Next Article in Journal
D2I and F9Y Mutations in the NS1 Protein of Influenza A Virus Affect Viral Replication via Regulating Host Innate Immune Responses
Next Article in Special Issue
Emergence of New Immunopathogenic Factors in Human Yellow Fever: Polarisation of the M1/M2 Macrophage Response in the Renal Parenchyma
Previous Article in Journal
Paraoxonase-1 Facilitates PRRSV Replication by Interacting with Viral Nonstructural Protein-9 and Inhibiting Type I Interferon Pathway
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Viruses
Volume 14
Issue 6
10.3390/v14061204
viruses-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Factors Involved in the Apoptotic Cell Death Mechanism in Yellow Fever Hepatitis

by
Jeferson da Costa Lopes
1,
Luiz Fábio Magno Falcão
2,
Arnaldo Jorge Martins Filho
1,
Marcos Luiz Gaia Carvalho
1,
Caio Cesar Henriques Mendes
2,
Fábio Alves Olímpio
3,
Vanessa do Socorro Cabral Miranda
1,
Lais Carneiro dos Santos
1,
Jannifer Oliveira Chiang
1,
Ana Cecilia Ribeiro Cruz
1,
Vanessa Costa Alves Galúcio
2,
Raimunda do Socorro da Silva Azevedo
1,
Lívia Caricio Martins
1,
Maria Irma Seixas Duarte
4,
Jorge Rodrigues de Sousa
1,2,†,
Pedro Fernando da Costa Vasconcelos
1,2,† and
Juarez Antônio Simões Quaresma
2,3,4,*,†
1
Evandro Chagas Institut, Ministry of Health, Ananindeua 67015-120, Brazil
2
Department of Pathology, State University of Para, Belem 66050-540, Brazil
3
Tripical Medicine Unit, Federal University of Para, Belem 66075-110, Brazil
4
Schhol of Medicine, Sao Paulo University, Sao Paulo 05508-070, Brazil
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Viruses 2022, 14(6), 1204; https://doi.org/10.3390/v14061204
Submission received: 26 March 2022 / Revised: 5 May 2022 / Accepted: 22 May 2022 / Published: 1 June 2022
(This article belongs to the Special Issue Arboviral Lifecycle)
Browse Figures
Versions Notes

Abstract

:
Yellow fever (YF), a non-contagious infectious disease, is endemic or enzootic to the tropical regions of the Americas and Africa. Periodic outbreaks or epidemics have a significant impact on public health. Programmed cell death, or apoptosis, is generally characterised by distinct morphological changes and energy-dependent biochemical pathways. In this study, we performed immunohistochemistry analysis to identify and quantify proteases and protein targets involved in the cascade that triggers apoptosis in YF virus (YFV)-infected human hepatocytes. Liver tissue samples were collected from 26 individuals, among whom 21 were diagnosed as YF-positive, and five were flavivirus-negative and died due to other causes. The histopathological alterations in YFV-positive cases were characterised by the presence of apoptotic bodies, steatosis, cellular swelling, and extensive necrosis and haemorrhage in the hepatic lobules. Additionally, we observed an abundance of inflammatory infiltrates in the portal tract. The expression of various apoptotic markers in the hepatic parenchyma, including CASPASE 3, CASPASE 8, BAX, FAS, FASL, GRANZYME B, and SURVIVIN, differed between YFV-positive cases and controls. Collectively, this study confirmed the complexity of YFV infection-induced apoptosis in situ. However, our data suggest that apoptosis in liver parenchyma lesions may significantly contribute to the pathogenesis of fatal YF in humans.

1. Introduction

Yellow fever (YF) is an endemic or enzootic non-contagious infectious disease that is prevalent in the tropical regions of the Americas and Africa. Periodic outbreaks or epidemics have a significant impact on public health. The disease is caused by the YF virus (YFV) (Flaviviridae–Flavivirus), which is vectorised by Aedes mosquitoes in Africa and is maintained in wild cycles by non-human primates (NHP) and hematophagous arthropods of the Haemagogus and Sabethes genera in South America. The re-emergence of the urban cycle in the Americas, which occurs between Aedes aegypti and susceptible humans, has been a concern for public authorities owing to the low vaccination coverage in non-endemic and endemic areas [1,2,3,4]. Epidemiologically, fatality in severe cases of YF is estimated to be approximately 31–45%, indicating the public health importance of this disease [5].
After entry into the host, viral replication occurs in the lymph nodes located near the area of the blood meal. The virus is then released into the bloodstream and is transported to multiple organs such as the kidney, heart, spleen, lung, and liver. The liver is the most affected organ in humans [5,6,7].
Among the possible mechanisms involved in the pathogenesis of YFV infection, apoptosis is seemingly crucial in inducing the death of hepatocytes. Apoptosis is a natural physiological process that maintains cell balance by killing undesirable cells. The apoptotic cascade is triggered through extrinsic or intrinsic mechanisms once the host detects a foreign aggressor [8,9]. The host immune response plays a fundamental role in triggering apoptosis, where pro- or anti-inflammatory cytokines contribute to the activation of genes encoding enzymes that participate in apoptosis. The expression of TNF-α and TGF-β could lead to cell cycle dysregulation and activate pro-apoptotic genes that cause apoptosis in parenchymal liver cells. Furthermore, cytokines are abundantly expressed in the areas of the liver with a substantial presence of apoptotic bodies, mainly in the medizonal zone [10,11]. During hepatic impairment, the relationship between the extrinsic and intrinsic pathways of apoptosis is seemingly vital in facilitating immune evasion by pathogens. However, several gaps exist in our understanding of this process, mainly concerning the expression of proteases, the action of pro- and anti-apoptotic regulators in situ, and the mechanism by which cell death triggers the destruction of hepatocytes through the interaction of the virus with the host defence system [12]. Thus, we investigated the activation of apoptosis via the extrinsic and intrinsic pathways in situ and its relationship with the development of injury in parenchymal liver cells in fatal human YF cases.

