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• W2920026340 abstract "•IFNAR−/−Axl−/− mice show Axl unnecessary for Zika virus replication in mice•Mice lacking Axl receptor are significantly resistant to Zika virus neuropathogenesis•IFNAR−/−Axl−/− mice have less ZIKV-driven caspase-dependent apoptosis in brain•Axl deficient mice have fewer apoptotic microglia after ZIKV infection The TAM receptor, Axl, has been implicated as a candidate entry receptor for Zika virus (ZIKV) infection but has been shown as inessential for virus infection in mice. To probe the role of Axl in murine ZIKV infection, we developed a mouse model lacking the Axl receptor and the interferon alpha/beta receptor (Ifnar−/−Axl−/−), conferring susceptibility to ZIKV. This model validated that Axl is not required for murine ZIKV infection and that mice lacking Axl are resistant to ZIKV pathogenesis. This resistance correlates to lower pro-interleukin-1β production and less apoptosis in microglia of ZIKV-infected mice. This apoptosis occurs through both intrinsic (caspase 9) and extrinsic (caspase 8) manners, and is age dependent, as younger Axl-deficient mice are susceptible to ZIKV pathogenesis. These findings suggest that Axl plays an important role in pathogenesis in the brain during ZIKV infection and indicates a potential role for Axl inhibitors as therapeutics during viral infection. The TAM receptor, Axl, has been implicated as a candidate entry receptor for Zika virus (ZIKV) infection but has been shown as inessential for virus infection in mice. To probe the role of Axl in murine ZIKV infection, we developed a mouse model lacking the Axl receptor and the interferon alpha/beta receptor (Ifnar−/−Axl−/−), conferring susceptibility to ZIKV. This model validated that Axl is not required for murine ZIKV infection and that mice lacking Axl are resistant to ZIKV pathogenesis. This resistance correlates to lower pro-interleukin-1β production and less apoptosis in microglia of ZIKV-infected mice. This apoptosis occurs through both intrinsic (caspase 9) and extrinsic (caspase 8) manners, and is age dependent, as younger Axl-deficient mice are susceptible to ZIKV pathogenesis. These findings suggest that Axl plays an important role in pathogenesis in the brain during ZIKV infection and indicates a potential role for Axl inhibitors as therapeutics during viral infection. First discovered in Africa in 1947 (Dick, 1952Dick G.W. Zika virus. II. Pathogenicity and physical properties.Trans. R. Soc. Trop. Med. Hyg. 1952; 46: 521-534Abstract Full Text PDF PubMed Scopus (485) Google Scholar, Dick et al., 1952Dick G.W. Kitchen S.F. Haddow A.J. Zika virus. I. Isolations and serological specificity.Trans. R. Soc. Trop. Med. Hyg. 1952; 46: 509-520Abstract Full Text PDF PubMed Scopus (1841) Google Scholar), Zika virus (ZIKV) is a positive-sense enveloped flavivirus that is primarily transmitted by the Aedes aegypti mosquito (Li et al., 2012Li M.I. Wong P.S. Ng L.C. Tan C.H. Oral susceptibility of Singapore aedes (Stegomyia) aegypti (Linnaeus) to zika virus.PLoS Negl. Trop. Dis. 2012; 6: e1792Crossref PubMed Scopus (174) Google Scholar). Unlike other flaviviruses, ZIKV is capable infecting the male reproductive organs, leading to testicular atrophy (Govero et al., 2016Govero J. Esakky P. Scheaffer S.M. Fernandez E. Drury A. Platt D.J. Gorman M.J. Richner J.M. Caine E.A. Salazar V. et al.Zika virus infection damages the testes in mice.Nature. 