Loading

Commentary Open Access
Volume 4 | Issue 3 | DOI: https://doi.org/10.33696/immunology.4.138

FLIP-expressing myeloid cells as driver of systemic immune disorders

  • 1Immunology section, Department of Medicine University and Hospital Trust of Verona, 37134 Verona, Italy
  • #Shared first authors
+ Affiliations - Affiliations

*Corresponding Author

Prof. Stefano Ugel, stefano.ugel@univr.it

Received Date: May 31, 2022

Accepted Date: June 23, 2022

Abstract

The role of FLIP as a moonlighting protein is becoming progressively evident since this protein is often involved in various processes correlated to aberrant immunological responses independently from its function as master anti-apoptotic regulator. It has been uncovered that FLIP drives the acquisition of immunosuppression and inflammation-associated pathways in myeloid cells. The clinical picture raised during SARSCoV- 2 pandemic has given the possibility to deeply investigate FLIP involvement in releasing a systemic cytokine storm, also linked to a chronic inflammatory syndrome associated with immune suppression and cancer progression. Indeed, a FLIP/STAT3 axis orchestrates an aberrant inflammatory program in myeloid cells of COVID-19 patients and SARS-CoV-2 infected hACE2 transgenic mice. Moreover, the same activated FLIP/STAT3 axis was confirmed in a chimeric vFLIP mouse model, where vFLIP overexpression was restricted exclusively in myeloid cells by using a tissue-specific CRE-driver (e.g., LysMCre mice), validating this model as a feasible platform to study the late phase of COVID-19 disease. The STAT3 pro-inflammatory pathway triggered by the aberrant expression of FLIP in myeloid cells well correlates to the outcome of the cytokine release syndrome (CRS) that is the latest and most severe phase in COVID-19 disease confirming FLIP-mediated myeloid reprogramming as a cornerstone of systemic immune disorders.

Keywords

c-FLIP (Cellular FLICE [FADD-like IL-1β-converting enzyme]-inhibitory protein), CRS (Cytokine Release Syndrome), COVID-19, Inflammation, Myeloid cells

 

Introduction

Cellular and viral FLICE (FADD-like IL-1β-converting enzyme)- inhibitory proteins (c-FLIP and vFLIP respectively) are traditionally recognized for their relevance in the anti-apoptotic activity, regulating cell survival and proliferation by inhibiting caspase-mediated cell death [1]. c-FLIP is encoded by CFLAR gene and it is translated into 3 different protein isoforms derived from 13 different variants of splicing. Among these isoforms, produced in diverse rates, it is possible to define the 55 kDa long form (c-FLIPL), the 26 kDa short form (c-FLIPS), and the 24kDa form of c-FLIP (c-FLIPR) [1,2]. These isoforms differ on the base of their primary structure and can differently exert their functions [3]. All of them contain 2 tandem N-terminal death effector domains (DEDs) responsible for protein-protein interaction properties, associated with c-FLIP ability in being recruited to the death signaling complex during apoptosis, and differ at the C-terminal domain. In particular, the long isoform includes a caspase-8 cleavage site that, cleaved upon heterodimerization with caspase 8, generates a cleaved-long isoform defined as p43FLIP [4]. This cleaved protein promotes the activation of nuclear factor kappa B (NF-κB) through their association with receptor-interacting protein 1 (RIP1) and tumor-necrosis-factor-receptor-associated factor 2 (TRAF2) [5,6]. Indeed, this proteolytic fragment has been reported to interact with NEMO (also known as IKKγ) by either a transient association with the ubiquitin binding domain of IKKγ [7] or a direct physical interaction when c-FLIP is overexpressed [8,9]. The vFLIP protein, which was initially identified in molluscum contagiosum virus and in several γ-herpesviruses [10], also activates IKK by forming a stable complex with the regulatory subunit IKKγ [7] leading to phenotypical features and the cytokine secretion of Kaposi sarcoma cells [11]. Last years, c-FLIP involvement in cellular processes apparently autonomous from its original role has raised more attention towards its alternative properties, and its recent description as a moonlighting protein[12] well attests the attention that such an unpredictable protein deserves. In fact, c-FLIP has been reported to be up-regulated in several cancer types, including pancreatic cancer [13] and lung cancer [14], stimulating tumor progression not only by enhancing cancer cells’ resistance to apoptosis [15] but also by impacting on immune cells regulatory activity favoring the development of a local immunosuppressive tumor microenvironment [3]. In this context, the enforced c-FLIP expression in human monocytes has been related to the marked regulation of genes encoding for proteins typical of immunosuppressive mechanisms, such as signal transducer and activator of transcription (STAT)3, interleukin (IL)-6, and programmed death-ligand 1 (PD-L1), possibly depending on the nuclear translocation of a protein complex formed by c-FLIP and nuclear factor kappa B p50 (NF- κB p50) [4] (Figure 1). On the contrary, FLIP genetic deletion completely abrogate the generation of monocytic-myeloid derived suppressor cells MDSCs [16], highlighting FLIP as a key regulator of this cell subset [17-19].

