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Correlation between Apoptosis and in Situ Immune Response in Fatal Cases of Microcephaly Caused by Zika Virus
2018; Elsevier BV; Volume: 188; Issue: 11 Linguagem: Inglês
10.1016/j.ajpath.2018.07.009
ISSN1525-2191
AutoresJorge Rodrigues de Sousa, Raimunda do Socorro da Silva Azevedo, Arnaldo Jorge Martins Filho, Marialva Tereza Ferreira de Araújo, Ermelinda R.C. Moutinho, Barbara Cristina Baldez Vasconcelos, Ana Cecília Ribeiro Cruz, Consuelo Silva de Oliveira, Lívia Carício Martins, Beatriz Helena Baldez Vasconcelos, Lívia Medeiros Neves Casseb, Jannifer Oliveira Chiang, Juarez Antônio Simões Quaresma, Pedro Fernando da Costa Vasconcelos,
Tópico(s)Infectious Encephalopathies and Encephalitis
ResumoZika virus (ZIKV) is a single-stranded positive-sense RNA flavivirus that possesses a genome approximately 10.7 Kb in length. Although pro-inflammatory and anti-inflammatory cytokines and apoptotic markers belonging to the extrinsic and intrinsic pathways are suggested to be involved in fatal cases of ZIKV-induced microcephaly, their exact roles and associations are unclear. To address this, brain tissue samples were collected from 10 individuals, five of whom were diagnosed as ZIKV positive with microcephaly and a further five were flavivirus-negative controls that died because of other causes. Examination of material from the fatal cases of microcephaly revealed lesions in the cerebral cortex, edema, vascular proliferation, neuronal necrosis, gliosis, neuronophagy, calcifications, apoptosis, and neuron loss. The expression of various apoptosis markers in the neural parenchyma, including FasL, FAS, BAX, BCL2, and caspase 3 differed between ZIKV-positive cases and controls. Further investigation of type 1 and 2 helper T-cell cytokines confirmed a greater anti-inflammatory response in fatal ZIKV-associated microcephaly cases. Finally, an analysis of the linear correlation between tumor necrosis factor-α, IL-1β, IL-4, IL-10, transforming growth factor-β, and IL-33 expression and various apoptotic markers suggested that the immune response may be associated with the apoptotic phenomenon observed in ZIKV-induced microcephaly. Zika virus (ZIKV) is a single-stranded positive-sense RNA flavivirus that possesses a genome approximately 10.7 Kb in length. Although pro-inflammatory and anti-inflammatory cytokines and apoptotic markers belonging to the extrinsic and intrinsic pathways are suggested to be involved in fatal cases of ZIKV-induced microcephaly, their exact roles and associations are unclear. To address this, brain tissue samples were collected from 10 individuals, five of whom were diagnosed as ZIKV positive with microcephaly and a further five were flavivirus-negative controls that died because of other causes. Examination of material from the fatal cases of microcephaly revealed lesions in the cerebral cortex, edema, vascular proliferation, neuronal necrosis, gliosis, neuronophagy, calcifications, apoptosis, and neuron loss. The expression of various apoptosis markers in the neural parenchyma, including FasL, FAS, BAX, BCL2, and caspase 3 differed between ZIKV-positive cases and controls. Further investigation of type 1 and 2 helper T-cell cytokines confirmed a greater anti-inflammatory response in fatal ZIKV-associated microcephaly cases. Finally, an analysis of the linear correlation between tumor necrosis factor-α, IL-1β, IL-4, IL-10, transforming growth factor-β, and IL-33 expression and various apoptotic markers