2016; 540: 438-442Crossref PubMed Scopus (367) Google Scholar, Ma et al., 2016Ma W. Li S. Ma S. Jia L. Zhang F. Zhang Y. Zhang J. Wong G. Zhang S. Lu X. et al.Zika virus causes testis damage and leads to male infertility in mice.Cell. 2016; 167: 1511-1524.e10Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, Uraki et al., 2017Uraki R. Hwang J. Jurado K.A. Householder S. Yockey L.J. Hastings A.K. Homer R.J. Iwasaki A. Fikrig E. Zika virus causes testicular atrophy.Sci. Adv. 2017; 3: e1602899Crossref PubMed Scopus (96) Google Scholar) and sexual transmission (Hastings and Fikrig, 2017Hastings A.K. Fikrig E. Zika virus and Sexual transmission: a new route of transmission for mosquito-borne flaviviruses.Yale J. Biol. Med. 2017; 90: 325-330PubMed Google Scholar). Most infections with this virus are asymptomatic (Duffy et al., 2009Duffy M.R. Chen T.H. Hancock W.T. Powers A.M. Kool J.L. Lanciotti R.S. Pretrick M. Marfel M. Holzbauer S. Dubray C. et al.Zika virus outbreak on Yap Island, Federated States of Micronesia.N. Engl. J. Med. 2009; 360: 2536-2543Crossref PubMed Scopus (2158) Google Scholar), and the majority of symptomatic infections result in a mild and self-limiting febrile illness (Simpson, 1964Simpson D.I. Zika virus infection in man.Trans. R. Soc. Trop. Med. Hyg. 1964; 58: 335-338Abstract Full Text PDF PubMed Scopus (339) Google Scholar, Bearcroft, 1956Bearcroft W.G. Zika virus infection experimentally induced in a human volunteer.Trans. R. Soc. Trop. Med. Hyg. 1956; 50: 442-448Abstract Full Text PDF PubMed Scopus (3) Google Scholar). Rare cases of more severe illness have been reported including Guillain-Barré syndrome (GBS), marked by subacute flaccid paralysis (Oehler et al., 2014Oehler E. Watrin L. Larre P. Leparc-Goffart I. Lastere S. Valour F. Baudouin L. Mallet H. Musso D. Ghawche F. Zika virus infection complicated by Guillain-Barre syndrome–case report, French Polynesia, December 2013.Euro Surveill. 2014; 19: 20720Crossref PubMed Google Scholar, Ioos et al., 2014Ioos S. Mallet H.P. Leparc Goffart I. Gauthier V. Cardoso T. Herida M. Current Zika virus epidemiology and recent epidemics.Med. Mal. Infect. 2014; 44: 302-307Crossref PubMed Scopus (440) Google Scholar) in infected adults, and infection of pregnant women has been associated with severe birth defects, including congenital malformations and severe birth defects in newborns (World Health Organization, 2016World Health Organization, 2016. Zika Situation Report.Google Scholar, Ventura et al., 2016Ventura C.V. Maia M. Bravo-Filho V. Gois A.L. Belfort Jr., R. Zika virus in Brazil and macular atrophy in a child with microcephaly.Lancet. 2016; 387: 228Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, Schuler-Faccini et al., 2016Schuler-Faccini L. Ribeiro E.M. Feitosa I.M. Horovitz D.D. Cavalcanti D.P. Pessoa A. Doriqui M.J. Neri J.I. Neto J.M. Wanderley H.Y. et al.Brazilian Medical Genetics Society–Zika Embryopathy Task ForcePossible association between Zika virus infection and microcephaly - Brazil, 2015.MMWR Morb. Mortal. Wkly. Rep. 2016; 65: 59-62Crossref PubMed Scopus (788) Google Scholar). Upon ZIKV infection, ZIKV is present in a variety of tissues and body fluids including the central nervous system (Tang et al., 2016Tang H. Hammack C. Ogden S.C. Wen Z. Qian X. Li Y. Yao B. Shin J. Zhang F. Lee E.M. et al.Zika virus infects human cortical neural progenitors and attenuates their growth.Cell Stem Cell. 2016; 18: 587-590Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar), saliva (Musso et al., 2015Musso D. Roche C. Nhan T.X. Robin E. Teissier A. Cao-Lormeau V.M. Detection of Zika virus in saliva.J. Clin. Virol. 2015; 68: 53-55Crossref PubMed Scopus (398) Google Scholar), blood (Musso et al., 2016Musso D. Stramer S.L. Busch M.P. Transfusion-Transmitted Diseases CommitteeInternational Society of Blood Transfusion Working Party on Transfusion-Transmitted Infectious DiseasesZika virus: a new challenge for blood transfusion.Lancet. 