydrorhodamine(DHR)-123(1/100°) before flow cytometry analysis.

612

613 **Statistical analyses.** Statistical analyses were conducted using GraphPad Prism on raw data. Data are 614 presented as mean ± SEM. The statistical values, including the number of replicates (n) and the 615 statistical test used, are detailed in the figure legends. \*p < 0.05, \*\*p < 0.005, \*\*\*p < 0.0005, \*\*\*\*p < 0.0001. For *in vitro* experiments, “n” refers to the number of separate experiments, while for *in vivo* 616 studies, “n” refers to the number of individual animals.

618

619 **DATA AVAILABILITY STATEMENT**
620 The datasets generated during and/or analysed during the current study are available from the
corresponding author on reasonable request.
621

622

623 **CONTRIBUTIONS DATA**

624 **AC** Data curation, Investigation, Conceptualization, Formal analysis, Supervision, Validation,
625 Visualization, Methodology, Writing - original draft; Writing - review & editing. **VV, AW, DD** Data
curation, Investigation, Formal analysis, Validation, review & editing, Methodology. **LG, SH**
626 Investigation, Methodology. **LC, FJ, CM, LPC** Investigation, Methodology, Resources, Writing –
627 review & editing. **RLG** Resources, Investigation, Validation, Writing - review & editing. **BDC**
628 Resources, Investigation, Validation. **AM, EH, FT** Resources, Validation, Methodology. **JBG, DT,**
629 **MGS** Resources, Investigation, Formal analysis, Validation. **TB** Investigation, Methodology,
630 Validation, review & editing. **DF** Investigation. **CP** Formal analysis, Resources, Validation; review &
631 editing, review & editing. **AG** Supervision, Validation, Writing - review & editing. **MS-T**
632 Conceptualization, Formal analysis, Resources, Validation, Supervision, Funding acquisition,
633 Supervision, Project administration, Writing - review & editing.
634

635

636

637 **FUNDING**

638 This work was partially supported by the following grants: Inserm, Université de Tours, Région
639 Centre-Val de Loire FLU-MET#2018-00124196 (to M.S.-T.), FEDER Euro-FERI (to M.S.-T. and
640 C.P.) and ANR-21-CE18-0061 ("SuccesS") (to M.S.-T.) and ANR-22-ASTR-0021
641 ("VIROMETABLOCK") grants (to M.S.-T., LPC and CM).

642

643 **ACKNOWLEDGMENTS**

644 The authors are grateful to Pr. Pieter Hiemstra (Laboratory for Respiratory Cell Biology and
645 Immunology of the Department of Pulmonology of the Leiden University Medical Center (LUMC))
646 for his collaboration in setting up the *ex vivo* culture model of primary human lung epithelial cells.
647 Also, we thank Benoit Briard and Sandra Khau (CEPR, Inserm, Tours) for their advice with the

648 Incucyte® and cell death assays.

649 DECLARATION OF COMPETING INTEREST: M.S-T and A.G. declared the deposit of a patent

650 application related to the anti-influenza activity of *cis*-aconitate (reference: WO 2024126742)
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867

868
869 **FIGURE LEGENDS**

870

871 **Figure 1. Anti-influenza properties of cis-aco among glycolysis and TCA cycle metabolites.**

872 **(a-d)** Human bronchial epithelial (BEAS-2B) cells were infected with influenza A/Scotland/20/74

873 (H3N2) virus at MOI=1 (IAV) or left uninfected (MOCK) for 4 h, then treated (Metabolite) or

874 untreated (Medium) with 3.4 mM of TCA cycle or glycolysis metabolites for 16h. **(a)** Metabolites

875 tested: (cis-aco (CA), itaconic acid (Ita), oxaloacetic acid (Oxa), isocitric acid (IsoC), citrate (Cit),

876 fumarate (Fum), pyruvate (Pyr) or glucose (Glc)). **(b)** Viral particle production assessed by

877 neuraminidase activity assay. **(c)** hIL-6 levels in cell supernatants measured by ELISA. **(d)** Immune

878 mediator levels in cell supernatants determined using a protein-array. Pink bars represent fold changes

879 in mediator secretion induced by IAV infection, and blue bars represent fold changes in IAV + CA

880 conditions relative to MOCK.

