Commentary on “Inhibition of cellular activation induced by platelet factor 4 via the CXCR3 pathway ameliorates Japanese encephalitis and dengue viral infections”

This edition of the Journal of Thrombosis and Haemostasis includes a paper by Singh et al. [1Singh A. Ghosh R. Asuru T.R. Prajapat S.K. Joshi G. Gaur K.K. et al.Inhibition of cellular activation induced by platelet factor 4 via the CXCR3 pathway ameliorates Japanese encephalitis and dengue viral infections.J Thromb Haemost. 2024; 22: 818-833Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar] entitled "Inhibition of platelet factor 4: CXCR3 pathway prevents JEV and DENV infection." In this article, the authors provide extensive evidence that both dengue virus (DENV) and Japanese encephalitis virus (JEV) infections of mice and cell cultures could be attenuated by a deficiency of the chemokine platelet factor 4 (PF4; chemokine [C-X-C motif] ligand 4) or inhibition of the PF4 via the C-X-C motif chemokine receptor 3 (CXCR3) pathway. The authors also undertook extensive experiments to begin to map out how PF4 impacts antiviral cytokine production (eg, interferon [IFN]-α and IFN-β) and autophagy flux in DENV and JEV infection models. Finally, the authors collected blood from DENV-infected patients during acute and convalescent phases and noted that patients had higher levels of PF4 and nonstructural protein (NS)+ monocytes in the acute phase compared to the convalescent phase. In this editorial, I will outline why this work mapping out the role that PF4 and CXCR3 play in DENV and JEV infections and disease is so important. Both DENV (Orthoflavivirus denguei) and JEV (Orthoflavivirus japonicum) are separate species within the genus Orthoflavivirus and family Flaviviridae. Humans can be infected with 4 serotypes (DENV-1,2,3,4) by mosquito vectors, which are primarily Aedes aegypti and Aedes albopictus. Serotypes may vary between regions and within regions over time, and in some regions, all 4 serotypes may circulate, with 1 being dominant [2Norshidah H. Vignesh R. Lai N.S. Updates on Dengue vaccine and antiviral: where are we heading?.Molecules. 2021; 26: 6768Crossref PubMed Scopus (23) Google Scholar]. In 2023, the World Health Organization (WHO) estimated that half of the world's population is at risk of DENV infection, and it is estimated that 100 to 400 million infections occur each year in tropical/subtropical regions and urban/semiurban settings. Most cases are asymptomatic or misdiagnosed. Between 2000 and 2019, the number of dengue cases reported to the WHO increased from 505 430 cases to 5.2 million in 2019 [3World Health OrganizationDengue and severe dengue.https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengueGoogle Scholar]. The epidemiology of DENV is expected to change in the face of climate change, and there are already signals for autochthonous DENV transmission in Western Europe [4Zatta M. Brichler S. Vindrios W. Melica G. Gallien S. Autochthonous Dengue outbreak, Paris region, France, September-October 2023.Emerg Infect Dis. 2023; 29: 2538-2540Crossref PubMed Scopus (3) Google Scholar]. Three main presentations may occur with infection: Dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, and these are described elsewhere [5Heymann D.L. Control of communicable diseases manual.21st ed. APHA Press, Washington, DC2022Google Scholar]. Currently, there are no specific antiviral agents for DENV infection. In the United States, Dengvaxia dengue vaccine (tetravalent live-attenuated dengue vaccine; Sanofi Pasteur Inc.) is approved in specific populations and has a good vaccine effectiveness profile. JEV transmission to humans is via the bite of infected Culex mosquitoes (mainly Culex tritaeniorhynchus), and a transmission cycle is maintained between birds, humans, mosquitoes, and pigs, usually in rural and periurban environments. Most individuals at risk for JEV infection live in 2 WHO regions (Southeast Asia and Western Pacific), where the population is estimated to be over 3 billion people. In 2011, the WHO estimated that there were 67 900 clinical JEV cases every year [6Campbell G.L. Hills S.L. Fischer M. Jacobson J.A. Hoke C.H. Hombach J.M. Marfin A.A. Solomon T. Tsai T.F. Tsu V.D. Ginsburg A.S. Estimated global incidence of Japanese encephalitis: a systematic review.Bull World Health Organ. 