2016; 387: 1993-1994Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), urine (Zhang et al., 2016Zhang F.C. Li X.F. Deng Y.Q. Tong Y.G. Qin C.F. Excretion of infectious Zika virus in urine.Lancet Infect. Dis. 2016; 16: 641-642Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and semen (Atkinson et al., 2016Atkinson B. Hearn P. Afrough B. Lumley S. Carter D. Aarons E.J. Simpson A.J. Brooks T.J. Hewson R. Detection of zika virus in semen.Emerg. Infect. Dis. 2016; 22: 940Crossref PubMed Scopus (279) Google Scholar), many of which are unique among flaviviruses. Similar to other flaviviruses, ZIKV targets dendritic cells and macrophages in the skin and other tissues for replication (Wu et al., 2000Wu S.J. Grouard-Vogel G. Sun W. Mascola J.R. Brachtel E. Putvatana R. Louder M.K. Filgueira L. Marovich M.A. Wong H.K. et al.Human skin Langerhans cells are targets of dengue virus infection.Nat. Med. 2000; 6: 816-820Crossref PubMed Scopus (528) Google Scholar, Jurado et al., 2016Jurado K.A. Simoni M.K. Tang Z. Uraki R. Hwang J. Householder S. Wu M. Lindenbach B.D. Abrahams V.M. Guller S. Fikrig E. Zika virus productively infects primary human placenta-specific macrophages.JCI Insight. 2016; 1: e88461Crossref PubMed Scopus (119) Google Scholar, Hamel et al., 2015Hamel R. Dejarnac O. Wichit S. Ekchariyawat P. Neyret A. Luplertlop N. Perera-Lecoin M. Surasombatpattana P. Talignani L. Thomas F. et al.Biology of zika virus infection in human skin cells.J. Virol. 2015; 89: 8880-8896Crossref PubMed Scopus (825) Google Scholar), and replication of the virus in the testes (Govero et al., 2016Govero J. Esakky P. Scheaffer S.M. Fernandez E. Drury A. Platt D.J. Gorman M.J. Richner J.M. Caine E.A. Salazar V. et al.Zika virus infection damages the testes in mice.Nature. 2016; 540: 438-442Crossref PubMed Scopus (367) Google Scholar, Ma et al., 2016Ma W. Li S. Ma S. Jia L. Zhang F. Zhang Y. Zhang J. Wong G. Zhang S. Lu X. et al.Zika virus causes testis damage and leads to male infertility in mice.Cell. 2016; 167: 1511-1524.e10Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, Uraki et al., 2017Uraki R. Hwang J. Jurado K.A. Householder S. Yockey L.J. Hastings A.K. Homer R.J. Iwasaki A. Fikrig E. Zika virus causes testicular atrophy.Sci. Adv. 2017; 3: e1602899Crossref PubMed Scopus (96) Google Scholar) and brain (Li et al., 2016aLi C. Xu D. Ye Q. Hong S. Jiang Y. Liu X. Zhang N. Shi L. Qin C.F. Xu Z. Zika virus disrupts neural progenitor development and leads to microcephaly in mice.Cell Stem Cell. 2016; 19: 672Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, Meertens et al., 2017Meertens L. Labeau A. Dejarnac O. Cipriani S. Sinigaglia L. Bonnet-Madin L. le Charpentier T. Hafirassou M.L. Zamborlini A. Cao-Lormeau V.-M. et al.Axl mediates Zika virus entry in human glial cells and modulates innate immune responses.Cell Rep. 2017; 18: 324-333Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) results in apoptosis of important cell types driving pathogenesis. This difference in tissue tropism for ZIKV compared with related flaviviruses has led to significant efforts to identify the entry receptor for this virus. One of the leading candidate proteins implicated as facilitating viral entry is a member of the TAM family of receptor tyrosine kinases, Axl (Hamel et al., 2015Hamel R. Dejarnac O. Wichit S. Ekchariyawat P. Neyret A. Luplertlop N. Perera-Lecoin M. Surasombatpattana P. Talignani L. Thomas F. et al.Biology of zika virus infection in human skin cells.J. Virol. 