881 **(e-h)** BEAS-2B cells were infected with: **(e)** influenza A/pandemic/2009 H1N1 (H1N1p) strain, **(f)**

882 A/Puerto Rico/8/1934 H1N1 (PR8) virus, **(g)** influenza B Yamagata (B/Paris/234/2013) virus (IBV) or

883 **(h)** A/Scotland/20/74 (H3N2) strain. At 4 hours p.i., cells were washed and treated or not for 16 h with

884 3.4 mM of cis-aco (CA). Production of infectious viral particles in cell supernatants was quantified by

885 plaque-forming unit assay.

886 **(i-j)** PBEC in two-dimensional (2D) liquid culture were infected with influenza A/Scotland/20/74

887 (H3N2) virus at MOI=1 for 4 h, then treated or not with varying concentrations of cis-aco (CA) for 44

888 h. At 48 h p.i., neuraminidase activity **(i)** and hIL-6 levels **(j)** were measured in cell supernatants to

889 assess viral particle production and pro-inflammatory cytokine release.

890 Data are presented as the mean ± SEM. Results represent cumulative data from 1 **(d)**, 7 **(b)**, 3 **(g)**, or 4

891 **(c, e, f, h, i, j)** independent experiments. PBEC data **(i-j)** are shown as the mean ± SEM from duplicate

892 samples of PBEC from 4 independent individuals. Statistical analyses were performed using the

893 Kruskal-Wallis test with Dunn's multiple comparison test or one-way ANOVA **(i-j)**.

894

895 **Figure 2. Cis-aco demonstrates antiviral and anti-inflammatory properties in human primary

896 bronchial epithelial cells (PBEC) and human organotypic lung cultures (OLCs) under air-liquid
897 **interface (ALI) conditions. (a-d)** PBEC in three-dimensional (3D) culture were infected with
898 influenza A/Scotland/20/74 (H3N2) virus at an MOI=1 for 4 h, followed by treatment with or without
899 5.7 mM *cis*-aco (CA) for 44 h. **(a)** At 48 h p.i., tissue composition was validated using 3D confocal
900 microscopy. Basal cells (yellow), goblet cells (green), ciliated cells (red) and nuclei (blue) were
901 labeled to confirm tissue structure. **(b)** Neuraminidase activity and hIL-6 levels were measured in
902 apical (c) or basal (d) cell supernatants.

903 (e-g) OLC were infected with 2 x 10⁴ pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and
904 treated or not with 3.4 mM of *cis*-aco at 2 h p.i.. (e) Alveolar structures were visualized using light
905 microscopy (scale bar: 500 µm). (f) IAV infection was confirmed via confocal microscopy, with viral
906 nucleoprotein (green), nuclei (blue) and α-tubulin (red) labeled at 48h p.i. (scale bar: 10 µm). (g) Viral
907 titers were measured in OLC supernatants at 48 h p.i..

908 Data are presented as the mean ± SEM. PBEC data (a: microscopy) represent duplicates from 3
909 independent patients analyzed in a single experiment. Panels (b-d) represent 4 independents
910 experiments. Representative images of OLC from 2 patients are shown in (e-f) and viral titers in (g)
911 were measured using pooled supernatants from 5 OLC derived from 2 patients. Statistical analyses
912 were conducted using the Wilcoxon test (b) or Kruskal-Wallis test (c-d).

913

914 **Figure 3.** *Cis-aco provides superior protection against influenza infection in mice compared to Oseltamivir, even with delayed treatment.* Seven weeks old female C57Bl/6 mice were intranasally 915 infected with 200 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated intranasally with 916 either 30 mg/kg of *cis*-aco (CA, blue) or 20 mg/kg of Oseltamivir (Osel, orange) at: (a-b) 20 min p.i. 917 or (c-d) 2 days p.i. Animal survival rates (a,c) and body weight loss (b,d) were monitored daily. Data 918 are presented as the mean ± SEM from at least 2 independent experiments. The number of mice in 919 each treatment group (“n”) is indicated. Statistical analysis was performed using the Log-rank 920 (Mantel-Cox) test. 921

922 **Figure 4.** *Cis-aco reduces viral load, lung inflammation and tissue damage in IAV-infected mice.*
923 (a-e) Seven-week-old female C57Bl/6 mice were intranasally infected with 200 pfu of influenza
924 A/Scotland/20/74 (H3N2) virus (IAV) and treated or not 2 days p.i. with 30 mg/kg of cis-aco (CA)
925 intranasally. Mice were euthanized at either 4 or 8 days p.i.. (a-b) At day 4 p.i., (a) the viral load in
926 lung tissues was quantified using a PFU assay, and (b) the levels of 50 mediators were measured in
927 BAL fluids. (c-e) At day 8 p.i., (c) the number and activation status of immune and inflammatory cells
928 in BAL fluids were determined by flow cytometry. (d-e) Lung sections were stained with H&E and
929 tissues lesions were assessed and scored (scale bar: 200 µm).