2011; 89: 766-774ECrossref PubMed Scopus (730) Google Scholar]. Most JEV infections are usually asymptomatic or mild (eg, fever and headache) or without apparent symptoms. However, 1 in 250 infections involve severe disease symptoms, and in those patients, the case fatality rate can be as high as 30%. There is no specific antiviral treatment for JEV; however, due to the role of inflammation in poor clinical outcomes, antiviral agents that also dampen inflammation may be candidates for study. Although current vaccines are highly effective and immunogenic, there are several challenges to JEV vaccine programs, including maintaining JEV vaccine coverage, maintaining a stable supply, and poor surveillance/vaccination data [7Vannice K.S. Hills S.L. Schwartz L.M. Barrett A.D. Heffelfinger J. Hombach J. Letson G.W. Solomon T. Marfin A.A. Japanese encephalitis vaccination experts panel. The future of Japanese encephalitis vaccination: expert recommendations for achieving and maintaining optimal JE control.NPJ Vaccines. 2021; 6: 82Crossref PubMed Scopus (32) Google Scholar]. In the last sentence of the conclusion of their article, Singh et al. state "Our studies suggest that the PF4-CXCR3 axis is a potential target to develop a treatment regimen against the flaviviruses, JEV and DENV." Modulating the PF4-CXCR3 axis to manage DENV and JEV infection would be a very different approach from the directly acting antiviral agents used to treat a distant relative, hepatitis C virus (species Hepacivirus C, genus Hepacivirus, family Flaviviridae) [8Martinello M. Naggie S. Rockstroh J.K. Matthews G.V. Direct-acting antiviral therapy for treatment of acute and recent hepatitis C virus infection: a narrative review.Clin Infect Dis. 2023; 77: S238-S244Crossref PubMed Scopus (8) Google Scholar]. Controlling CXCR3-mediated disease processes has been a proposed approach for other disease processes. Although CXCR3 is potentially druggable, there have been no successful CXCR3 antagonist clinical trials to date. One agent AMG487 (N-[(1R)-1-[3-(4-ethoxyphenyl)-3,4-dihydro-4-oxopyrido[2,3-d]pyrimidin-2-yl]ethyl]-N-(3-pyridinylmethyl)-4-(trifluoromethoxy)-benzeneacetamide; CAS number 473719-41-4) progressed to phase II clinical trials focused on psoriasis but was withdrawn due to a lack of efficacy [9National Center for Advancing Translational SciencesInxight drugs: AMG-487.https://drugs.ncats.io/substances?q=%22AMG-487%20METABOLITE%20M3%22&facet=Substance%20Form%2FPrincipal%20FormGoogle Scholar]. A review by Pease [10Pease J.E. Designing small molecule CXCR3 antagonists.Expert Opin Drug Discov. 2017; 12: 159-168Crossref PubMed Scopus (16) Google Scholar] highlights potential problems in moving CXCR3 antagonist through clinical trials and emphasizes that researchers undertaking discovery and development work need to prove "beyond reasonable doubt that the target is critical for disease pathogenesis and that effective blockade will be therapeutic." Thus, the complex interactions mapped out by Singh et al. are key in determining if the interaction between PF4 and CXCR3 in the context of DENV and JEV is a hopeful target for future therapeutics discovery and development. DENV infections can activate platelets and lead to the release of α-granules containing PF4. The role of JEV in platelet activation remains