2015; 89: 8880-8896Crossref PubMed Scopus (825) Google Scholar, Liu et al., 2016Liu S. Delalio L.J. Isakson B.E. Wang T.T. AXL-mediated productive infection of human endothelial cells by zika virus.Circ. Res. 2016; 119: 1183-1189Crossref PubMed Scopus (111) Google Scholar, Retallack et al., 2016Retallack H. di Lullo E. Arias C. Knopp K.A. Laurie M.T. Sandoval-Espinosa C. Mancia Leon W.R. Krencik R. Ullian E.M. et al.Zika virus cell tropism in the developing human brain and inhibition by azithromycin.Proc. Natl. Acad. Sci. U S A. 2016; 113: 14408-14413Crossref PubMed Scopus (329) Google Scholar, Meertens et al., 2017Meertens L. Labeau A. Dejarnac O. Cipriani S. Sinigaglia L. Bonnet-Madin L. le Charpentier T. Hafirassou M.L. Zamborlini A. Cao-Lormeau V.-M. et al.Axl mediates Zika virus entry in human glial cells and modulates innate immune responses.Cell Rep. 2017; 18: 324-333Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, Savidis et al., 2016Savidis G. Mcdougall W.M. Meraner P. Perreira J.M. Portmann J.M. Trincucci G. John S.P. Aker A.M. Renzette N. Robbins D.R. et al.Identification of zika virus and dengue virus dependency factors using functional genomics.Cell Rep. 2016; 16: 232-246Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). However, work from this group (Hastings et al., 2017Hastings A.K. Yockey L.J. Jagger B.W. Hwang J. Uraki R. Gaitsch H.F. Parnell L.A. Cao B. Mysorekar I.U. Rothlin C.V. et al.TAM receptors are not required for zika virus infection in mice.Cell Rep. 2017; 19: 558-568Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and others (Wang et al., 2017Wang Z.Y. Wang Z. Zhen Z.D. Feng K.H. Guo J. Gao N. Fan D.Y. Han D.S. Wang P.G. An J. Axl is not an indispensable factor for Zika virus infection in mice.J. Gen. Virol. 2017; 98: 2061-2068Crossref PubMed Scopus (56) Google Scholar) has shown that Axl is dispensable in a murine model of ZIKV infection, and genetic ablation of Axl in human neural progenitor cells and cerebral organoids does not prevent ZIKV infection (Wells et al., 2016Wells M.F. Salick M.R. Wiskow O. Ho D.J. Worringer K.A. Ihry R.J. Kommineni S. Bilican B. Klim J.R. Hill E.J. et al.Genetic ablation of <em>AXL</em> does not protect human neural progenitor cells and cerebral organoids from zika virus infection.Cell Stem Cell. 2016; 19: 703-708Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). ZIKV infects several cell types that express high levels of Axl (Lemke and Burstyn-Cohen, 2010Lemke G. Burstyn-Cohen T. TAM receptors and the clearance of apoptotic cells.Ann. N. Y. Acad. Sci. 2010; 1209: 23-29Crossref PubMed Scopus (164) Google Scholar, Nowakowski et al., 2016Nowakowski T.J. Pollen A.A. di Lullo E. Sandoval-Espinosa C. Bershteyn M. Kriegstein A.R. Expression analysis highlights AXL as a candidate Zika virus entry receptor in neural stem cells.Cell Stem Cell. 2016; 18: 591-596Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, Ma et al., 2016Ma W. Li S. Ma S. Jia L. Zhang F. Zhang Y. Zhang J. Wong G. Zhang S. Lu X. et al.Zika virus causes testis damage and leads to male infertility in mice.Cell. 2016; 167: 1511-1524.e10Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, Tabata et al., 2016Tabata T. Petitt M. Puerta-Guardo H. Michlmayr D. Wang C. Fang-Hoover J. Harris E. Pereira L. Zika virus targets different primary human placental cells, suggesting two routes for vertical transmission.Cell Host Microbe. 2016; 20: 155-166Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, Rothlin et al., 2015Rothlin C.V. Carrera-Silva E.A. Bosurgi L. Ghosh S. TAM receptor signaling in immune homeostasis.Annu. Rev. Immunol. 2015; 33: 355-391Crossref PubMed Scopus (274) Google Scholar), and signaling of this protein contributes to infection of astrocytes by downregulating type I interferon (IFN) signaling (Chen et al., 2018Chen J. Yang Y.-F. Yang Y. Zou P. Chen J. He Y. Shui S.-L. Cui Y.-R. Bai R. Liang Y.-J. et al.AXL promotes Zika virus infection in astrocytes by antagonizing type I interferon signalling.Nat. Microbiol. 