930 (f-g) NF-κB transgenic Balb/c mice (n=9) were intranasally infected with 300 pfu of IAV and treated
931 2 days p.i. with 30 mg/kg CA. At 8 days p.i., mice were anesthetized, and luciferin was administered
932 intranasally (0.75 mg/kg). Bioluminescence was quantified using the IVIS imaging system.

933 Statistical analyses were performed using the one-way ANOVA test (c), or the Mann-Whitney test (a,
934 e, g). Data are presented as the mean ± SEM and include results from 1 experiment (b,f) or 3 (a) or 2
935 (c,d,e,g) independent experiments.

936

937 **Figure 5. Anti-influenza virus properties of cis-aco involve the inhibition of viral polymerase**
938 **activity.** BEAS-2B cells were infected with influenza A/Scotland/20/74 (H3N2) virus at a MOI=5 (a)
939 or MOI=1 (b-f) for 4 h, then washed and treated with 3.4 mM of cis-aco (CA) or left untreated
940 (Medium). (a): Representative images from transmission electron microscopy (upper panel) and
941 scanning electron microscopy (lower panel) show IAV particles budding at 20 h p.i., indicated by
942 arrows (scale bar: 1 µm).

943 (b-e) At 8 h p.i., viral protein (green) expression and trafficking were analyzed by (b-c) confocal
944 microscopy (scale bar: 20 µm) and (d-e) Western blotting to detect viral NP, NS1, and PA proteins. (c)
945 Raw integrated density (RawIntDen), calculated as the sum of all pixel values in the region of interest,
946 was measured and normalized to the mean of the IAV condition for each experiment. (e) Relative
947 protein levels were normalized to the mean value of “IAV condition” samples, with β-actin as a
948 loading control.

949 (f) At 6 h p.i., IAV transcription was quantified by RT-qPCR, measuring M1 viral mRNA levels. (g) A
950 minigenome assay was performed in HEK-293T cells to test the effect of cis-aco on viral polymerase
951 activity. Cells were transfected with plasmids encoding PA, PB1, PB2, NP, and the reporter plasmid
952 pPolI-WSN-NA-firefly luciferase. At 20 h post-transfection, cells were treated with 0 or 3.4 mM cis-
953 aco (CA) and luciferase activity was measured at 48h post-transfection.

954 Results are presented as the mean ± SEM from 3 (a, b, c), 4 (d, e, g), or 5 (f) independent experiments.

955 Statistical analyses were performed using the Kruskal-Wallis test with Dunn's multiple comparison
956 test (g), the Mann-Whitney test (c), or the Wilcoxon matched-pairs rank test (e,f).

957

958 **Figure 6. Cis-aco reduces pro-inflammatory responses and signaling.** BEAS-2B cells were
959 infected or not with the influenza A/Scotland/20/74 (H3N2) virus at an MOI=1 for 4 h, and
960 subsequently treated or not with 3.4 mM of cis-aco (CA) for 16 h (a-b). (a) Representative Western-
961 blotting showing phosphorylated forms (P) of ERK1/2, AKT and p65 proteins with β-actin as a
962 loading control. (b) Signal quantification of proteins and normalization was performed for each
963 experiment.

964 (c) BEAS-2B cells were stimulated or not (MOCK) with 2 µg/mL Poly(I:C) (PIC), 50 nM Phorbol 12-
965 myristate 13-acetate (PMA), or 20 ng/mL Tumor necrosis factor alpha (TNFα) for 4 h, followed by
966 treatment with increasing doses of cis-aco (CA) for 16 h. IL-6 levels in cell supernatants were
967 measured by ELISA.

968 (d-f) NF-κB transgenic Balb/c mice were instilled with 10µg LPS and treated intranasally with either
969 PBS or 30 mg/kg cis-aco (CA) 15 min post-stimulation. At 24h post-instillation, bioluminescence was
970 measured using the IVIS imaging system after intranasal administration of luciferin.

971 Data are presented as the mean ± SEM. Results reflect cumulative data from a single experiment (d-
972 f; n=4 mice per condition) or at least 4 independent experiments (a-c). Statistical analyses were
973 performed using the Friedman test (b), Kruskal-Wallis test (c), or Mann-Whitney test (e-f)

974

975 **Figure 7. Anti-influenza activity of cis-aco is independent from itaconate. (a-e) BEAS-2B cells**
976 were transfected with either cis-aconitate decarboxylase (CAD) or control (scramble) siRNA. At 48 h
977 post-transfection, cells were infected or not with A/Scotland/20/74 (H3N2) virus (IAV) at an MOI=1
978 for 4 h, and subsequently treated or not with 3.4 mM cis-aco (CA) for 16h. (a) CAD catalyzes the
979 decarboxylation of cis-aco to produce itaconate. (b) Gene knockdown was confirmed by RT-qPCR. (c,
980 d) IAV particles production was measured by a plaque-forming units (pfu) assay (c) and a
981 neuraminidase activity assay (d). (e) IL-6 levels in cell supernatants were quantified by ELISA. (f)
982 CAD-deficient and wild-type (WT) mice were infected intranasally with 100 pfu of A/Scotland/20/74
983 (H3N2) virus (IAV) and treated intranasally or not with 30 mg/kg of cis-aco (CA) 20 min p.i.. Animal
984 survival was monitored daily.

985 Data are presented as the mean ± SEM and are cumulative from a single experiment (f), or 3 (b), or 4
986 (c-e) independent experiments. Statistical analyses were performed using the Kruskal-Wallis test with
987 Dunn's multiple comparison test (a-e), or the Log-rank (Mantel-Cox) test (f).

988 **Figure 8.** *Cis-aco protects mice from influenza infection within a clinically relevant time frame.*

990 (a) Time between the symptoms onset and the first hospital admission in patients hospitalized for
991 community acquired pneumonia (CAP). Each dot represents an individual patient with the median
992 indicated by the black line. (b) Seven-week-old female mice were intranasally infected with 200 pfu of
993 influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated intranasally 4 and 5 days p.i. with 30
994 mg/kg cis-aco (CA, blue) or 20mg/kg Oseltamivir (Osel, orange), or left untreated (pink). Survival
995 was monitored daily. Data in (b) are presented as the mean ± SEM and are cumulative from 3
996 independent experiments; the number of mice (“n”) is indicated. Statistical analysis was performed
997 using the Log-rank (Mantel-Cox) test.

998 **Figure 9.** Anti□influenza mechanism of action of *cis*-aco. *Cis*-aco (CA) impairs IAV polymerase
1000 activity, reducing viral mRNA expression and protein synthesis, thereby preventing effective virus
1001 replication. Additionally, *cis*-aco downregulates inflammatory pathways triggered by various stimuli.
1002 *In vivo*, these combined antiviral and anti-inflammatory effects decrease viral load and mitigate
1003 excessive inflammation, providing superior protection against mortality compared to the reference
1004 anti-influenza drug Oseltamivir.

1005
1006 EXPANDED VIEW CONTENT

1007 **Figure EV1. Tolerance of bronchial epithelial cells to metabolite exposure.** (a) BEAS-2B cells were treated with medium alone or with 3.4 mM *cis*-aco (CA), itaconate (Ita), oxaloacetate (Oxa), isocitrate (IsoC), citrate (Cit), fumarate (Fum), pyruvate (Pyr) or glucose (Glc) for 16h. Cytotoxicity was assessed using the MTS assay. (b–e) BEAS-2B cells were treated (blue) or not (white) with 1.2 or 2.3 or 3.4 mM *cis*-aco for 20 h (b) or with 3.4 mM *cis*-aco for 6 or 24h (c–e). Effects on cell viability (b), proliferation (c), mitochondrial labeling (d), and ROS production (d) were analyzed using Live Dead, Ki67, mitotracker and DHR123 staining respectively.

1014 (f) PBEC were treated with 2.3, 3.4 or 5.7 mM *cis*-aco for 16h, and cytotoxicity was assessed using the MTS assay.

1016 Data are presented as the mean ± SEM from 4 (b–f) or 5 (a) independent experiments. Statistical analyses were performed using the Kruskal-Wallis test with Dunn's multiple comparison test (a–c) or the Friedman test with Dunn's multiple comparison test (d–f).

1019

1020 **Figure EV2. Comparison of anti-influenza effects of *cis*-aco and *trans*-aco.** BEAS-2B cells were incubated with medium alone or with increasing concentrations of *cis*-aconitic (CA) or *trans*-aconitic acid (TA) for 20 h. The chemical structures of these metabolites are shown in (a). (b) Cell viability was assessed using the MTS assay. A viability threshold of 80% was set (red dashed line).

1024 (c, d) BEAS-2B cells were infected with influenza A/Scotland/20/74 (H3N2) virus (IAV) at an MOI=1 (pink bars) or left uninfected (NI) for 4 h, followed by treatment or not with increasing concentrations of CA (blue) or TA (green) for 16h. (c) Viral particle production was assessed using a neuraminidase activity assay. (d) IL-6 levels in cell supernatants were measured by ELISA.

1028 Data are presented as the mean ± SEM and represent cumulative results from 4 or 5 independent experiments. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison test.

1031
1032 **Figure EV3.** *Cis-aco* protects against IAV-induced cell death in human primary bronchial
1033 epithelial cells (PBEC). (**a-b**) PBEC were infected with virus A/Scotland/20/74 (H3N2) at an MOI=1,
1034 and treated or not with 3.4 mM *cis-aco* (CA) 10 min p.i.. SYTOX™ labelling was monitored over 18
1035 h. Representative images (**a**); scale bar: 200 µm) and quantification of labeling (**b**) at 18 h p.i. were
1036 used to assess cell death.

1037 Data are presented as the mean ± SEM from duplicate PBEC derived from 4 independent patients
1038 analyzed. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison
1039 test.

1040

1041 **Figure EV4. Tolerance and safety of cis-aco in vivo.** Seven-week-old female and male C57Bl/6
1042 mice were intranasally instilled with PBS or 30 mg/kg *cis-aco* (CA) for 15 days, according to the
1043 schedule outlined. (**a**) Body weight was monitored throughout the study. (**b**) Mice were euthanized on
1044 day 15 and serum was collected to determine ALAT activity levels. (**c**) Microbiota alterations were
1045 assessed in mouse fecal pellets, after genomic DNA extraction and 16S rRNA sequencing. Microbial
1046 community diversity was quantified using Aitchison distance values, a measure of beta diversity. Each
1047 point represents the Aitchison distance between a CA-treated mouse and a PBS-treated mouse at day
1048 0, 7, or 14. (**d**) The number of immune cells in BAL fluids was assessed by flow cytometry. (**e**) Blood
1049 cell analysis was also conducted.

1050 Data from (e) represent pooled results from 3 mice. All other data are presented as the mean ± SEM
1051 and are cumulative (**a-c**) or representative (**d**) of 3 independent experiments. Statistical analysis was
1052 performed using the Kruskal-Wallis test with Dunn's multiple comparison test.

1053

1054 **Figure EV5. Confirmation of the protective effects of cis-aco in IAV-infected Balb/c mice.** To
1055 validate the findings observed in C57Bl/6 mice (see Figures 3 a-d) in a different mouse strain and sex,
1056 we conducted an experiment using male Balb/c mice. These (NF-κB transgenic) Balb/c animals were
1057 infected with 300 PFU of A/Scotland/20/74 (H3N2) IAV and treated intranasally with 30 mg/kg cis-
1058 aco (CA) 2 days p.i.. Survival (**a**) and body weight (**b**) were monitored daily. All data are presented as
1059 the mean ± SEM. Statistical analysis was performed using the Log-rank (Mantel-Cox) test.
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Cezard *et al.*</figcaption></figure>
<caption>**Figure 2.** Cezard *et al.*</caption>

bioRxiv preprint doi: https://doi.org/10.1101/2025.06.18.660279; this version posted June 24, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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bioRxiv preprint doi: https://doi.org/10.1101/2025.06.18.660279; this version posted June 24, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 international license.

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0 days

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PBS or IAV

PBS or CA

Viral titration (a)

Flow cytometry in BAL (c)

Protein array in BAL (b)

Histological analysis (d-e)

NF-κB-luc measurement (f-g)

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<caption>Figure 5. Cezard *et al.*</caption>

bioRxiv preprint doi: https://doi.org/10.1101/2025.06.18.660279; this version posted June 24, 2025. The copyright holder for this preprint (which was not certified by Peer review) has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 international license.

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bioRxiv preprint doi: https://doi.org/10.1101/2025.06.18.660279; this version posted June 24, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.

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<caption>**Figure 7.** Cezard *et al.*</caption>

bioRxiv preprint doi: https://doi.org/10.1101/2025.06.18.660279; this version posted June 24, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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bioRxiv preprint doi: https://doi.org/10.1101/2025.06.18.660279; this version posted June 24, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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bioRxiv preprint doi: https://doi.org/10.1101/2025.06.18.660279; this version posted June 24, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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