less clear, and treatment of platelets with increasing doses of JEV did not significantly activate platelets in one set of experiments [11Ojha A. Nandi D. Batra H. Singhal R. Annarapu G.K. Bhattacharyya S. Seth T. Dar L. Medigeshi G.R. Vrati S. Vikram N.K. Guchhait P. Platelet activation determines the severity of thrombocytopenia in dengue infection.Sci Rep. 2017; 741697Crossref Scopus (118) Google Scholar] (Table). In a prior series of experiments, Ojha et al. [12Ojha A. Bhasym A. Mukherjee S. Annarapu G.K. Bhakuni T. Akbar I. Seth T. Vikram N.K. Vrati S. Basu A. Bhattacharyya S. Guchhait P. Platelet factor 4 promotes rapid replication and propagation of Dengue and Japanese encephalitis viruses.EBioMedicine. 2019; 39: 332-347Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar] showed that PF4 could enhance the in vivo and in vitro propagation of both DENV and JEV. This most recent work further elucidates the relationship between PF4-CXCR3 and the role of this interaction in DENV and JEV infection. It also prompts us to think about future studies that could be undertaken (Table).TableKey elements raised in this study and how they might support further work targeting PF4-CXCR3 and downstream processes as druggable targets in DENV and JEV infections and disease.Key element identified by Singh et al.Why this is importantWhat else can be doneA receptor-ligand interaction is key: an interaction between PF4 and CXCR3 enhances DENV and JEV propagation in infection models.Provides further context to prior research on the role of PF4-CXCR3 in flavivirus infections; describes virus-specific protocols and biomarkers for future PF4-CXCR3 studies in cell lines and mouse models infected with flaviviruses (eg, cell/protein staining, nucleic acid testing, and mouse viability); describes potential controls (eg, AMG487) for other small-molecule studies.Understand the role of PF4 in JEV infection; generate a more detailed understanding of CXCR3 and PF4 binding kinetics in the context of flavivirus infection; study other pathways that might synergize with PF-CXCR3 in flavivirus infection; screen for small molecules or other antibodies that could block PF4-CXCR3 interactions.Lysosome degradation: The interaction between PF4 and CXCR3 blocks lysosome degradation and promotes DENV and JEV replication.Fulfills guideline recommendations for autophagy studies; provides protocols for lysosome degradation of flaviviruses; identifies controls (eg, AM487) for other small-molecule inhibitors of lysosome degradation of flaviviruses; identifies PF4-mediated targets in mTOR-p38-ULK1 axis that might block phagosome-lysosome fusion and prevent DENV degradation.Compare/contrast role of the mTOR-p38-ULK1 axis important in JEV infection; clarify crosstalk with proinflammatory/anti-inflammatory signal transduction pathways; use small-molecule inhibitors to block ULK1, p38, and mTOR serine/threonine kinase activity.IFN-α/β expression blockage: PF4 and CXCR3 interactions lead to reduced IFN-α/β expression in parallel to reduced autophagosome-lysosome fusion in DENV and JEV infection models.Identifies potential crosstalk pathways between cytokine expression and autophagy pathways.Can these signaling pathways be targeted with small-molecule inhibitors? Can these linkages between autophagy and cytokine pathways be clarified?DENV, dengue virus; IFN, interferon; JEV, Japanese encephalitis virus. Open table in a new tab DENV, dengue virus; IFN, interferon; JEV, Japanese encephalitis virus. It is still not clear where PF4 is binding and activating CXCR3 to promote DENV and JEV replication (Table). CXCR3 is a transmembrane G protein–coupled receptor with an N-terminus extracellular domain, 3 extracellular loop (ECL) structures, 3 intracellular loop structures, and an intracellular C-terminal domain that is widely expressed on human cells (eg, activated CD8+ T cells, B cells, dendritic cells, endothelial cells, macrophages, mast cells, natural killer cells, and Th1 cells). In humans, the CXCR3 gene is transcribed into 3 splice variants: CXCR3A, CXCR3B, and CXCR3-alt. Monomers of PF4 (PF4 is a tetramer in solution) likely bind to the ECL2 and N-terminal domain of CXCR3A and CXCR3B [13Liu Y.H. Chuang C.H. Lee Y.Z. Lee E.T. Lo C.L. Wu C.Y. Huang L.K. Bikfalvi A. Sue S.C. Structural properties of CXCL4L1 and its recognition of the CXCR3 N-terminus.Biochemistry. 