suggested that the immune response may be associated with the apoptotic phenomenon observed in ZIKV-induced microcephaly. Zika virus (ZIKV) is a member of the single-stranded positive-sense RNA virus Flaviviridae family1Azevedo R.S.S. de Sousa J.R. Araujo M.T.F. Martins Filho A.J. de Alcantara B.N. Araujo F.M.C. Queiroz M.G.L. Cruz A.C.R. Vasconcelos B.H.B. Chiang J.O. Martins L.C. Casseb L.M.N. da Silva E.V. Carvalho V.L. Vasconcelos B.C.B. Rodrigues S.G. Oliveira C.S. Quaresma J.A.S. Vasconcelos P.F.C. In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus.Sci Rep. 2018; 8: 1Crossref PubMed Scopus (165) Google Scholar that was first isolated from the blood of a rhesus macaque (Macaca mulatta) in 1947 in the Zika Forest, Uganda, during a sentinel study.2Dick 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 (1854) Google Scholar Like many other flaviviruses, ZIKV was initially thought to be transmitted solely through the bite of infective mosquitoes of the Aedes genus (primarily Aedes aegypti in tropical regions). Recently, however, ZIKV has been found to be transmissible via congenital and sexual routes,3Desclaux A. de Lamballerie X. Leparc-Goffart I. Vilain-Parcé A. Coatleven F. Fleury H. Malvy D. Probable sexually transmitted Zika virus infection in a pregnant woman.N Engl J Med. 2018; 378: 1458-1460Crossref PubMed Scopus (13) Google Scholar and to infect a wide group of cells across different organs and tissues.4Miner J.J. Diamond M.S. Zika virus pathogenesis and tissue tropism.Cell Host Microbe. 2017; 21: 134-142Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar The symptoms presented during infection are similar to those presented by other viruses, such as dengue (DENV) and chikungunya, although ZIKV is defined by an abrupt onset of mild fever, myalgia, rash, conjunctival hyperemia, joint edema, and headache.5Zorrilla C.D. García I. García Fragoso L. De La Vega A. Zika virus infection in pregnancy: maternal, fetal, and neonatal considerations.J Infect Dis. 2017; 216 Suppl 10: S891-S896Crossref Scopus (32) Google Scholar In recent years, ZIKV has become a global concern because of increased incidences of Guillain-Barré syndrome, microcephaly, and other congenital malformations.1Azevedo R.S.S. de Sousa J.R. Araujo M.T.F. Martins Filho A.J. de Alcantara B.N. Araujo F.M.C. Queiroz M.G.L. Cruz A.C.R. Vasconcelos B.H.B. Chiang J.O. Martins L.C. Casseb L.M.N. da Silva E.V. Carvalho V.L. Vasconcelos B.C.B. Rodrigues S.G. Oliveira C.S. Quaresma J.A.S. Vasconcelos P.F.C. In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus.Sci Rep. 2018; 8: 1Crossref PubMed Scopus (165) Google Scholar In 2007, ZIKV was first reported outside of its original endemic area during an outbreak on Yap Island, Federated States of Micronesia.6Duffy 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. Guillaumot L. Griggs A. Bel M. Lambert A.J. Laven J. Kosoy O. Panella A. Biggerstaff B.J. Fischer M. Hayes E.B. Zika virus outbreak on Yap Island, Federated States of Micronesia.N Engl J Med. 2009; 360: 2536-2543Crossref PubMed Scopus (2171) Google Scholar, 7Musso D. Nilles E.J. Cao-Lormeau V.M. Rapid spread of emerging Zika virus in the Pacific area.Clin Microbiol Infect. 2014; 20: O595-O596Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar By October 2013, ZIKV had reached French Polynesia in the South Pacific, and by April 2014, an estimated 30,000 individuals were infected.8Cao-Lormeau V.M. Roche C. Teissier A. Robin E. Berry A.L. Mallet H.P. Sall A.A. Musso D. Zika virus, French Polynesia, South Pacific, 2013.Emerg Infect Dis. 2014; 20: 1085-1086Crossref PubMed Scopus (55) Google Scholar, 9Jouannic J.M. Friszer S. Leparc-Goffart I. Garel C. Eyrolle-Guignot D. Zika virus infection in French Polynesia.Lancet. 