 

 

Figure 1: The c-FLIP/NF-κB role in STAT3 activation. Stimulation by different pathogens or proinflammatory cytokines (e.g., TNF) causes phosphorylation and degradation of inhibitor of NF-κBα (IκBα), allowing NF-κB to translocate inside the nucleus and to exert its function as a transcription factor. Previous data [4] describe the nuclear colocalization of a c-FLIP/NF-κB p50 protein complex, which is associated with up-regulation of pro-inflammatory genes such as STAT3 and IL6. In addition, the JAK-STAT3 signaling pathway can be also activated by IL-6 stimulation of IL-6 receptor binding gp130, promoting together with NF-κB the regulation of a transcriptional program favoring the expression of several inflammatory mediators and an immunosuppressive state.

The Role of FLIP/STAT3 Axis in COVID-19 Patients

Traditionally c-FLIP is studied as an anti-apoptotic protein, since it is capable of blocking caspase-mediated cell death [1]. During infection, this mechanism is exploited by several viruses to prevent host cell death supporting viral replication [20]. The SARS-CoV-2 outcome has focused the attention on viral-induced cellular alterations able to favor viral latency and, specifically, the correlation between SARSCoV- 2 infection and c-FLIP alteration was analyzed in lung autopsy samples by Musiu and colleagues [21]. Pulmonary immunohistochemistry has revealed a heterogeneous composition of myeloid cells including alveolar macrophages, monocytes/interstitial macrophages, and histiocytes, identified as CD68-expressing cells, that overexpressed c-FLIP and pSTAT3. Interestingly, COVID-19 patients, who present high frequency of CD68+ FLIP+ pSTAT3+ myeloid cells in lungs, had a significantly reduced survival rate. To strengthen the correlation between an aberrant STAT3 activation and c-FLIP expression, the same myeloid infiltrate was detected in lung autopsy samples isolated from SARS-CoV-2-infected mice transgenic for hACE2. Interestingly, circulating CD14+c-FLIP+ cells isolated from COVID-19 patients showed an enforced PDL1 expression, similar to what was displayed by monocytes isolated from PDAC patients with worst clinical outcome4 as well as pSTAT3+ monocytes isolated from PDAC patients were identified as MDSCs22 since they displayed arginase- 1-dependent T cell inhibition [23]. Besides, STAT3-mediated inflammatory pathway stimulated by FLIP plays a central role in inducing a fatal cytokine release syndrome (CRS), one of the latest and most severe hallmarks of SARS-CoV-2 infection [24] and generally associated with dramatic clinical aspects [25] (Figure 2).

 

 

Figure 2: CRS pathophysiological progression. Cytokine storm can be caused by different sources, including iatrogenic or pathogeninduced causes, cancer, and monogenic/autoimmune disorders. The failure of physiological negative regulatory mechanisms allows a deleterious hyper-activation of the immune system leading to the uncontrolled release of proinflammatory cytokines, implying the unbalanced activation of signaling pathways such as NF-κB and JAK-STAT3, which can in turn fuel a positive loop overstressing the inflammatory response. As a consequence, CRS-induced pathological condition could evolve towards a dramatic multi-organ failure and, in the worst case, patient’s death.

