Targeting necroptosis prevents viral-induced lung damage

You have full access to this article via your institution. Download PDF A recent study featured in _Nature_ reveals a promising drug candidate, UH15-38, that significantly reduces lung

damage and increases survival rates in mice with influenza A virus (IAV) infection by inhibiting necroptosis, a type of inflammatory cell death [1]. The research team led by Gregory Cuny,

Paul Thomas, Alexei Degterev and Siddharth Balachandran showed that UH15-38 specifically targets and deactivates RIPK3, an essential kinase in the necroptosis pathway. This discovery not

only unveils new pathways involved in inflammatory diseases, such as severe influenza A infection, but also highlights the potential of this drug to treat a wide array of inflammatory viral

or non-viral conditions. Apoptosis was considered the only form of cell death, but novel mechanisms, such as necroptosis, has broadened our comprehension of cell death [2]. Necroptosis plays

a role in several pathophysiological scenarios, such as development, immune responses, and inflammatory conditions [3]. Necroptosis involves a cascade of signaling events that culminate in

necrotic-like death. At the heart of this process is the activation of receptor-interacting protein kinase-1 (RIPK1) [4] and RIPK3 [5], forming the necrosome complex, that, in turn,

activates the Mixed-Lineage-Kinase-Domain-Like protein (MLKL), causing it to oligomerize on the plasma membrane, where it leads to cell lysis [6]. The initiation of necroptosis is carefully

regulated by various elements, including death receptors like tumor necrosis factor receptor 1 (TNFR1) and the level of caspase-8, which normally prevents necroptosis by deactivating RIPK1

and RIPK3. However, necroptosis can occur without restraint under specific conditions, e.g. low caspase-8 activity or when certain triggers are present. This is in fact the case in

IAV-infected lung epithelial cells, in which caspase-8 is dramatically reduced [7]. In lung epithelial cells infected by IAV, Z-form nucleic acid binding protein 1 (ZBP1) binds viral Z-RNA,

which then activates RIPK3, culminating in necroptosis, triggered in RIPK3-dependent manner, and apoptosis, induced by RIPK3 acting as a scaffold for caspase-8 activation [8, 9]. Apoptosis

is sufficient for viral clearance. On the other hand, necroptosis is crucial in causing tissue damage, but is not essential for virus elimination or the antiviral actions of CD8+ T cells;

therefore, targeting RIPK3 could be a promising strategy for treating IAV-induced lung damage [1]. IAV is a highly transmissible respiratory pathogen that significantly endangers global

health. It attacks lung epithelial cells, causing tissue harm and dysfunction. The immune response to infection, which includes programmed cell death pathways such as apoptosis and

necroptosis, is vital for fighting the virus. Yet, unregulated or excessive death of alveolar epithelial cells can irreversibly compromise lung gas exchange function, leading to mortality

despite virus clearance. The damage from necroptosis, in particular, can lead to the release of damage-associated molecular-patterns [10], intensifying inflammation and attracting more

immune cells, causing additional tissue harm. Furthermore, the delayed clearance of necroptotic cells can extend inflammation and hinder tissue repair, leading to worse clinical outcomes.

Therefore, necroptosis blockade offers a new therapeutic approach to reduce damage and improve recovery during severe IAV infection. Existing RIPK3 inhibitors have been somewhat ineffective

due to limited cellular potency or their propensity to induce apoptosis at higher concentrations [11]. However, a newly developed inhibitor, UH15-38 has shown greater efficacy in blocking

necroptosis without triggering apoptosis. UH15-38 binds robustly to RIPK3, and distributes effectively across various organs in mice, while exhibiting negligible adverse effects (Fig. 1).

Before assessing the effectiveness of UH15-38 in reducing IAV-induced lung damage, the study found that the primary cell types in the lungs undergo RIPK3-dependent necroptosis during

infection. Indeed, type I Alveolar-Epithelial-Cells (AECs) as the main site of IAV replication and the major targets for UH15-38. These cells showed significantly higher levels of IAV mRNA

and were crucial for the virus’s replication. Type I AECs were found to undergo ZBP1-dependent necroptosis post-IAV infection, and UH15-38 effectively protected them from IAV-induced

necroptosis without affecting virus-induced apoptosis. This selective inhibition was confirmed across various cell types and with different IAV strains. UH15-38 inhibited necroptosis in a

virus-independent system and in both human and mouse cells, and in human donor lung tissues infected with IAV. These findings underscore UH15-38’s potency and specificity in blocking