2016; 387: 1051-1052Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar In 2015, there was an outbreak in Brazil, with estimated ZIKV cases ranging from 440,000 to 1,300,000.10Hennessey M. Fischer M. Staples J.E. Zika virus spreads to new areas-region of the Americas, May 2015-January 2016.MMWR Morb Mortal Wkly Rep. 2016; 65: 55-58Crossref PubMed Scopus (309) Google Scholar, 11Heukelbach J. Alencar C.H. Kelvin A.A. de Oliveira W.K. Pamplona de Góes Cavalcanti L. Zika virus outbreak in Brazil.J Infect Dev Ctries. 2016; 10: 116-120Crossref PubMed Scopus (130) Google Scholar Accordingly, an increase in the number of Guillain-Barré syndrome cases was also reported in the country. More important, higher rates of microcephaly in neonates and greater mortality in patients with autoimmune disorders were directly associated with ZIKV infection for the first time during the outbreak.12Araujo L.M. Ferreira M.L. Nascimento O.J. Guillain-Barré syndrome associated with the Zika virus outbreak in Brazil.Arq Neuropsiquiatr. 2016; 74: 253-255Crossref PubMed Scopus (111) Google Scholar, 13Azevedo R.S. Araujo M.T. Martins Filho A.J. Oliveira C.S. Nunes B.T. Cruz A.C. Nascimento A.G. Medeiros R.C. Caldas C.A. Araujo F.C. Quaresma J.A. Vasconcelos B.C. Queiroz M.G. da Rosa E.S. Henriques D.F. Silva E.V. Chiang J.O. Martins L.C. Medeiros D.B. Lima J.A. Nunes M.R. Cardoso J.F. Silva S.P. Shi P.Y. Tesh R.B. Rodrigues S.G. Vasconcelos P.F. Zika virus epidemic in Brazil, I: fatal disease in adults: clinical and laboratorial aspects.J Clin Virol. 2016; 85: 56-64Crossref PubMed Scopus (58) Google Scholar Since the identification of this association, research has been undertaken to understand the development of microcephaly in neonates and the role of the immune response in ZIKV pathogenesis.1Azevedo R.S.S. de Sousa J.R. Araujo M.T.F. Martins Filho A.J. de Alcantara B.N. Araujo F.M.C. Queiroz M.G.L. Cruz A.C.R. Vasconcelos B.H.B. Chiang J.O. Martins L.C. Casseb L.M.N. da Silva E.V. Carvalho V.L. Vasconcelos B.C.B. Rodrigues S.G. Oliveira C.S. Quaresma J.A.S. Vasconcelos P.F.C. In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus.Sci Rep. 2018; 8: 1Crossref PubMed Scopus (165) Google Scholar, 14Ali A. Wahid B. Rafique S. Idrees M. Advances in research on Zika virus.Asian Pac J Trop Med. 2017; 10: 321-331Crossref PubMed Scopus (27) Google Scholar It is currently believed that the first cells infected by ZIKV in the brain are astrocytes, microglial cells, and neural progenitor cells. Infection of these cells may be facilitated by the presence of T-cell/transmembrane, Ig, and mucin and Tyro3, AXL, and Mertk receptors that mediate ZIKV entry, particularly the receptor tyrosine kinase AXL.1Azevedo R.S.S. de Sousa J.R. Araujo M.T.F. Martins Filho A.J. de Alcantara B.N. Araujo F.M.C. Queiroz M.G.L. Cruz A.C.R. Vasconcelos B.H.B. Chiang J.O. Martins L.C. Casseb L.M.N. da Silva E.V. Carvalho V.L. Vasconcelos B.C.B. Rodrigues S.G. Oliveira C.S. Quaresma J.A.S. Vasconcelos P.F.C. In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus.Sci Rep. 2018; 8: 1Crossref PubMed Scopus (165) Google Scholar, 15Meertens L. Labeau A. Dejarnac O. Cipriani S. Sinigaglia L. Bonnet-Madin L. Le Charpentier T. Hafirassou M.L. Zamborlini A. Cao-Lormeau V.M. Coulpier M. Missé D. Jouvenet N. Tabibiazar R. Gressens P. Schwartz O. Amara A. 