2. Materials and Method

2.1. Ethics Statement

An analytical cross-sectional study was conducted using retrospective samples from the Pathology Sector of Instituto Evandro Chagas, Ananindeua, Pará, Brazil, from 2001 to 2016. We analysed human liver fragments obtained by viscerotomy, collected from fatal cases, and confirmed the diagnosis by histopathological analysis, immunohistochemistry, and viral molecular biology. Among the 26 cases evaluated, 21 were fatal YFV-positive cases, and five were included in the control group. The cases in the control group showed preservation of the liver parenchyma and tested negative for hepatotropic viruses, based on viscerotomy-derived immunohistochemistry and molecular biology analyses. This study was approved (CAAE 04920918.4.0000.0019) by the Research Ethics Committee (CEP) of the Instituto Evandro Chagas.

2.2. Histopathology and Immunohistochemistry

For histopathological analysis, 5 μm sections were obtained from liver fragments embedded in paraffin and stained with haematoxylin–eosin (HE). Tissue immunostaining with specific antibodies against apoptotic markers (Table 1) was performed based on the biotin–streptavidin–peroxidase technique.
Sections were deparaffinised in xylene and hydrated in decreasing ethyl alcohol series (90%, 80%, and 70%). Endogenous peroxidase was blocked with 3% hydrogen peroxide (H2O2) for 45 min. The antigen was retrieved using citrate buffer (pH 6.0) for 20 min at 90 °C. Nonspecific proteins were blocked with 10% concentrated skim milk for 30 min. Histological sections were then incubated overnight with primary antibodies diluted in 1% bovine serum albumin (BSA).
The slides were then immersed in a 1× phosphate-buffered saline (PBS) and incubated with a biotinylated secondary antibody (labelled streptavidin–biotin (LSAB); DakoCytomation, Glostrup, Denmark) in an oven for 30 min at 37 °C. Subsequently, the slides were immersed in 1× PBS and incubated with streptavidin peroxidase (LSAB; DakoCytomation) for 30 min at 37 °C. The slides were developed using a chromogen solution composed of 0.03% diaminobenzidine (DAB) and 3% H2O2. The slides were then washed in distilled water and counterstained with Harris haematoxylin for 1 min before being dehydrated in increasing ethyl alcohol series (70%, 80%, and 90%). The slides were then cleaned with xylene.

2.3. Quantitative Analysis and Photodocumentation

Histological analysis was performed using an Axio Imager Z1 microscope (model 456006, Zeiss, Oberkochen, Germany) at 400× magnification. The immunomarkers were quantitatively analysed by selecting 10 fields under 400× magnification in areas I, II, and III of Rappaport and the portal tract (PT) (Figure 1) [8]. Five liver acinar units were detected in each sample, totalling 400 fields in each case. Each field was subdivided into 10 × 10 areas delimited by the microscope lens, comprising an area of 0.0625 mm2.

2.4. Statistical Analysis

The results obtained were stored in electronic spreadsheets and analysed using the GraphPad Prism 5.0 software. Numerical variables were analysed based on the measures of central tendency and dispersion. The hypotheses were evaluated using ANOVA and Tukey’s post-hoc test. Significance was set at p ≤ 0.05.

3. Results

3.1. Histopathology

The changes in the histopathological patterns were highly intense in the liver lobules, characterised mainly by macro- and micro-vesicular steatosis, cell swelling (cells are typically swollen, with compression or displacement of adjacent structures), the appearance of apoptotic bodies, and focal points of lytic necrosis (showing absence of intact cell morphology due to membrane lysis), coagulative necrosis (anucleated cells with preserved cell outlines), hyperplasia of Kupffer cells (increase in the number of Kupffer cells), and extensive haemorrhagic foci (Figure 2A–C). Infiltrates of lymphocytes, neutrophils, and macrophages were observed during the inflammatory response in the PT (Figure 2D).