CRS Origins and Pathophysiology

CRS definition covers a wide immune pathologies embodying acute systemic inflammation and immune dysfunction resulting in multi-organ failure. Since CRS definition still represents a debated question, Fajgenbaum et al. proposed 3 specific criteria for CRS’s characterization: elevated circulating cytokine levels, acute systemic inflammatory symptoms, and secondary organ dysfunction [26]. Regarding the clinical symptoms of CRS early phase, patients display a high grade of fever in several cases, as well as headache, fatigue, diarrhea, and myalgia [27]. Worsening condition can lead to a second phase of CRS symptoms including catastrophic hemorrhages, hypotension, vasodilatory shock, and vascular occlusion, culminating in acute respiratory distress syndrome (ARDS) in lungs, renal failure, liver injury, and death at worst. Notably, pre-existing pathological chronic conditions (e.g., hypertension, obesity, diabetes) were found to exacerbate CRS development. At root of CRS rising, it is possible to define 3 main sources. Latrogenic cytokine storm founds its bases on specific drug treatments like immunotherapy that can alter the homeostatic cytokine levels. Differently, pathogen-induced cytokine storm is strictly correlated to pathogen infection, in which sepsis condition can be correlated to many cytokine productions that can cause undesirable effects more serious than the pathogen itself. Finally, the last source of CRS could be monogenic or autoimmune [26] (Figure 2). Among the various markers useful to identify CRS, high levels of C-reactive protein (CRP), blood count abnormalities, anemia, leukopenia, thrombocytopenia, and elevated ferritin and D-dimer levels are the most relevant ones, together with increased levels of circulating inflammatory cytokines such as interferon (IFN)-γ, IL-1, IL-6, IL-18, and TNF [28,29]. Consequently, cytokines play the main role in CRS progression since they can finely regulate the entire immunological framework driving acute inflammation. Indeed, abnormal levels of pro-inflammatory cytokines like IL-1 can initiate the NF-κB intracellular signaling pathway resulting in hyper-activation of inflammatory genes transcription, whereas IL-6, upon IL-6 receptor binding gp130, triggers the JAK-STAT3 signaling pathway leading to systemic hyper-inflammation [30] (Figure 1). Inside the myeloid cell population implicated in innate immunological response, neutrophils, monocytes, and macrophages are the main source of cytokine production. Specifically, during netosis, neutrophils can generate a neutrophil extracellular trap (NET) that can participate in thrombi formation and magnify the intensity of cytokine storm. On the other hand, during CRS, over-stimulated monocytes and macrophages secrete an atypical amount of pro-inflammatory cytokines causing immune imbalance. Treating CRS by targeting all the involved pro-inflammatory pathways represent the main therapeutic goal, even if to discriminating between physiological and aberrant inflammation still remains an open challenge.

vFLIP Mouse Model as an Optimal Platform to Develop CRS-Targeting Approach to Restrain Severe COVID-19 Disease

In order to get an in vivo model suitable to mimic the pathophysiological characteristics of CRS in COVID-19 disease, a chimeric mouse model expressing v-FLIP in myeloid cells was generated. As detected in COVID-19 patients, an analogous lung myeloid infiltrate composition, characterized by the presence of pSTAT3 in mononuclear phagocytes (defined by CD68+, F4/80+ or Ly6C+) and neutrophils (defined by CD68+, NE+ or Ly6G+), was found in vFLIP mice displaying diffused lung tissue damage. Remarkably, an increasing number of myeloid cells expressing pSTAT3 and, more specifically, a significant number of cytokine-producing monocytes was detected in spleen of vFLIP mice, supporting a SARSCoV- 2-like inflammatory landscape. Deepening further the structural organization of vFLIP lung myeloid infiltrate, singlecell RNA sequencing (scRNA-seq) data analysis unveiled lymphocytopenia in parallel with an increasing number of neutrophils in vFLIP model compared to wild type (WT) mice. Moreover, a gene set enrichment analysis (GSEA) underlined a significant upregulation of genes involved in inflammatory response, in accordance with STAT3 upregulation. These findings well correlate with the late phase of COVID-19 disease [31].