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 (283) Google Scholar However, the exact mechanisms involved in ZIKV infection, especially the entry pathway, remain unclear and somewhat controversial. Indeed, it has recently been shown that AXL may not be essential for ZIKV entry into cells of the central nervous system (CNS). Instead, AXL may promote ZIKV infection in astrocytes by antagonizing type I interferon signaling.16Chen J. Yang Y.F. Yang Y. Zou P. Chen J. He Y. Shui S.L. Cui Y.R. Bai R. Liang Y.J. Hu Y. Jiang B. Lu L. Zhang X. Liu J. Xu J. AXL promotes Zika virus infection in astrocytes by antagonizing type I interferon signalling.Nat Microbiol. 2018; 3: 302-309Crossref PubMed Scopus (105) Google Scholar With regards to the development of microcephaly, apoptosis appears to contribute to pathology via the elimination of infected cells, with little tissue damage.17Teng Y. Liu S. Guo X. Liu S. Jin Y. He T. Bi D. Zhang P. Lin B. An X. Feng D. Mi Z. Tong Y. An integrative analysis reveals a central role of P53 activation via MDM2 in Zika virus infection induced cell death.Front Cell Infect Microbiol. 2017; 7: 327Crossref PubMed Scopus (23) Google Scholar, 18Ho C.Y. Ames H.M. Tipton A. Vezina G. Liu J.S. Scafidi J. Torii M. Rodriguez F.J. du Plessis A. DeBiasi R.L. Differential neuronal susceptibility and apoptosis in congenital Zika virus infection.Ann Neurol. 2017; 82: 121-127Crossref PubMed Scopus (27) Google Scholar Because of the close relationship between the immune response and apoptosis, activation of various death receptors, including the first apoptosis signal receptor (FAS) and tumor necrosis factor-α receptor, can induce activation of FAS-associated protein with death domain (alias MORT1) and tumor necrosis factor receptor type 1–associated death domain proteins. Such activation can lead to the development of a caspase cascade that culminates in programed cell death.19Shrestha B. Diamond M.S. Fas ligand interactions contribute to CD8+ T-cell-mediated control of West Nile virus infection in the central nervous system.J Virol. 2007; 81: 11749-11757Crossref PubMed Scopus (76) Google Scholar, 20Tsao C.H. Su H.L. Lin Y.L. Yu H.P. Kuo S.M. Shen C.I. Chen C.W. Liao C.L. Japanese encephalitis virus infection activates caspase-8 and -9 in a FADD-independent and mitochondrion-dependent manner.J Gen Virol. 2008; 89: 1930-1941Crossref PubMed Scopus (35) Google Scholar In particular, toll-like receptor 3 has been shown to recognize pathogen-associated molecular patterns specific to ZIKV, leading to deregulation of the cellular cycle and activation of proapoptotic genes. After expression, these proteins promote the death of progenitor neurons.21Faizan M.I. Abdullah M. Ali S. Naqvi I.H. Ahmed A. Parveen S. Zika virus-induced microcephaly and its possible molecular mechanism.Intervirology. 2016; 59: 152-158Crossref PubMed Scopus (51) Google Scholar In the brain, and primarily the cerebral cortex, increased expression of caspase 3 has been shown to associate with the death of cortical neurons.1Azevedo R.S.S. de Sousa J.R. Araujo M.T.F. Martins Filho A.J. de Alcantara B.N. Araujo F.M.C. Queiroz M.G.L. Cruz A.C.R. Vasconcelos B.H.B. Chiang J.O. Martins L.C. Casseb L.M.N. da Silva E.V. Carvalho V.L. Vasconcelos B.C.B. Rodrigues S.G. Oliveira C.S. Quaresma J.A.S. Vasconcelos P.F.C. In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus.Sci Rep. 2018; 8: 1Crossref PubMed Scopus (165) Google Scholar