3.2. Immunoexpression of Apoptotic Markers

Immunohistochemical analysis of the lobular regions (Z3, Z2, Z1, and PT) tested positive for FAS, FASL, CASPASE 3, CASPASE 8, BAX, FAS, FASL, GRANZYME B, and SURVIVIN, whose expression levels were significantly different compared to those of the controls (Table 2 and Figure 3). All markers showed significantly intense immunostaining in Z2 compared to other lobular regions (p < 0.05, Table 2 and Figure 3). Generally, pro and anti-apoptotic markers were predominant in the hepatocytes and inflammatory infiltrates compared to those in the controls (Figure 4 and Figure 5).

4. Discussion

In this study, the histopathological investigation suggested that the main alterations in the hepatic lobes were consistent with extensive tissue destruction in the hepatic lobules, characterised by haemorrhagic foci (Figure 2A–C), areas of lytic and coagulative necrosis, and apoptotic bodies. The viral tropism by resident cells and the intense liver damage resulting from a disproportionate immune response directly correlated with the degree of liver parenchymal involvement [13,14,15]. These findings corroborate with those of the previous studies on YF pathogenesis. In this follow-up study, liver cell damage seemingly occurred from a series of mechanisms that cause tissue hypoxia, influencing the stress response of the endoplasmic reticulum and mitochondrial dysfunction [16,17,18]. In the pathophysiology of YFV infection, virus-induced apoptosis is considered one of the immunological escape strategies, modulating the inflammatory process in situ and consequently the organ tissue damage [19]. Understanding protein interactions and their biological significance during viral infection are paramount to correlating the viral pathogenesis mechanism, as viruses use the host cell machinery for their replication [20,21]. Therefore, we focused on the markers involved in the apoptosis of YFV-infected hepatocytes to further elucidate the YF pathogenesis mechanism.
In the liver, severe apoptosis results in acute organ failure, whereas persistent apoptosis is often associated with fibrinogenesis and chronic liver dysfunction [22,23,24]. In addition to YFV, apoptotic cell death has been described in other flaviviral infections such as dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) [25,26,27,28]. However, these apoptotic events are not exclusive to flaviviruses. We observed the expression of FAS and FASL proteins in all liver lobes, particularly in hepatocytes, which were higher in the zonal mid-region. The FAS-FASL binding drives a Fas-associated death domain (FADD) protein cluster that recruits CASPASE 8 and CASPASE 10 to form a death-inducing signalling complex (DISC), leading to a mitochondrial-independent cell death signalling cascade. As death receptor-mediated apoptotic signals are usually insufficient to initiate the cascade in hepatocytes, the participation of the mitochondria-mediated pathway is also required for its initiation. This occurs when DISC production is insufficient, causing the activated CASPASE 8 to proteolytically cleave the proapoptotic Bcl-2 homology 3 (BH3) protein of the Bcl-2 family, transforming BH3-interacting domain death agonist (Bid) to tBid, and activating Bcl-2-associated X (Bax) and Bcl-2 homologous antagonist killer (Bak) proteins [29,30,31]. Therefore, this appears to be a mitochondrial apoptotic pathway activated via an extrinsic mechanism in situ, resulting from the death receptor activation in fatal cases of YFV in humans.
Our investigation revealed a high degree of CASPASE 3 and CASPASE 8 expressions, mainly in the Z2 region. In addition, BAX protein immunoexpression was the highest among all our analysed markers, with the highest degree observed in Z2. This may indicate the preference for mitochondrial apoptotic pathways in YFV-infected hepatocytes initially and preferentially mediated by the intrinsic pathway. Notably, in this process, the intrahepatic infiltrating NK and CD8 T cells cause hepatic cell death in different phases of DENV infection. In fatal cases of ZIKV-induced microcephaly, FAS, FASL, and tumour necrosis factor (TNF)-α were also overexpressed and directly regulated the mechanisms of neural parenchymal cell injury [32,33]. The action of pro-inflammatory cytokines tends to generate tissue hypoxia, which in turn increases oxidative stress. In addition, the intrinsic changes in the mitochondrial membrane caused by the Bcl-2 protein family activation may result in the activation of CASPASE 3, which was found to be highly expressed in our study.
We investigated the immunoexpression of GRANZYME B and observed that this protein was expressed in all regions of the lobes, predominantly in the Z2 region, similar to other markers. These results indicate that liver infection by VFA causes liver damage by infiltration of cytotoxic T lymphocytes, which, in addition to expressing FASL on their surface, release GRANZYME B and consequently lead to apoptosis of hepatocytes. GRANZYME B-induced cell death depends on caspases and causes permeabilisation of the mitochondrial outer membrane, mediated by BAX, Bak, and Bid, releasing cytochrome c and other apoptogenic factors into the cytosol. Such mechanisms can also be observed in infections by flaviviruses [10,20,34]. Finally, given that the SURVIVIN protein possesses important anti-apoptotic activity, we also quantified this marker in our samples, which was expressed in all areas of the liver lobes, with the highest expression level being in the Z2 region, compared to the controls. The anti-apoptotic activity of SURVIVIN occurs through the inhibition of effector caspases 3, 7, and 9 [35,36]. Furthermore, this marker is known to inhibit important proteins involved in the activation of caspases; thus, increasing cell survival both directly and indirectly. However, SURVIVIN expression in the environment cannot negatively regulate the progression of cell injury and directly affects the immunopathogenecity of the disease. Collectively, our study demonstrated the complexity of apoptosis in situ caused by YFV infection, and the data suggest that apoptosis in liver parenchymal lesions may significantly contribute to tissue damage in fatal cases of YF in humans. Therefore, additional in vitro and in vivo studies are required to establish the relationship between apoptosis and YFV pathology in the liver.