2018; 3: 302-309Crossref PubMed Scopus (102) Google Scholar). Axl is a member of the TAM family of tyrosine kinase receptors. These receptors bind the ligands, Gas6 and Protein S, which recognize phosphatidylserine present on enveloped viruses and dying cells (Shimojima et al., 2007Shimojima M. Ikeda Y. Kawaoka Y. The mechanism of Axl-mediated Ebola virus infection.J. Infect. Dis. 2007; 196: S259-S263Crossref PubMed Scopus (86) Google Scholar, Lemke and Burstyn-Cohen, 2010Lemke G. Burstyn-Cohen T. TAM receptors and the clearance of apoptotic cells.Ann. N. Y. Acad. Sci. 2010; 1209: 23-29Crossref PubMed Scopus (164) Google Scholar). Type I IFN signaling upregulates TAM receptors, which are part of a negative feedback loop for inflammatory responses and inhibits the Toll-like receptor pathway (Rothlin et al., 2007Rothlin C.V. Ghosh S. Zuniga E.I. Oldstone M.B. Lemke G. TAM receptors are pleiotropic inhibitors of the innate immune response.Cell. 2007; 131: 1124-1136Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, Carrera Silva et al., 2013Carrera Silva E.A. Chan P.Y. Joannas L. Errasti A.E. Gagliani N. Bosurgi L. Jabbour M. Perry A. Smith-Chakmakova F. Mucida D. et al.T cell-derived protein S engages TAM receptor signaling in dendritic cells to control the magnitude of the immune response.Immunity. 2013; 39: 160-170Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In dendritic cells, this inhibition is dependent on a physical interaction with the type I IFN receptor (Ifnar) (Rothlin et al., 2007Rothlin C.V. Ghosh S. Zuniga E.I. Oldstone M.B. Lemke G. TAM receptors are pleiotropic inhibitors of the innate immune response.Cell. 2007; 131: 1124-1136Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar). In addition, these receptors contribute to the clearance of apoptotic cells and the differentiation of natural killer cells (Bosurgi et al., 2013Bosurgi L. Bernink J.H. Delgado Cuevas V. Gagliani N. Joannas L. Schmid E.T. Booth C.J. Ghosh S. Rothlin C.V. Paradoxical role of the proto-oncogene Axl and Mer receptor tyrosine kinases in colon cancer.Proc. Natl. Acad. Sci. U S A. 2013; 110: 13091-13096Crossref PubMed Scopus (94) Google Scholar, Caraux et al., 2006aCaraux A. Kim N. Bell S.E. Zompi S. Ranson T. Lesjean-Pottier S. Garcia-Ojeda M.E. Turner M. Colucci F. Phospholipase C-gamma2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells.Blood. 2006; 107: 994-1002Crossref PubMed Scopus (110) Google Scholar, Caraux et al., 2006bCaraux A. Lu Q. Fernandez N. Riou S. di Santo J.P. Raulet D.H. Lemke G. Roth C. Natural killer cell differentiation driven by Tyro3 receptor tyrosine kinases.Nat. Immunol. 2006; 7: 747-754Crossref PubMed Scopus (113) Google Scholar, Paolino et al., 2014Paolino M. Choidas A. Wallner S. Pranjic B. Uribesalgo I. Loeser S. Jamieson A.M. Langdon W.Y. Ikeda F. Fededa J.P. et al.The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells.Nature. 2014; 507: 508-512Crossref PubMed Scopus (307) Google Scholar). ZIKV infection is controlled by type I IFN signaling (Lazear et al., 2016Lazear H.M. Govero J. Smith A.M. Platt D.J. Fernandez E. Miner J.J. Diamond M.S. A mouse model of Zika virus pathogenesis.Cell Host Microbe. 2016; 19: 720-730Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar) and is capable of using its NS5 protein to degrade human STAT2 and inhibit this signaling, but not mouse STAT2 (Grant et al., 2016Grant A. Ponia S.S. Tripathi S. Balasubramaniam V. Miorin L. Sourisseau M. Schwarz M.C. Sánchez-Seco M.P. Evans M.J. Best S.M. García-Sastre A. Zika virus targets human STAT2 to inhibit type I interferon signaling.Cell Host Microbe. 