2023; 62: 722-734Crossref PubMed Scopus (1) Google Scholar]. What happens next is unclear in this model of infection, but CXCR3 binding to other agonists (eg, CXCL9, CXCL10, and CXCL11) uses a multistep approach to ligand binding and receptor activation [14Xanthou G. Williams T.J. Pease J.E. Molecular characterization of the chemokine receptor CXCR3: evidence for the involvement of distinct extracellular domains in a multi-step model of ligand binding and receptor activation.Eur J Immunol. 2003; 33: 2927-2936Crossref PubMed Scopus (78) Google Scholar]. One question arises as to whether PF4 can synergize with other pathways to impact DENV and JEV infections (Table). Another study has shown that PF4, in the absence of DENV or JEV, can synergize with toll-like receptor 8 (TLR8) to signal through receptor-interacting serine/threonine-protein kinase to activate interleukin-1 expression in monocytes [15Yang C. Bachu M. Du Y. Brauner C. Yuan R. Ah Kioon M.D. Chesi G. Barrat F.J. Ivashkiv L.B. CXCL4 synergizes with TLR8 for TBK1-IRF5 activation, epigenomic remodeling and inflammatory response in human monocytes.Nat Commun. 2022; 13: 3426Crossref PubMed Scopus (13) Google Scholar]. This is intriguing as TLR8 can also interact with flavivirus RNA. Where AMG487 disrupts PF4-CXCR3 interactions and promotes DENV and JEV replication is also unclear (Table). For other studies, we understand that AMG487 can prevent binding of CXCL10 and CXCL11 to CXCR3 [16Johnson M. Li A.R. Liu J. Fu Z. Zhu L. Miao S. Wang X. Xu Q. Huang A. Marcus A. Xu F. Ebsworth K. Sablan E. Danao J. Kumer J. Dairaghi D. Lawrence C. Sullivan T. Tonn G. Schall T. et al.Discovery and optimization of a series of quinazolinone-derived antagonists of CXCR3.Bioorg Med Chem Lett. 2007; 17: 3339-3343Crossref PubMed Scopus (81) Google Scholar]. Binding of CXCL10 and CXCL11 is thought to occur at the ECL1 region and the N-terminal region of CXCR3. CXCL10 binding is also thought to occur at the Asn22 residue near the N-terminal region of CXCR3 [17Satarkar D. Patra C. Evolution, expression and functional analysis of CXCR3 in neuronal and cardiovascular diseases: a narrative review.Front Cell Dev Biol. 2022; 10882017Crossref PubMed Scopus (16) Google Scholar]. Therefore, it is possible that AMG487 could reduce PF4 binding to a region between the N-terminus and ECL2 regions of CXCR4. In their work, Singh et al. thoroughly followed the principles of guidelines developed for using and interpreting autophagy assays. These guidelines, although not mentioned in the study, are an excellent resource and provide a glossary of molecules involved in autophagy. The role of DENV in subverting autophagy in a variety of cell lines was described in 2008 [18Lee Y.R. Lei H.Y. Liu M.T. Wang J.R. Chen S.H. Jiang-Shieh Y.F. Lin Y.S. Yeh T.M. Liu C.C. Liu H.S. Autophagic machinery activated by dengue virus enhances virus replication.Virology. 2008; 374: 240-248Crossref PubMed Scopus (301) Google Scholar]. In the current study by Singh et al., PF4 treatment of DENV-infected cells increased phosphorylation of phosphorylated (P)-unc-51 like autophagy activating kinase (ULK) 1, and P-p38 (Table). ULK1 has 3 major domains: a kinase domain, a serine proline-rich domain, and a C-terminal domain. The interaction of ULK1 with p38 is complex and may activate ULK1 or be a substrate of ULK1. The serine proline-rich domain of ULK interacts with microtubule-associated protein 1 light chain 3/LC3 (LC3). Conjugation of cytosolic LC3-I to phosphatidylethanolamine allows for generation of LC3-II associated phagosomes, which promote phagosome maturation and processing of the engulfed cargo. PF4 treatment of DENV-infected cells also increased phosphorylation of mechanistic target of rapamycin (mTOR) kinase. In mammals, mTOR interacts with binding partners to form 2 complexes, mTORC1 and mTORC2, with TORC1 directly regulating autophagy [19Klionsky D.J. Abdel-Aziz A.K. Abdelfatah S. Abdellatif M. Abdoli A. Abel S. Abeliovich H. Abildgaard M.H. Abudu Y.P. Acevedo-Arozena A. Adamopoulos I.E. Adeli K. Adolph T.E. Adornetto A. Aflaki E. Agam G. Agarwal A. Aggarwal B.B. Agnello M. Agostinis P. et al.Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).Autophagy. 2021; 17: 1-382Crossref PubMed Scopus (1323) Google Scholar]. The association of ULK-1, mTOR, and p38 serine/threonine kinase activity in preventing lysosome-phagosome fusion in models of DENV infection provides a rationale for identifying these kinases in inhibitor studies [20Blázquez A.B. Saiz J.C. Potential for protein kinase pharmacological regulation in Flaviviridae infections.Int J Mol Sci. 2020; 21: 9524Crossref PubMed Scopus (8) Google Scholar]. Involvement of serine/threonine kinases leads to questions as to whether there could be further crosstalk between the autophagy process and other proinflammatory or anti-inflammatory pathways in DENV infection. It is also unclear whether JEV would interact with this process in the same manner as DENV (Table). DENV can evade the innate immune response by blocking type I IFN [21Castillo Ramirez J.A. Urcuqui-Inchima S. Dengue virus control of type I IFN responses: a history of manipulation and control.J Interferon Cytokine Res. 2015; 35: 421-430Crossref PubMed Scopus (58) Google Scholar]. In a prior study, members of the study team noted that treatment of infected monocytes with PF4 increased P-p38, decreased P-STAT2, and decreased interferon regulatory factor 9 (IRF9) and IFN-α production. Both anti-PF4 and AMG487 reversed the effect of PF4 by decreasing P-p38, increasing P-STAT2, and increasing IRF9 and IFN-α expression [12Ojha A. Bhasym A. Mukherjee S. Annarapu G.K. Bhakuni T. Akbar I. Seth T. Vikram N.K. Vrati S. Basu A. Bhattacharyya S. Guchhait P. Platelet factor 4 promotes rapid replication and propagation of Dengue and Japanese encephalitis viruses.EBioMedicine. 2019; 39: 332-347Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. In this study, Singh et al. showed that IRF3/7/9 and IFN-α/β expression in DENV-2 and JEV infection models occurred in parallel to decreased autophagosome-lysosome fusion. This leads to an interesting question as to whether there is crosstalk between signal transduction pathways and whether multiple pathways involved in DENV and JEV pathophysiology could be blocked by small molecules. DENV and JEV are viruses of global importance that can lead to substantial burdens on health systems in affected countries. Although there are vaccines for both viruses, there are no specific treatments for either DENV or JEV. Infection models such as those used by Singh et al. identify how host immune components or processes, such as autophagy, the mTOR-p38-ULK1 axis, and antiviral cytokine production, can be subverted during DENV and JEV infections. The work also provides further evidence on how the PF4-CXCR3 axis might act as a target for new antiviral agents or therapies. One question arises as to whether PF4 can synergize with other signaling pathways (eg, TLR8 and receptor-interacting serine/threonine-protein kinase) to blunt antiviral IFN response and sustain DENV and JEV infections.