Author Contributions

Designed the study: J.R.d.S., M.I.S.D., J.A.S.Q. and P.F.d.C.V. Performed lab tests: J.d.C.L., L.F.M.F., A.J.M.F., M.L.G.C., C.C.H.M., F.A.O., V.d.S.C.M., L.C.d.S., J.O.C., A.C.R.C., V.C.A.G., R.d.S.d.S.A. and L.C.M. Furnished reagents: M.I.S.D., J.A.S.Q. and P.F.d.C.V. Drafted the manuscript: J.R.d.S., M.I.S.D., J.A.S.Q. and P.F.d.C.V. Final version review and agreed for submission: all authors. All authors have read and agreed to the published version of the manuscript.

Funding

Brazilian Council of Scientific and Technological Development Agency for financial support to PFCV (grants 457664/2013-4 and 303999/2016-0).

Institutional Review Board Statement

This study was approved (CAAE 04920918.4.0000.0019) by the Research Ethics Committee (CEP) of the Instituto Evandro Chagas.

Informed Consent Statement

The use of the samples was authorized by the formal consent of the responsible institution (Instituto Evandro Chagas). Patient data were preserved and not disclosed.

Data Availability Statement

The data used to support the fidings of this study are included within the article.

Acknowledgments

We are grateful to the Brazilian Council for Scientific and Technologic Development Agency (CNPq) for financial support to PFCV (grants 457664/2013-4 and 303999/2016-0) and PAPQ program (UFPA). We thank Camilla Costa Silva for the technical support.

Conflicts of Interest

The authors declare that there is no conflict of interest.