2016; 19: 882-890Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar), requiring the use of immune-deficient mice for assessment of infection in the mouse model. To further assess the role of Axl, we generated an Ifnar/Axl double knockout mouse, which is susceptible to infection, and tested ZIKV replication and pathogenesis in this mouse model. To probe the function of the TAM receptor Axl in a holistic murine infection model lacking this protein (Ifnar−/−Axl−/−), we bred together two existing mouse models. The first, an IFN-αβ receptor knockout (Ifnar−/−), will render this model susceptible to ZIKV infection, and the second, an Axl knockout (Axl−/−), will allow us to probe the role of Axl in ZIKV pathogenesis and replication in specific tissues (Figure 1A). To determine if Axl is involved in replication of ZIKV in this model, we subcutaneously inoculated Ifnar1−/−Axl−/− and Ifnar1−/− mice and show that Axl is not required for ZIKV replication in the blood at days 2, 4, and 6 as measured by qRT-PCR (Figure 1B), in the brain at day 6 as measured by qRT-PCR (Figure 1C), or in plaque assay (Figure 1D). Interestingly, in mice lacking Axl expression ZIKV pathogenesis is significantly decreased, with infected animals showing a slight decrease in weight around days 6–8 (Figure 1E) but most recovering fully, whereas the virus is 100% lethal in mice expressing Axl (Figure 1F). The pathogenesis in this model appeared to be driven by replication of virus in the brain, as the mice that died had hindlimb paralysis and unsteadiness on their feet. This phenotype appears to be age dependent, as Axl knockout weanling mice (3 weeks old) show no difference in ZIKV pathogenesis compared with those expressing Axl (Figures 1G and 1H). These data indicate that although ZIKV does not require Axl to replicate in the mouse model, this protein is important for driving severe disease in vivo. To attempt to determine the mechanism underlying the increased pathogenesis of ZIKV in Axl-competent mice, we collected brains from Ifnar1−/− and Ifnar1−/−Axl−/− mice and isolated RNA to determine the expression of important inflammatory cytokines during viral infection. Using qRT-PCR, we show that the levels of transforming growth factor-β, tumor necrosis factor-α, IFN-γ, and IL-6 are not different between Axl−/− and Axl wild-type mice (Figures 2B–2E), but that Ifnar1−/−Axl−/− mice had significantly lower pro-IL-1β levels when compared with Ifnar1−/− mice (Figure 2A). Interestingly, no differences in the expression of inflammatory cytokines were observed between 3-week-old IFNAR−/−Axl−/− mice and Axl-competent mice of the same age during ZIKV infection (Figure S1). To determine if this increased inflammatory response in 6-week-old Axl knockout mice is a result of enhanced inflammatory cell recruitment or proliferation in the brains of ZIKV-infected mice, we examined the number of immune cells in the brain at day 6 of ZIKV infection using flow cytometry (Figure S2). There is a robust expansion or infiltration of monocytes (Figure 3A), neutrophils (Figure 3B), macrophages (Figure 3C), dendritic cells (Figure 3D), microglia (Figure 3E), and both CD4+ (Figure 3G) and CD8+ (Figure 3H) T cells in infected mice, whereas there are no significant differences between Ifnar1−/− and Ifnar1−/−Axl−/− mice (Figure 3). Intriguingly, there was a trend toward slightly fewer CD11c+ activated microglia in Ifnar1−/−Axl−/− mice (Figure 3F), but this difference was not significant. IL-1β is highly expressed during apoptosis, so we hypothesized that Axl could instead be playing a role in driving apoptosis during ZIKV infection. To examine if Axl plays a role in driving apoptosis in the 6-week-old animals, we dissected brains from ZIKV-infected Ifnar1−/− or Ifnar1−/−Axl−/− mice at