To make consistence between the mouse model and COVID-19 disease, scRNA-seq data obtained from lungs of vFLIP mice and bronchoalveolar lavage fluids (BAL) of patients affected by severe COVID-19 were clustered and consequently compared in order to generate two different maps useful to identify shared cell populations in lung myeloid infiltrate. The analysis revealed a conserved arrangement of myeloid cells between the two different species, and, among myeloid populations, monocytes presented a similar profile of active inflammatory pathways such as tumor necrosis factor (TNF)-α via NF-κB and JAK-STAT3 signaling. These data clearly support vFLIP mouse as a representative model of CRS in severe COVID-19 patients.

Since clear evidence correlate FLIP up-regulation with an enforced aberrant STAT3 activation, it is linear to consider STAT3 as an appealing target in attempt to control the immunosuppressive activity of myeloid cells and therefore, several drugs such as baricitinib or silibinin and short hairpin RNAs (e.g., shSTAT3) delivery [32,33] have been developed and tested on vFLIP mice. Firstly, STAT3 inhibitors seem to soften the inflammatory condition in localized tissue as well as systemically, though maintaining the physiological antiviral response. Secondly, recovery of lymphocyte proliferation was observed upon STAT3 inhibition by restraining the myeloid immunosuppressive phenotype triggered by SARS-CoV-2 infection. Finally, pointing to STAT3 as the most important player in FLIP-expressing myeloid cells during CRS, targeting this specific transcription factor might greatly contribute to preventing immunopathological disorders and tissue damage [21].

Conclusions

The evolution of c-FLIP protein towards a key regulator in immunosuppression in diverse contexts, apart from its physiological anti-apoptotic one, has raised the curtain for a better comprehension of immune molecular mechanisms in both tumor microenvironment and pro-inflammatoryinduced pathologies, as CRS. Actually, FLIP was found to be involved in modulating some key immunoregulatory pathways, such as NF-κB and JAK-STAT3, and transcription of genes typically related to immunosuppressive mechanisms, such as IL6, and CD274 encoding for PD-L1. A specular FLIP activity was observed also in SARS-CoV-2 infected patients. Indeed, monocytes isolated from COVID-19 patients displayed immunoregulatory function that correlated with the expression of PD-L1. At the same time, FLIP over-expression in monocytes modulates pro-inflammatory pathways, conducing to aberrant cytokine production and severe CRS manifestation. Therefore, FLIP may be a crucial target to develop more effective targeting approaches to either improve cancer immunotherapy or control aberrant immune response that fuel CRS. For instance, FLIP promotes the up-regulation of indoleamine 2,3-dioxygenase 1 (IDO1) [4], which play a critical role on T cell inhibition by depleting L-tryptophan availability in tumor microenvironment [34,35], consequently the protumoral effect of immunosuppressive myeloid cells can be potentially mitigated by limiting specific FLIP-dependent products [36]. At the same time reprogramming myeloid cells by enforcing FLIP expression may be a critical step to establish innovative therapy based on immunosuppressive cell-based tools able to fight immunopathologies originating from enhanced immune reactivity against host-self antigens in the course of transplantation (e.g., graft-versus host disease, GvHD) and autoimmunity (e.g., multiple sclerosis, MS). FLIPrelated knowledge needs further examination to clarify its untraditional features linked to the alteration of cellular transcriptional program, possibly by acting as a transcriptional factor or cofactor in collaboration with transcriptional machinery.

Author Contributions

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Disclosure of Potential Conflict of Interest

The authors disclose no potential conflict of interest.

Funding

This work was supported by the PRIN programs of the Italian Ministry of Education, University and Research (MIUR, CUP: B38D19000140006) Fondazione Associazione Italiana per la Ricerca sul Cancro (AIRC, Project: 21509) and Fondazione Cariverona (Enact project).

Acknowledgments

We thank all components of Immunology section of University of Verona (Bronte lab; #Immunologia1) for providing feedback on this manuscript. We would like to thank Biorender for the artwork used in the figure of this manuscript (https:// biorender.com, accessed on 24 August 2021).