References

  1. Vasconcelos, P.F.; Bryant, J.E.; Da Rosa, A.P.T.; Tesh, R.B.; Rodrigues, S.G.; Barrett, A.D. Genetic Divergence and Dispersal of Yellow Fever Virus, Brazil. Emerg. Infect. Dis. 2004, 10, 1578–1584. [Google Scholar] [CrossRef] [Green Version]
  2. Reed, W.; Carroll, J. Yellow Fever: 100 Years of Discovery the Etiology of Yellow Fever: An Additional Note. JAMA Class. 2008, 36, 1–3. [Google Scholar]
  3. Mutebi, J.-P.; Wang, H.; Li, L.; Bryant, J.; Barrett, A.D.T. Phylogenetic and Evolutionary Relationships among Yellow Fever Virus Isolates in Africa. J. Virol. 2001, 75, 6999–7008. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Couto-Lima, D.; Madec, Y.; Bersot, M.I.; Campos, S.S.; Motta, M.D.A.; Santos, F.; Vazeille, M.; Vasconcelos, P.F.D.C.; Lourenço-De-Oliveira, R.; Failloux, A.-B. Potential risk of re-emergence of urban transmission of Yellow Fever virus in Brazil facilitated by competent Aedes populations. Sci. Rep. 2017, 7, 4848. [Google Scholar] [CrossRef] [PubMed]
  5. Servadio, J.L.; Muñoz-Zanzi, C.; Convertino, M. Estimating case fatality risk of severe Yellow Fever cases: Systematic literature review and meta-analysis. BMC Infect. Dis. 2021, 21, 89. [Google Scholar] [CrossRef]
  6. Ferreira, M.S.; Bezerra Júnior, P.S.; Cerqueira, V.D.; Rivero, G.R.C.; Oliveira Júnior, C.A.; Castro, P.H.G.; da Silva, G.A.; da Silva, W.B.; Imbeloni, A.A.; Sousa, J.R.; et al. Experimental Yellow Fever Virus Infection in the Squirrel Monkey (Saimiri Spp.) I: Gross Anatomical and Histopathological Findings in Organs at Necropsy. Mem. Inst. Oswaldo Cruz 2020, 115, 1–8. [Google Scholar] [CrossRef]
  7. de Oliveira, G.M.M.; Ferreira, R.M. Yellow Fever and Cardiovascular Disease: An Intersection of Epidemics. Arq. Bras. Cardiol. 2018, 110, 207–210. [Google Scholar] [CrossRef]
  8. Quaresma, J.A.; Barros, V.L.; Pagliari, C.; Fernandes, E.R.; Guedes, F.; Takakura, C.F.; Andrade, H.F., Jr.; Vasconcelos, P.F.; Duarte, M.I. Revisiting the liver in human yellow fever: Virus-induced apoptosis in hepatocytes associated with TGF-β, TNF-α and NK cells activity. Virology 2006, 345, 22–30. [Google Scholar] [CrossRef] [Green Version]
  9. Monath, T.P.; Vasconcelos, P.F. Yellow fever. J. Clin. Virol. 2015, 64, 160–173. [Google Scholar] [CrossRef]
  10. Quaresma, J.A.; Duarte, M.I.; Vasconcelos, P.F. Midzonal lesions in yellow fever: A specific pattern of liver injury caused by direct virus action and in situ inflammatory response. Med. Hypotheses 2006, 67, 618–621. [Google Scholar] [CrossRef]
  11. Quaresma, J.A.S.; Pagliari, C.; Medeiros, D.B.A.; Duarte, M.I.S.; Vasconcelos, P.F.C. Immunity and immune response, pathology and pathologic changes: Progress and challenges in the immunopathology of yellow fever. Rev. Med. Virol. 2013, 23, 305–318. [Google Scholar] [CrossRef] [PubMed]
  12. Clem, R.J. Arboviruses and apoptosis: The role of cell death in determining vector competence. J. Gen. Virol. 2016, 97, 1033–1036. [Google Scholar] [CrossRef] [PubMed]
  13. Holanda, G.M.; Casseb, S.M.M.; Quaresma, J.A.S.; Vasconcelos, P.F.C.; Cruz, A.C.R. Yellow fever virus modulates cytokine mRNA expression and induces activation of caspase 3/7 in the human hepatocarcinoma cell line HepG2. Arch. Virol. 2019, 164, 1187–1192. [Google Scholar] [CrossRef] [PubMed]
  14. Lefeuvre, A.; Contamin, H.; Decelle, T.; Fournier, C.; Lang, J.; Deubel, V.; Marianneau, P. Host-cell interaction of attenuated and wild-type strains of yellow fever virus can be differentiated at early stages of hepatocyte infection. Microbes Infect. 2006, 8, 1530–1538. [Google Scholar] [CrossRef] [PubMed]
  15. Gerold, G.; Brüning, J.; Weigel, B.; Pietschmann, T. Protein Interactions during the Flavivirus and Hepacivirus Life Cycle. Mol. Cell. Proteom. 2017, 16, S75–S91. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Quaresma, J.A.S.; Barros, V.L.R.S.; Fernandes, E.R.; Pagliari, C.; Guedes, F.; Vasconcelos, P.F.D.C.; Junior, H.F.D.A.; Duarte, M.I.S. Immunohistochemical examination of the role of Fas ligand and lymphocytes in the pathogenesis of human liver yellow fever. Virus Res. 2006, 116, 91–97. [Google Scholar] [CrossRef]
  17. Galluzzi, L.; Brenner, C.; Morselli, E.; Touat, Z.; Kroemer, G. Viral Control of Mitochondrial Apoptosis. PLOS Pathog. 2008, 4, e1000018. [Google Scholar] [CrossRef] [Green Version]
  18. Malhi, H.; Gores, G.J. Cellular and Molecular Mechanisms of Liver Injury. Gastroenterology 2008, 134, 1641–1654. [Google Scholar] [CrossRef] [Green Version]
  19. Malhi, H.; Guicciardi, M.E.; Gores, G.J. Hepatocyte Death: A Clear and Present Danger. Physiol. Rev. 2010, 90, 1165–1194. [Google Scholar] [CrossRef] [Green Version]
  20. Pan, Y.; Cheng, A.; Wang, M.; Yin, Z.; Jia, R. The Dual Regulation of Apoptosis by Flavivirus. Front. Microbiol. 2021, 12, 574. [Google Scholar] [CrossRef]
  21. Okamoto, T.; Suzuki, T.; Kusakabe, S.; Tokunaga, M.; Hirano, J.; Miyata, Y.; Matsuura, Y. Regulation of Apoptosis during Flavivirus Infection. Viruses 2017, 9, 243. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Shojaie, L.; Iorga, A.; Dara, L. Cell Death in Liver Diseases: A Review. Int. J. Mol. Sci. 2020, 21, 9682. [Google Scholar] [CrossRef]