day 6, and performed a TUNEL assay, which selectively marks cells with breaks in DNA indicating apoptosis, on fixed brain sections. It is evident that Axl-competent mice have markedly increased apoptosis across several regions of the brain, including the ventral striatum (Figure 4A), the cerebellum (Figure 4B), and the hippocampus (Figure 4C). By co-staining these tissue sections with TUNEL and NeuN (neurons, Figure 5A), glial fibrillary acidic protein (GFAP) (astrocytes, Figure 5B), or Iba1 (microglia, Figure 5C), we can clearly show that microglia represent the main apoptotic cell type present in the brain. As these cells are capable of phagocytosing dead cells, we cannot rule out the possibility that some of the microglial cells appear TUNEL positive owing to phagocytosis of other cell types; however, the majority of these cells show TUNEL staining in the nucleus (co-localizing with DAPI staining) and not in the cytoplasm (Figure 5C), which indicates that these cells are undergoing apoptosis themselves. When TUNEL+ microglia were quantified, significantly more TUNEL+ microglia were observed in the brains of Axl-competent mice compared with Axl−/− mice (Figure 5D), further suggesting a role of Axl in driving apoptosis in these cells. The canonical apoptosis pathway is caspase 3 dependent and can be either extrinsic through death receptor (like tumor necrosis factor-related apoptosis-inducing ligand receptor) signaling and caspase 8, or intrinsic through mitochondrial-associated Bcl-2 homology proteins and caspase 9. Downstream of both these pathways is cleavage of poly (ADP-ribose) polymerase (PARP), which is responsible for repairing DNA. Six-week-old Axl-deficient mice have significantly lower levels of cleaved PARP shown by western blot analysis of whole ZIKV-infected brain homogenates of ZIKV-infected mice, and there was significantly higher caspase 8 and cleaved caspase 9, and a trend toward higher caspase 3, cleaved caspase 3, and caspase 9, in the Ifnar1−/− mice when compared with the Axl-deficient mice, which indicates that the observed apoptosis is induced both through the intrinsic and extrinsic pathways (Figures 6 and S3). Caspase 12 is involved in a less common apoptotic pathway, and whereas less caspase 12 expression was seen in the Axl-deficient group (Figure 6H), no change in cleaved caspase 12 was observed (Figure 6I). This suggests that Axl signals through a caspase-dependent PARP-dependent manner leading to apoptosis in microglia. Axl has been shown to be involved in the immune response during ZIKV infection and has been implicated as an entry factor for specific cell types. To further examine the role of Axl in replication and pathogenesis in mice, we generated a mouse lacking Axl that was also deficient in the Ifnar protein, rendering these animals highly susceptible to ZIKV infection. Using this model, we demonstrate that although, similar to our previous work, ZIKV replication levels are unaffected by the lack of Axl, the loss of this molecule protected mice from severe viral pathogenesis in the brain. This protection coincided with significantly lower inflammatory pro-IL-1β expression and a decrease in apoptosis, specifically in glial cells, in the brains of Axl-deficient mice. These data suggest that Axl present on microglia is involved in facilitating ZIKV pathogenesis by inducing apoptosis in these cells. Small-molecule inhibition of Axl in human cell lines (Hamel et al., 2015Hamel R. Dejarnac O. Wichit S. Ekchariyawat P. Neyret A. Luplertlop N. Perera-Lecoin M. Surasombatpattana P. Talignani L. Thomas F. et al.Biology of zika virus infection in human skin cells.J. Virol. 