Despite these many studies, a full understanding of the roles of pro-inflammatory and anti-inflammatory cytokines, and apoptotic markers, in the pathogenesis of ZIKV remains unclear. Therefore, our study examined apoptosis and the in situ immune response in the neural parenchyma of fatal cases of microcephaly caused by ZIKV relative to uninfected controls. These data were used to investigate the mechanisms and associations that may underlie cell injury because of the virus. We also examined the relationship between in situ apoptosis markers and the expression of various type 1 and 2 helper T-cell cytokines, in addition to any gross changes in histopathology. Patient samples were collected and processed under an emergency context during the recent ZIKV epidemic, defined by the Ministry of Health, Brazil. The study was approved by the research human ethic committee of the Evandro Chagas Institute, Brazil (opinion number 1.888.946). In total, 10 patients were included in the study. Five comprised the ZIKV-positive group and presented with microcephaly (three were neonates, whereas two were stillbirths). The control group consisted of two newborns and three stillbirths who had no evidence of ZIKV infection, nor that of any other flavivirus or alfavirus infections that circulate in Brazil, such as DENV, yellow fever virus, and chikungunya. Each of the five patients of the control group had preserved neural architecture samples suitable for comparison with the ZIKV group. All control group samples were submitted to the same laboratory tests of the patients [ie, real-time quantitative RT-PCR (RT-qPCR), attempts of virus isolation or cell culture, and immunohistochemical assay]. All cases were obtained during the ZIKV epidemic in Brazil in 2016. Detailed information on patients and controls is shown in Table 1.Table 1Details and Distributions of ZIKV-Positive Microcephaly Cases and Controls without Evidence of ZIKV InfectionCaseCategoryLifetimeSexCase informationIHC (ZIKV)Real-time RT-qPCR (ZIKV)Microcephaly 1Newborn2 hoursMMicrocephaly and other malformations (nasopalatine palate and sindactyly, club foot)PositiveNegative 2Newborn27 daysMMicrocephaly and other congenital malformationsPositivePositive 3Newborn2 daysMMicrocephalyPositiveNegative 4StillbirthNAFMicrocephaly detected at birthPositiveNR 5StillbirthNAFMicrocephalyPositiveNegativeControl 6Newborn10 daysFNo microcephaly; death 10 days after birth without evidence of ZIKV infectionNegativeNegative 7StillbirthNAMMicrocephaly without evidence ZIKV infectionNegativeNegative 8StillbirthNAFMicrocephaly without evidence ZIKV infectionNegativeNR 9StillbirthNAFMicrocephaly without evidence ZIKV infectionNegativeNR 10Newborn19 hoursFMicrocephaly case without evidence of ZIKV infection; mother with hyperthyroidism and pregnancy-induced by hypertensionNegativeNegativeF, female; M, male; IHC, immunohistochemistry; NA, not applicable; NR, not performed; RT-qPCR, quantitative RT-PCR; ZIKV, Zika virus. Open table in a new tab F, female; M, male; IHC, immunohistochemistry; NA, not applicable; NR, not performed; RT-qPCR, quantitative RT-PCR; ZIKV, Zika virus. Viruses were initially isolated using C6/36 cell culture, as described in a previous study.22Tesh R.D. An improved method for the isolation and identification of dengue viruses, using mosquito cell cultures.Am J Trop Med Hyg. 1979; 6: 1053-1059Crossref Scopus (141) Google Scholar Briefly, a solution of phosphate-buffered saline containing 10% fetal bovine serum and antibiotics was used to triturate the blood and tissue samples. Next, 100-μL aliquots of each supernatant were inoculated into 25-cm2 tissue culture flasks preseeded with a monolayer of C6/36 Aedes albopictus cells. After 2 hours' incubation at 28°C, more medium was added and the cells were incubated at room temperature (approximately 25°C) for 10 days. ZIKV RNA was detected by real-time RT-qPCR using a previously described protocol.23Faye O. Faye O. Dupressoir A. Weidmann M. Ndiaye M. Alpha Sall A. One-step RT-PCR for detection of Zika virus.J Clin Virol. 2008; 43: 96-101Crossref PubMed Scopus (183) Google Scholar, 24Lanciotti R.S. Kosoy O.L. Laven J.J.J. Velez O. Lambert A.J. Johnson A.J. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007.Emerg Infect Dis. 2008; 14: 1232-1239Crossref PubMed Scopus (1607) Google Scholar For the final assessment, RNA was isolated from tissue homogenate supernatants and sera using a TRIzol Plus RNA Extraction Kit (Ambion; Thermo Fisher Scientific, Waltham, MA), according to the manufacturer's instructions. Real-time RT-qPCR was performed using a 7500 Real Time PCR System (Thermo Fisher Scientific) and a Superscript III Platinum One-Step RT-qPCR Kit (Thermo Fisher Scientific). The detection of specific regions of the ZIKV genome was performed using various primer/probe sets that targeted the NS523Faye O. Faye O. Dupressoir A. Weidmann M. Ndiaye M. Alpha Sall A. One-step RT-PCR for detection of Zika virus.J Clin Virol. 2008; 43: 96-101Crossref PubMed Scopus (183) Google Scholar and E24Lanciotti R.S. Kosoy O.L. Laven J.J.J. Velez O. Lambert A.J. Johnson A.J. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007.Emerg Infect Dis. 2008; 14: 1232-1239Crossref PubMed Scopus (1607) Google Scholar sequences. Real-time PCR was performed independently for all cases, despite cell culture results obtained during attempts of virus isolation. In general, the specimens sent to RT-PCR were blood and CNS tissue samples. All CNS specimens were obtained from autopsy performed in the place of the case report. For all laboratory protocols, the patient and control samples were run together in the same assay, which in general were performed in triplicate. Mother samples were not equally received. To avoid autolysis of the tissue, immediately after autopsy, the material was conserved in 10% neutral-buffered formalin to be processed. For the histopathological analysis, sections (5 μm thick) were cut from paraffin-embedded tissue samples and stained with hematoxylin and eosin. The immunohistochemical study used a streptavidin-alkaline phosphatase method that was adapted to detect viral antigens using a polyclonal anti-ZIKV antibody produced by the Evandro Chagas Institute/Ministry of Health, Brazil.13Azevedo R.S. Araujo M.T. Martins Filho A.J. Oliveira C.S. Nunes B.T. Cruz A.C. Nascimento A.G. Medeiros R.C. Caldas C.A. Araujo F.C. Quaresma J.A. Vasconcelos B.C. Queiroz M.G. da Rosa E.S. Henriques D.F. Silva E.V. Chiang J.O. Martins L.C. Medeiros D.B. Lima J.A. Nunes M.R. Cardoso J.F. Silva S.P. Shi P.Y. Tesh R.B. Rodrigues S.G. Vasconcelos P.F. Zika virus epidemic in Brazil, I: fatal disease in adults: clinical and laboratorial aspects.J Clin Virol. 2016; 85: 56-64Crossref PubMed Scopus (58) Google Scholar It is important to emphasize that in serologic tests, the ZIKV-antibody presents cross-reactivity mainly with DENV; however, in the immunohistochemical assays, the reaction was clearly monotypic to ZIKV-antibody in the tissues and cross-reactivity with other flaviviruses, such as DENV and yellow fever virus, was not observed. The investigation of apoptosis markers and cytokines (Table 2) was based on a previously published biotin-streptavidin-peroxidase method.25Hsu S.M. Raine L. Protein A, avidin, and biotin in immunohistochemistry.J Histochem Cytochem. 1981; 29: 1349-1353Crossref PubMed Scopus (541) Google Scholar Briefly, histologic sections were first deparaffinized in xylene and hydrated in a decreasing ethanol series (90%, 80%, and 70%). Endogenous peroxidase was then blocked with 3% hydrogen peroxide for 45 minutes. Finally, antigenic recovery was performed using a citrate buffer (pH 6.0) for 20 minutes at 90°C. Nonspecific protein interactions were blocked by incubating the sections in 10% skim milk for 30 minutes. Histologic sections were incubated overnight with primary antibodies (Abcam, Paris, France) diluted in 1% bovine serum albumin (Table 2). Next, the slides were immersed in 1× phosphate-buffered saline and incubated with a secondary biotinylated antibody (labeled streptavidin-biotin; DakoCytomation, Glostrup, Denmark) in an oven for 30 minutes at 37°C. The slides were then again immersed in 1× phosphate-buffered saline and incubated with streptavidin peroxidase (labeled streptavidin-biotin) for 30 minutes at 37°C. Finally, the reactions were developed with 0.03% diaminobenzidine and 3% hydrogen peroxide as a chromogenic solution. The slides were washed in distilled water and counterstained with Harris hematoxylin for 1 minute before being dehydrated in an increasing ethanol series and cleared in xylene.Table 2Antibodies Used in the Study of Apoptosis and Immune Response in Fatal Cases of ZIKV-Associated MicrocephalyMarkersReferenceDilutionFasLAbcam (Ab 15285)1:50FASAbcam (Ab 82419)1:50BAXAbcam (Ab 32503)1:50BCL2Abcam (Ab 59348)1:50RIP1Abcam (Ab 72139)1:50Caspase 3Abcam (Ab 4051)1:50TNF-αAbcam (Ab 6671)1:50IL-1βAbcam (Ab 9722)1:50IL-4Abcam (Ab 9622)1:50IL-10Abcam (Ab 34843)1:50TGF-βAbcam (Ab 190503)1:50IL-33Abcam (Ab 118503)1:50TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; ZIKV, Zika virus. Open table in a new tab TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; ZIKV, Zika virus. Histologic sections were analyzed using an Axio Imager Z1 microscope (Zeiss, Oberkochen, Germany). Quantification of staining was assessed across a random selection of 10 fields at high magnification (×400) from the parenchyma of the cerebral cortex of ZIKV-positive or ZIKV-negative cases. Each field was subdivided into 10 × 10 areas, delimited by a grid that comprised an area of 0.0625 mm2. Statistical analysis was performed in GraphPad Prism 5.0 (GraphPad, La Jolla, CA) using t-tests and Pearson's correlation tests. For both tests, the threshold level of significance was set at 5% (P ≤ 0.05). An initial analysis of the intraparenchymal environment showed various differences in brain structure between ZIKV-infected and control samples. Specifically, there were lesions in the cerebral cortex of fatal cases of ZIKV-associated microcephaly, in addition to edema and vascular proliferation (Figure 1, A and B ), neuronal necrosis (Figure 1C), gliosis, neuronophagy (Figure 1D), calcifications (Figure 1F), apoptosis (Figure 1H), and neuron loss, suggestive of neuronophagy (Figure 1H). Detection of viral antigen revealed that ZIKV infected several different cell types in the neural parenchyma, including neurons and glial cells (Figure 1, C–H). The study of different markers for apoptosis, including FasL, FAS, BAX, BCL2, RIP1, and