  23. Kisseleva, T.; Brenner, D. Molecular and cellular mechanisms of liver fibrosis and its regression. Nat. Rev. Gastroenterol. Hepatol. 2020, 18, 151–166. [Google Scholar] [CrossRef] [PubMed]
  24. Guicciardi, M.E.; Malhi, H.; Mott, J.L.; Gores, G.J. Apoptosis and Necrosis in the Liver. Compr. Physiol. 2013, 3, 977–1010. [Google Scholar] [CrossRef] [Green Version]
  25. Garber, C.; Soung, A.; Vollmer, L.L.; Kanmogne, M.; Last, A.; Brown, J.; Klein, R.S. T cells promote microglia-mediated synaptic elimination and cognitive dysfunction during recovery from neuropathogenic flaviviruses. Nat. Neurosci. 2019, 22, 1276–1288. [Google Scholar] [CrossRef]
  26. Uno, N.; Ross, T.M. Dengue virus and the host innate immune response. Emerg. Microbes Infect. 2018, 7, 1–11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. de Sousa, J.R.; Azevedo, R.D.S.D.S.; Quaresma, J.A.S.; Vasconcelos, P.F.D.C. Cell Death And Zika Virus: An Integrated Network Of The Mechanisms Of Cell Injury. Infect. Drug Resist. 2019, 12, 2917–2921. [Google Scholar] [CrossRef] [Green Version]
  28. Wang, Q.; Xin, X.; Wang, T.; Wan, J.; Ou, Y.; Yang, Z.; Yu, Q.; Zhu, L.; Guo, Y.; Wu, Y.; et al. Japanese Encephalitis Virus Induces Apoptosis and Encephalitis by Activating the PERK Pathway. J. Virol. 2019, 93, e00887. [Google Scholar] [CrossRef] [Green Version]
  29. Suwanmanee, S.; Luplertlop, N. Immunopathogenesis of Dengue Virus-Induced Redundant Cell Death: Apoptosis and Pyroptosis. Viral Immunol. 2017, 30, 13–19. [Google Scholar] [CrossRef]
  30. Roy, S.G.; Sadigh, B.; Datan, E.; Lockshin, A.R.; Zakeri, Z. Regulation of cell survival and death during Flavivirus infections. World J. Biol. Chem. 2014, 5, 93–105. [Google Scholar] [CrossRef]
  31. Lee, A.; Hirabayashi, Y.; Kwon, S.-K.; Lewis, T.L.; Polleux, F. Emerging roles of mitochondria in synaptic transmission and neurodegeneration. Curr. Opin. Physiol. 2018, 3, 82–93. [Google Scholar] [CrossRef] [PubMed]
  32. Sung, J.-M.; Lee, C.-K.; Wu-Hsieh, B.A.-Y. Intrahepatic Infiltrating NK and CD8 T Cells Cause Liver Cell Death in Different Phases of Dengue Virus Infection. PLoS ONE 2012, 7, e46292. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Azevedo, R.S.S.; De Sousa, J.R.; Araujo, M.T.F.; Filho, A.J.M.; De Alcantara, B.N.; Araujo, F.M.C.; Queiroz, M.G.L.; Cruz, A.C.R.; Vasconcelos, B.H.B.; Chiang, J.O.; et al. In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus. Sci. Rep. 2018, 8, 1. [Google Scholar] [CrossRef] [PubMed]
  34. Grifoni, A.; Tian, Y.; Sette, A.; Weiskopf, D. Transcriptomic immune profiles of human flavivirus-specific T-cell responses. Immunology 2019, 160, 3–9. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Johnson, M.E.; Howerth, E.W. Survivin: A Bifunctional Inhibitor of Apoptosis Protein. Veter. Pathol. 2004, 41, 599–607. [Google Scholar] [CrossRef]
  36. Nassar, A.; Lawson, D.; Cotsonis, G.; Cohen, C. Survivin and Caspase-3 Expression in Breast Cancer: Correlation with Prognostic Parameters, Proliferation, Angiogenesis, and Outcome. Appl. Immunohistochem. Mol. Morphol. 2008, 16, 113–120. [Google Scholar] [CrossRef]
Viruses 14 01204 g001 550
Figure 1. Illustrative diagram demonstrating the hepatic lobule constituted by acini and the division scheme between zone 1 (Z1), zone 2 (Z2), and zone 3 (Z3) located around the portal tract (PT) (Z1), midzonal area (Z2), and around the central lobular vein (Z3). (This figure was created with biorender.com)
Figure 1. Illustrative diagram demonstrating the hepatic lobule constituted by acini and the division scheme between zone 1 (Z1), zone 2 (Z2), and zone 3 (Z3) located around the portal tract (PT) (Z1), midzonal area (Z2), and around the central lobular vein (Z3). (This figure was created with biorender.com)
Viruses 14 01204 g001
Viruses 14 01204 g002 550
Figure 2. Histopathological analysis of the acinar zones in the liver of humans with fatal yellow fever and normal controls (NC). Z3, Z2, Z1, and PT in the hepatic parenchyma of fatal cases affected by YFV and NC. (A–D) Necrotic areas with haemorrhagic foci in A-Z3, B-Z2, and C-Z1 (black circles) characterised by areas of presence of red blood cells and absence of nuclei with the presence of cell debris. The massive presence of inflammatory infiltrates in the PT (black circle) D-PT. (E–H) Preservation of the hepatic parenchyma (Z3, Z2, Z1, and PT) in NC cases (black circles) E-Z3, F-Z2, G-Z1, H-PT. Z3: centrolobular zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract. NC: normal control. 400× magnification (scale: 20 µm).
Figure 2. Histopathological analysis of the acinar zones in the liver of humans with fatal yellow fever and normal controls (NC). Z3, Z2, Z1, and PT in the hepatic parenchyma of fatal cases affected by YFV and NC. (A–D) Necrotic areas with haemorrhagic foci in A-Z3, B-Z2, and C-Z1 (black circles) characterised by areas of presence of red blood cells and absence of nuclei with the presence of cell debris. The massive presence of inflammatory infiltrates in the PT (black circle) D-PT. (E–H) Preservation of the hepatic parenchyma (Z3, Z2, Z1, and PT) in NC cases (black circles) E-Z3, F-Z2, G-Z1, H-PT. Z3: centrolobular zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract. NC: normal control. 400× magnification (scale: 20 µm).