References

1. Safa AR. c-FLIP, a master anti-apoptotic regulator. Experimental Oncology. 2012 Oct;34(3):176-84.

2. Safa AR. Roles of c-FLIP in apoptosis, necroptosis, and autophagy. Journal of Carcinogenesis & Mutagenesis. 2013.

3. Humphreys L, Espona-Fiedler M, Longley DB. FLIP as a therapeutic target in cancer. The FEBS Journal. 2018 Nov;285(22):4104-23.

4. Fiore A, Ugel S, De Sanctis F, Sandri S, Fracasso G, Trovato R, et al. Induction of immunosuppressive functions and NF-κB by FLIP in monocytes. Nature Communications. 2018 Dec 5;9(1):1-3.

5. Koenig A, Buskiewicz IA, Fortner KA, Russell JQ, Asaoka T, He YW, et al. The c-FLIPL cleavage product p43FLIP promotes activation of extracellular signal-regulated kinase (ERK), nuclear factor κB (NF-κB), and caspase-8 and T cell survival. Journal of Biological Chemistry. 2014 Jan 10;289(2):1183-91.

6. Budd RC, Yeh WC, Tschopp J. cFLIP regulation of lymphocyte activation and development. Nature Reviews Immunology. 2006 Mar;6(3):196-204.

7. Liu L, Eby MT, Rathore N, Sinha SK, Kumar A, Chaudhary PM. The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the IκB kinase complex. Journal of Biological Chemistry. 2002 Apr 19;277(16):13745- 51.

8. Golks A, Brenner D, Krammer PH, Lavrik IN. The c-FLIP–NH2 terminus (p22-FLIP) induces NF-κB activation. The Journal of Experimental Medicine. 2006 May 15;203(5):1295-305.

9. Neumann L, Pforr C, Beaudouin J, Pappa A, Fricker N, Krammer PH, et al. Dynamics within the CD95 death-inducing signaling complex decide life and death of cells. Molecular systems biology. 2010;6(1):352.

10. Li FY, Jeffrey PD, Jong WY, Shi Y. Crystal structure of a viral FLIP: insights into FLIP-mediated inhibition of death receptor signaling. Journal of Biological Chemistry. 2006 Feb 3;281(5):2960-8.

11. Grossmann C, Podgrabinska S, Skobe M, Ganem D. Activation of NF-κB by the latent vFLIP gene of Kaposi’s sarcoma-associated herpesvirus is required for the spindle shape of virus-infected endothelial cells and contributes to their proinflammatory phenotype. Journal of Virology. 2006 Jul 15;80(14):7179-85.

12. Adamo A, Frusteri C, Pallotta MT, Pirali T, Sartoris S, Ugel S. Moonlighting proteins are important players in cancer immunology. Frontiers in Immunology. 2021 Jan 18;11:613069.

13. Haag C, Stadel D, Zhou S, Bachem MG, Möller P, Debatin KM, et al. Identification of c-FLIPL and c-FLIPS as critical regulators of death receptor-induced apoptosis in pancreatic cancer cells. Gut. 2011 Feb 1;60(2):225-37.

14. Rao-Bindal K, Rao CK, Yu L, Kleinerman ES. Expression of c-FLIP in pulmonary metastases in osteosarcoma patients and human xenografts. Pediatric Blood & Cancer. 2013 Apr;60(4):575-9.

15. Djerbi M, Screpanti V, Catrina AI, Bogen B, Biberfeld P, Grandien A. The inhibitor of death receptor signaling, FLICE-inhibitory protein defines a new class of tumor progression factors. The Journal of Experimental Medicine. 1999 Oct 4;190(7):1025-32.

16. Haverkamp JM, Smith AM, Weinlich R, Dillon CP, Qualls JE, Neale G, et al. Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways. Immunity. 2014 Dec 18;41(6):947-59.

17. Bronte V, Brandau S, Chen SH, Colombo MP, Frey AB, Greten TF, et al. Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nature Communications. 2016 Jul 6;7(1):12150.

18. Ugel S, Canè S, De Sanctis F, Bronte V. Monocytes in the tumor microenvironment. Annual Review of Pathology: Mechanisms of Disease. 2021 Jan 24;16:93-122.

19. De Sanctis F, Bronte V, Ugel S. Tumor-induced myeloid-derived suppressor cells. Microbiology Spectrum. 2016 May 6;4(3):4-3.

20. Thome M, Schneider P, Hofmann K, Fickenscher H, Meinl E, Neipel F, et al. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature. 1997 Apr;386(6624):517-21.

21. Musiu C, Caligola S, Fiore A, Lamolinara A, Frusteri C, Del Pizzo FD, et al. Fatal cytokine release syndrome by an aberrant FLIP/STAT3 axis. Cell Death & Differentiation. 2022 Feb;29(2):420-38.

22. Ugel S, De Sanctis F, Mandruzzato S, Bronte V. Tumor-induced myeloid deviation: when myeloid-derived suppressor cells meet tumor-associated macrophages. The Journal of Clinical Investigation. 2015 Sep 1;125(9):3365-76.

23. Trovato R, Fiore A, Sartori S, Canè S, Giugno R, Cascione L, et al. Immunosuppression by monocytic myeloid-derived suppressor cells in patients with pancreatic ductal carcinoma is orchestrated by STAT3. Journal for Immunotherapy of Cancer. 2019 Dec;7(1):255.

24. Pum A, Ennemoser M, Adage T, Kungl AJ. Cytokines and chemokines in SARS-CoV-2 infections—therapeutic strategies targeting cytokine storm. Biomolecules. 2021 Jan 12;11(1):91.

25. Ferraccioli G, Gremese E, Goletti D, Petrone L, Cantini F, Ugel S, et al. Immune-guided therapy of COVID-19. Cancer Immunology Research. 2022 Apr 1;10(4):384-402.

26. Fajgenbaum DC, June CH. Cytokine Storm. New England Journal of Medicine. 2020 Dec 3;383(23):2255-73.

27. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor–modified T cells for acute lymphoid leukemia. New England Journal of Medicine. 2013 Apr 18;368(16):1509-18.

28. Lee DW, Gardner R, Porter DL, Louis CU, Ahmed N, Jensen M, et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood, The Journal of the American Society of Hematology. 2014 Jul 10;124(2):188-95.

29. Doherty GM, Lange JR, Langstein HN, Alexander HR, Buresh CM, Norton JA. Evidence for IFN-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha. The Journal of Immunology. 1992 Sep 1;149(5):1666-70.

30. Kang S, Tanaka T, Narazaki M, Kishimoto T. Targeting interleukin-6 signaling in clinic. Immunity. 2019 Apr 16;50(4):1007-23.

31. Bost P, De Sanctis F, Canè S, Ugel S, Donadello K, Castellucci M, et al. Deciphering the state of immune silence in fatal COVID-19 patients. Nature Communications. 2021 Mar 5;12(1):1428.

32. Zilio S, Vella JL, Adriana C, Daftarian PM, Weed DT, Kaifer A, et al. 4PD functionalized dendrimers: a flexible tool for in vivo gene silencing of tumor-educated myeloid cells. The Journal of Immunology. 2017 May 15;198(10):4166-77.

33. De La Fuente A, Zilio S, Caroli J, Van Simaeys D, Mazza EM, Ince TA, et al. Aptamers against mouse and human tumor-infiltrating myeloid cells as reagents for targeted chemotherapy. Science Translational Medicine. 2020 Jun 17;12(548):eaav9760.

34. Hofer F, Di Sario G, Musiu C, Sartoris S, De Sanctis F, Ugel S. A complex metabolic network confers immunosuppressive functions to myeloid-derived suppressor cells (MDSCs) within the tumour microenvironment. Cells. 2021 Oct 9;10(10):2700.

35. Mondanelli G, Bianchi R, Pallotta MT, Orabona C, Albini E, Iacono A, et al. A relay pathway between arginine and tryptophan metabolism confers immunosuppressive properties on dendritic cells. Immunity. 2017 Feb 21;46(2):233-44.

36. Serafini M, Torre E, Aprile S, Grosso ED, Gesù A, Griglio A, et al. Discovery of highly potent benzimidazole derivatives as indoleamine 2, 3-dioxygenase-1 (IDO1) inhibitors: from structure-based virtual screening to in vivo pharmacodynamic activity. Journal of Medicinal Chemistry. 2020 Mar 9;63(6):3047-65.

Author Information X