caspase 3, showed that extrinsic and intrinsic pathways are activated, where expression of proteins (FasL, FAS, BAX, BCL2, and RIP1) and caspase 3 is more intense in ZIKV-positive cases compared with the control (Table 3 and Figure 2).Table 3Quantitative Analysis of Apoptotic Markers and Cytokines in the Neural Parenchyma of Fatal ZIKV-Associated Microcephaly Cases Compared with ControlsMarkersParenchymaControlP valueFasL14.60 ± 2.7025.000 ± 2.5500.0004∗∗∗∗FAS15.80 ± 0.8364.200 ± 1.9240.0001∗∗∗∗BAX15.00 ± 1.8716.800 ± 2.3870.0003∗∗∗∗BCL210.80 ± 1.4835.600 ± 2.0740.0018∗∗∗RIP117.40 ± 3.7827. 000 ± 3.5360.0020∗∗∗Caspase 325.20 ± 5.2156.400 ± 2.9660.0001∗∗∗∗TNF-α31.20 ± 4.14711.40 ± 2.8810.0001∗∗∗∗IL-1β28.00 ± 1.5819.400 ± 2.8810.0001∗∗∗∗IL-435.40 ± 2.9669.600 ± 4.6150.0001∗∗∗∗IL-1040.20 ± 3.1149.000 ± 4.7430.0001∗∗∗∗TGF-β33.20 ± 1.3046.600 ± 2.7020.0001∗∗∗∗IL-3352.40 ± 2.51011.00 ± 1.5810.0001∗∗∗∗∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 (t-test).TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; ZIKV, Zika virus. Open table in a new tab ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 (t-test). TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; ZIKV, Zika virus. The expression of several cytokines was also analyzed in the neural parenchyma, including IL-1β, TNF-α, IL-4, IL-10, transforming growth factor (TGF)-β, and IL-33. This demonstrated statistically significant differences in the expression of both pro-inflammatory (IL-1β and TNF-α) (Figure 3) and anti-inflammatory cytokines between cases and controls (Table 3 and Figure 3). Immunostaining for type 2 helper T-cell cytokines (IL-4, IL-10, TGF-β, and IL-33) was more intense in areas of the cerebral cortex where histopathology revealed extensive cell damage, particularly for IL-33 (Figure 3). Finally, a correlation analysis indicated a positive association between the FasL/FAS, BAX/BCL2, and caspase 3 markers and pro-inflammatory and anti-inflammatory cytokines in the neural parenchyma (Table 4).Table 4Linear Correlation between Markers of Apoptosis and Cytokines in Fatal ZIKV-Associated Microcephaly CasesCorrelationr valueP valueFasL × FAS0.50870.3814FasL × BAX0.89030.0429∗P < 0.05 (Pearson correlation).FasL × BCL2−058640.2986FasL × caspase 30.68130.2053FAS × caspase 30.64170.2431BAX × BCL2−0.72070.1695BAX × caspase 30.69180.1955BCL2 × RIP1−0.73990.1528BCL2 × caspase 3−0.89840.0382∗P < 0.05 (Pearson correlation).RIP1 × caspase 30.80620.0993TNF-α × FasL0.92370.0250∗P < 0.05 (Pearson correlation).TNF-α × FAS0.59080.2941TNF-α × BAX0.83780.0765TNF-α × RIP10.50370.3869TNF-α × caspase 30.89920.0378∗P < 0.05 (Pearson correlation).IL-1β × FasL0.87780.0503IL-1β × FAS0.75590.1393IL-1β × BAX0.67610.2101IL-1β × caspase 30.84890.0689IL-4 × FasL0.52400.3647IL-4 × FAS0.84610.0707IL-4 × BAX0.54060.3469IL-4 × RIP10.87360.0529IL-4 × caspase 30.89840.0689IL-10 × FasL0.57640.3090IL-10 × FAS0.69080.1965IL-10 × BAX0.72940.1619IL-10 × RIP10.86180.0603IL-10 × caspase 30.79730.1061TGF-β × FAS0.50420.3864TGF-β × RIP10.74030.1525TGF-β × caspase 30.83820.0761IL-33 × FasL0.80360.1013IL-33 × BAX0.95830.0101∗P < 0.05 (Pearson correlation).IL-33 × RIP10.69010.1971IL-33 × caspase 30.83270.0800TNF-α × IL-1β0.95310.0121∗P < 0.05 (Pearson correlation).IL-4 × IL-100.90920.0324∗P < 0.05 (Pearson correlation).IL-10 × IL-330.85070.0676IL-4 × IL-330.71120.1774TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; ZIKV, Zika virus.∗ P < 0.05 (Pearson correlation). 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