Viruses 14 01204 g002
Viruses 14 01204 g003 550
Figure 3. Quantitative analysis of immunohistochemical data positive for (A) FAS, (B) FASL, (C) BAX, (D) CASPASE 3, (E) CASPASE 8, (F) GRANZYME B, and (G) SURVIVIN in zones Z3, Z2, Z1, and PT in the hepatic parenchyma of fatal yellow fever cases (blue) and negative controls (red). Z3: centrolobular zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract; one-way ANOVA; *** p < 0.0001. Tukey’s post-hoc test; *** p < 0.0001.
Figure 3. Quantitative analysis of immunohistochemical data positive for (A) FAS, (B) FASL, (C) BAX, (D) CASPASE 3, (E) CASPASE 8, (F) GRANZYME B, and (G) SURVIVIN in zones Z3, Z2, Z1, and PT in the hepatic parenchyma of fatal yellow fever cases (blue) and negative controls (red). Z3: centrolobular zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract; one-way ANOVA; *** p < 0.0001. Tukey’s post-hoc test; *** p < 0.0001.
Viruses 14 01204 g003
Viruses 14 01204 g004 550
Figure 4. Immunostaining for FAS (A–D) (Z3-A, Z2-B, Z1-C), FASL (F–I) (Z3-F, Z2-G, Z1-H), BAX (K–N) (Z3-K, Z2-L, Z1-M), CASPASE 3 (P–S) (Z3-P, Z2-Q, Z1-R), in the hepatocytes (black circle) and D-PT, I-PT, N-PT, and S-PT in the inflammatory infiltrates (black circles). Preservation of parenchyma hepatic in control cases and minimal expression of FAS (E), FASL (J), BAX (O), and CASPASE 3 (T). 400× magnification (scale: 20 µm). Z3: centrolobular zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract; NC: normal control.
Figure 4. Immunostaining for FAS (A–D) (Z3-A, Z2-B, Z1-C), FASL (F–I) (Z3-F, Z2-G, Z1-H), BAX (K–N) (Z3-K, Z2-L, Z1-M), CASPASE 3 (P–S) (Z3-P, Z2-Q, Z1-R), in the hepatocytes (black circle) and D-PT, I-PT, N-PT, and S-PT in the inflammatory infiltrates (black circles). Preservation of parenchyma hepatic in control cases and minimal expression of FAS (E), FASL (J), BAX (O), and CASPASE 3 (T). 400× magnification (scale: 20 µm). Z3: centrolobular zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract; NC: normal control.
Viruses 14 01204 g004
Viruses 14 01204 g005 550
Figure 5. Immunostaining for CASPASE 8 (A–D) (Z3-A, Z2-B, Z1-C), GRANZYME B (F–I) (Z3-F, Z2-G, Z1-H), (K–N) SURVIVIN (Z3-K, Z2-L, Z1-M), in the hepatocytes (black circles) and D-PT, I-PT, N-PT in the inflammatory infiltrates (black circles). Preservation of hepatic parenchyma in control cases and minimal expression of CASPASE 8 (E), GRANZYME B (J), SURVIVIN (O). 400× magnification (scale: 20 µm). Z3: centrolobular zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract; NC: normal control.
Figure 5. Immunostaining for CASPASE 8 (A–D) (Z3-A, Z2-B, Z1-C), GRANZYME B (F–I) (Z3-F, Z2-G, Z1-H), (K–N) SURVIVIN (Z3-K, Z2-L, Z1-M), in the hepatocytes (black circles) and D-PT, I-PT, N-PT in the inflammatory infiltrates (black circles). Preservation of hepatic parenchyma in control cases and minimal expression of CASPASE 8 (E), GRANZYME B (J), SURVIVIN (O). 400× magnification (scale: 20 µm). Z3: centrolobular zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract; NC: normal control.
Viruses 14 01204 g005
Table
Table 1. Antibodies used to monitor apoptosis in fatal human YF cases.
Table 1. Antibodies used to monitor apoptosis in fatal human YF cases.
MarkersReferenceDilution
BAXAbcam/ab325031:100
SURVIVINNovus/NB500-2011:100
FASDako/m35551:100
FASLAbcam/ab152851:100
GRANZYME BDako/m72351:100
CASPASE 3Abcam/ab40511:100
CASPASE 8Abcam/ab259011:100
Table
Table 2. Quantitative analysis of apoptotic markers in the hepatic parenchyma (Z3, Z2, Z1, and PT) in fatal YFV cases.
Table 2. Quantitative analysis of apoptotic markers in the hepatic parenchyma (Z3, Z2, Z1, and PT) in fatal YFV cases.
Markers (Cells/mm2)Z3Tukey (p ≤ 0.05)Z2Tukey (p ≤ 0.05)Z1Tukey (p ≤ 0.05)TPTukey (p ≤ 0.05)ANOVA (p ≤ 0.05)
BAX68.53 ± 8.36***89.9 ± 4.9***59.64 ± 4.37***54.24 ± 5.54******
Control17.28 ± 4.517.9 ± 9.018.56 ± 6.3616.0 ± 3.9
GRANZYME B50.92 ± 6.9***61.64 ± 4.25***49.22 ± 5.35***43.7 ± 5.7******
Control17.92 ± 3.216.96 ± 3.315.36 ± 3.8515.68 ± 3.64
FAS45.56 ± 4.99***55.38 ± 3.3***41.9 ± 4.3***40.97 ± 4.4******
Control14.4 ± 4.015.36 ± 8.215.36 ± 3.812.16 ± 3.8
FASL61.56 ± 3.9***84.13 ± 7.5***60.34 ± 5.3***57.37 ± 4.0******
Control17.28 ± 3.816.64 ± 6.318.8 ± 3.617.2 ± 2.8
SURVIVIN46.1 ± 7.2***54.32 ± 5.2***44.8 ± 6.0***44.11 ± 3.7******
Control16.28 ± 3.416.96 ± 3.315.68 ± 2.613.44 ± 3.1
CASPASE 360.65 ± 7.6***75.57 ± 9.9***49.45 ± 6.1***48.06 ± 4.7******
Control15.0 ± 4.019.8 ± 3.814.4 ± 2.214.76 ± 2.8
CASPASE 861.94 ± 6.0***80.91 ± 7.9***60.95 ± 9.9***54.78 ± 4.6******
Control14.08 ± 2.322.72 ± 4.216.0 ± 2.914.4 ± 2.9
One-way ANOVA; *** p < 0.0001. Tukey’s post-hoc test; *** p < 0001.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