IAV-triggered necroptosis in both mouse and human settings. Notably, UH15-38 did not induce apoptosis at doses significantly higher than those needed to block necroptosis, indicating a clear

distinction between its necroptosis-inhibitory activity and the potential to trigger apoptosis. Hence, UH15-38 as a promising candidate for mitigating IAV-induced pathology. Intraperitoneal

UH15-38 injections starting one day post-infection with a lethal dose of IAV, significantly improved survival rates and reduced weight loss at doses as low as 7.5 mg/kg/day, with optimal

protection observed at 30 mg/kg/day. This protected 80% of the mice from death and mitigated weight loss, allowing for complete recovery within three weeks. Shortening the treatment duration

to two days or delaying the start of treatment by up to two days post-infection still significantly reduced weight loss and prevented death in a substantial number of mice. Remarkably,

UH15-38 offered 100% protection against a less lethal dose of IAV (closer to the dose lethal to 60% of mice) even at a dose as low as 1 mg/kg/day, demonstrating its efficacy against both

highly lethal and more clinically relevant doses of IAV. UH15-38’s protective effect was absent in knockout mice lacking RIPK3 or MLKL. Interestingly, UH15-38 was more effective than the

genetic deletion of MLKL in preventing IAV-induced lethality, suggesting that early necroptosis might play a beneficial role in initiating antiviral immune responses or maintaining immune

homeostasis, which could be compromised with permanent MLKL ablation. Mice infected with a lethal dose of IAV were treated with UH15-38, which significantly reduced necroptosis in lung

cells, specifically in type I AECs. UH15-38 treatment also prevented the release of inflammatory cytokines and chemokines from infected AECs both in vitro and in vivo, leading to a reduction

in inflammatory responses, such as decreased neutrophil infiltration in the lungs. Hence, necroptosis play a key role in boosting inflammation. Further, UH15-38 treatment resulted in lower

levels of lung damage, including reduced alveolar damage, hyaline membrane formation, bronchiolar denudation, and fibrosis, indicating that the protective effect of UH15-38 on IAV-infected

lungs is systemic. This was supported by the reduced release of pro-fibrotic mediators and dampened inflammatory responses in the lungs. Additionally, UH15-38 did not affect virus

replication, spread, or clearance, nor did it impair the immune response against IAV, as evidenced by the maintenance of IAV-specific cytotoxic T cell responses and improved functional

profile of these cells. Lung damage is also crucial in other viral infections, such as Covid-19 where the cytokine storm activates a powerful inflammatory response, that, in presence of a

leaky blood-brain barrier, involves also serious brain damage [12]. In the initial phases of the COVID-19 outbreak, it was suggested that the Hyaluronan Synthase 2 blocker,

4-methylumbelliferone (4-MU)—clinically used for functional and obstructive biliary tract spasms—could alleviate lung congestion [13]. Trials in patients with severe COVID-19 indicated an

89% improvement rate when treated with 4-MU, compared to a 42% improvement rate in those receiving standard care [14]. Based on these findings, it appears plausible to consider the

combination of 4-MU with UH15-38 as a potential therapy for lung damage. In summary, RIPK3 is a novel powerful target for preventing lung inflammation and injury during viral infections.

UH15-38 effectively dampens IAV-induced lung injury by selectively inhibiting necroptosis and reducing inflammatory and fibrotic responses, without compromising the host’s ability to clear

the virus or mount an effective immune response. This suggests that UH15-38 could be a promising therapeutic agent for managing not only IAV-induced lung injury, but also other cause, such

as COVID-19, induced lung damages or even other inflammation related diseases. REFERENCES * Gautam A, Boyd DF, Nikhar S, Zhang T, Siokas I, Van de Velde L-A, et al. Necroptosis blockade

prevents lung injury in severe influenza. Nature. 2024. https://doi.org/10.1038/s41586-024-07265-8. * Vitale I, Pietrocola F, Guilbaud E, Aaronson SA, Abrams JM, Adam D, et al. Apoptotic

cell death in disease-Current understanding of the NCCD 2023. Cell Death Differ. 2023;30:1097–154. Article  PubMed  PubMed Central  Google Scholar  * Ye K, Chen Z, Xu Y. The double-edged

functions of necroptosis. Cell Death Dis. 2023;14:163. Article  PubMed  PubMed Central  Google Scholar  * Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, et al. Fas triggers

an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol. 2000;1:489–95. Article  CAS  PubMed  Google Scholar  * Snyder AG, Hubbard NW,

Messmer MN, Kofman SB, Hagan CE, Orozco SL, et al. Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity. Sci Immunol.

2019;4:eaaw2004. Article  CAS  PubMed  PubMed Central  Google Scholar  * Galluzzi L, Kepp O, Chan FK, Kroemer G. Necroptosis: Mechanisms and Relevance to Disease. Annu Rev Pathol.

2017;12:103–30. Article  CAS  PubMed  Google Scholar  * Othumpangat S, Beezhold DH, Noti JD. Influenza virus infection modulates the death receptor pathway during early stages of infection

in human bronchial epithelial cells. Physiol Genomics. 2018;50:770–9. Article  CAS  PubMed  Google Scholar  * Thapa RJ, Ingram JP, Ragan KB, Nogusa S, Boyd DF, Benitez AA, et al. DAI Senses

Influenza A Virus Genomic RNA and Activates RIPK3-Dependent Cell Death. Cell Host Microbe. 2016;20:674–81. Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhang T, Yin C, Boyd DF,

Quarato G, Ingram JP, Shubina M, et al. Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis. Cell. 2020;180:1115–29.e1113. Article  CAS  PubMed  PubMed Central  Google Scholar  *

Balachandran S, Rall GF. Benefits and Perils of Necroptosis in Influenza Virus Infection. J Virol. 2020;94:e01101–19. Article  CAS  PubMed  PubMed Central  Google Scholar  * Mandal P, Berger

SB, Pillay S, Moriwaki K, Huang C, Guo H, et al. RIP3 induces apoptosis independent of pronecrotic kinase activity. Mol Cell. 2014;56:481–95. Article  CAS  PubMed  PubMed Central  Google

Scholar  * Lloreda CL. COVID’s toll on the brain: new clues emerge. Nature. 2024;628:20–20. Article  Google Scholar  * Shi Y, Wang Y, Shao C, Huang J, Gan J, Huang X, et al. COVID-19

infection: the perspectives on immune responses. Cell Death Differ. 2020;27:1451–4. Article  CAS  PubMed  PubMed Central  Google Scholar  * Yang S, Ling Y, Zhao F, Li W, Song Z, Wang L, et

al. Hymecromone: a clinical prescription hyaluronan inhibitor for efficiently blocking COVID-19 progression. Signal Transduct Target Ther. 2022;7:91. Article  CAS  PubMed  PubMed Central 

Google Scholar  * Solon M, Ge N, Hambro S, Haller S, Jiang J, Baca M, et al. ZBP1 and TRIF trigger lethal necroptosis in mice lacking caspase-8 and TNFR1. Cell Death Differ. 2024.

https://doi.org/10.1038/s41418-024-01286-6. Download references FUNDING Funding This work has been supported by the European Union NextGenerationEU via MUR-PNRR M4C2-II.3 PE6 project

PE00000019 Heal Italia (CUP: E83C22004670001) to GM., the Jiangsu Province International Joint Laboratory for Regenerative Medicine Fund and Suzhou Foreign Academician Workstation Fund

(SWY202202) to YS. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * The Third Affiliated Hospital of Soochow University, Institutes for Translational Medicine, State Key Laboratory of Radiation

Medicine and Protection, Medical College of Soochow University, Soochow University, Suzhou, 215000, China Yufang Shi, Peishan Li & Jun Zhou * Department of Experimental Medicine, TOR,

University of Rome Tor Vergata, 00133, Rome, Italy Yufang Shi & Gerry Melino Authors * Yufang Shi View author publications You can also search for this author inPubMed Google Scholar *

Peishan Li View author publications You can also search for this author inPubMed Google Scholar * Jun Zhou View author publications You can also search for this author inPubMed Google

Scholar * Gerry Melino View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS GM and YS conceived and supervised the project; all authors wrote

the manuscript; PL and JZ prepared Fig. 1. CORRESPONDING AUTHORS Correspondence to Yufang Shi or Gerry Melino. ETHICS DECLARATIONS COMPETING INTERESTS YS and GM are Editorial Board Member of

Cell Death Differentiation. The authors declare no other competing interests. CONSENT FOR PUBLICATION All of the authors have approved this submitted version. ADDITIONAL INFORMATION

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RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Shi, Y., Li, P., Zhou, J. _et al._ Targeting necroptosis prevents viral-induced lung damage. _Cell Death

Differ_ 31, 541–543 (2024). https://doi.org/10.1038/s41418-024-01299-1 Download citation * Received: 08 April 2024 * Revised: 17 April 2024 * Accepted: 17 April 2024 * Published: 27 April

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