2015; 89: 8880-8896Crossref PubMed Scopus (825) Google Scholar, Liu et al., 2016Liu S. Delalio L.J. Isakson B.E. Wang T.T. AXL-mediated productive infection of human endothelial cells by zika virus.Circ. Res. 2016; 119: 1183-1189Crossref PubMed Scopus (111) Google Scholar, Retallack et al., 2016Retallack H. di Lullo E. Arias C. Knopp K.A. Laurie M.T. Sandoval-Espinosa C. Mancia Leon W.R. Krencik R. Ullian E.M. et al.Zika virus cell tropism in the developing human brain and inhibition by azithromycin.Proc. Natl. Acad. Sci. U S A. 2016; 113: 14408-14413Crossref PubMed Scopus (329) Google Scholar, Meertens et al., 2017Meertens L. Labeau A. Dejarnac O. Cipriani S. Sinigaglia L. Bonnet-Madin L. le Charpentier T. Hafirassou M.L. Zamborlini A. Cao-Lormeau V.-M. et al.Axl mediates Zika virus entry in human glial cells and modulates innate immune responses.Cell Rep. 2017; 18: 324-333Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, Savidis et al., 2016Savidis G. Mcdougall W.M. Meraner P. Perreira J.M. Portmann J.M. Trincucci G. John S.P. Aker A.M. Renzette N. Robbins D.R. et al.Identification of zika virus and dengue virus dependency factors using functional genomics.Cell Rep. 2016; 16: 232-246Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), small interfering RNA knockdown of Axl human alveolar basal epithelial carcinoma cells (A549) (Hamel et al., 2015Hamel R. Dejarnac O. Wichit S. Ekchariyawat P. Neyret A. Luplertlop N. Perera-Lecoin M. Surasombatpattana P. Talignani L. Thomas F. et al.Biology of zika virus infection in human skin cells.J. Virol. 2015; 89: 8880-8896Crossref PubMed Scopus (825) Google Scholar), and CRISPR knockout of Axl in human cervical adenocarcinoma cells (HeLa) (Savidis et al., 2016Savidis G. Mcdougall W.M. Meraner P. Perreira J.M. Portmann J.M. Trincucci G. John S.P. Aker A.M. Renzette N. Robbins D.R. et al.Identification of zika virus and dengue virus dependency factors using functional genomics.Cell Rep. 2016; 16: 232-246Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), a human glioblastoma line (U87) (Retallack et al., 2016Retallack H. di Lullo E. Arias C. Knopp K.A. Laurie M.T. Sandoval-Espinosa C. Mancia Leon W.R. Krencik R. Ullian E.M. et al.Zika virus cell tropism in the developing human brain and inhibition by azithromycin.Proc. Natl. Acad. Sci. U S A. 2016; 113: 14408-14413Crossref PubMed Scopus (329) Google Scholar), a human microglial cell line (CHME3) (Meertens et al., 2017Meertens L. Labeau A. Dejarnac O. Cipriani S. Sinigaglia L. Bonnet-Madin L. le Charpentier T. Hafirassou M.L. Zamborlini A. Cao-Lormeau V.-M. et al.Axl mediates Zika virus entry in human glial cells and modulates innate immune responses.Cell Rep. 2017; 18: 324-333Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar), and human embryonic kidney cells (293T) (Liu et al., 2016Liu S. Delalio L.J. Isakson B.E. Wang T.T. AXL-mediated productive infection of human endothelial cells by zika virus.Circ. Res. 2016; 119: 1183-1189Crossref PubMed Scopus (111) Google Scholar) has been shown to block or reduce ZIKV infection, but Axl is not necessary for replication of ZIKV in the mouse model (Hastings et al., 2017Hastings A.K. Yockey L.J. Jagger B.W. Hwang J. Uraki R. Gaitsch H.F. Parnell L.A. Cao B. Mysorekar I.U. Rothlin C.V. et al.TAM receptors are not required for zika virus infection in mice.Cell Rep. 2017; 19: 558-568Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, Wang et al., 2017Wang Z.Y. Wang Z. Zhen Z.D. Feng K.H. Guo J. Gao N. Fan D.Y. Han D.S. Wang P.G. An J. Axl is" @default.
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• W2920026340 title "Loss of the TAM Receptor Axl Ameliorates Severe Zika Virus Pathogenesis and Reduces Apoptosis in Microglia" @default.
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