da Costa Lopes, J.; Falcão, L.F.M.; Martins Filho, A.J.; Carvalho, M.L.G.; Mendes, C.C.H.; Olímpio, F.A.; do Socorro Cabral Miranda, V.; dos Santos, L.C.; Chiang, J.O.; Cruz, A.C.R.; et al. Factors Involved in the Apoptotic Cell Death Mechanism in Yellow Fever Hepatitis. Viruses 2022, 14, 1204. https://doi.org/10.3390/v14061204

AMA Style

da Costa Lopes J, Falcão LFM, Martins Filho AJ, Carvalho MLG, Mendes CCH, Olímpio FA, do Socorro Cabral Miranda V, dos Santos LC, Chiang JO, Cruz ACR, et al. Factors Involved in the Apoptotic Cell Death Mechanism in Yellow Fever Hepatitis. Viruses. 2022; 14(6):1204. https://doi.org/10.3390/v14061204

Chicago/Turabian Style

da Costa Lopes, Jeferson, Luiz Fábio Magno Falcão, Arnaldo Jorge Martins Filho, Marcos Luiz Gaia Carvalho, Caio Cesar Henriques Mendes, Fábio Alves Olímpio, Vanessa do Socorro Cabral Miranda, Lais Carneiro dos Santos, Jannifer Oliveira Chiang, Ana Cecilia Ribeiro Cruz, and et al. 2022. "Factors Involved in the Apoptotic Cell Death Mechanism in Yellow Fever Hepatitis" Viruses 14, no. 6: 1204. https://doi.org/10.3390/v14061204

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop