Inflammasome Activation in Human Liver Parenchyma During Severe Yellow Fever Inflammasome in Fatal Yellow Fever

Inflammasome Activation in Human Liver Parenchyma During Severe Yellow Fever Inflammasome in Fatal Yellow Fever | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 4 December 2025 V1 Latest version Share on Inflammasome Activation in Human Liver Parenchyma During Severe Yellow Fever Inflammasome in Fatal Yellow Fever Authors : Vanessa do Socorro Cabral Miranda , Luiz Fábio Magno Falcão , Luis Arthur Moreira Ferreira , Lucas Corrêa Modesto , Pablo Rodrigues Nunes de Souza , Rafael Malcher Meira Rocha , Arnaldo Jorge Martins Filho , … Show All … , Jorge Rodrigues de Sousa , Mayumi Duarte Wakimoto , Pedro Fernando da Costa Vasconcelos , Carla Pagliari 0000-0001-6210-6917 , Hellen Fuzii , and Juarez Antônio Simões Quaresma [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.176482281.11650381/v1 196 views 138 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Yellow fever virus (YFV) infection causes severe liver damage, yet the specific immunopathological mechanisms remain partially unclear. This study investigated the contribution of the inflammasome complex to the pathogenesis of fatal yellow fever (YF) in human liver tissue. Methods: We analyzed liver tissue samples from 21 patients with fatal YFV infection and five flavivirus-negative controls. Immunohistochemical analysis was performed to detect receptors (NLRP1, NLRP3, AIM2), cytokines (IL-1β, IL-18, IL-33), and enzymes (caspase 1, iNOS, arginase 1). Results: Upregulation of NLRP1, NLRP3, and AIM2 receptors, along with increased expression of IL-1β, IL-18, IL-33, caspase 1, iNOS, and arginase 1, was observed in infected tissues compared to controls. These markers were predominantly expressed in the midzonal region. Statistical analysis revealed strong correlations among these markers across hepatic zones. Conclusion: The findings demonstrate that inflammasome activation is associated with a robust inflammatory response in fatal human YF cases. The midzonal zone acts as a critical area for the production of inflammatory markers, suggesting a key role for the inflammasome in YFV-induced liver pathology. Inflammasome Activation in Human Liver Parenchyma During Severe Yellow Fever Inflammasome in Fatal Yellow Fever Vanessa do Socorro Cabral Miranda a , Luiz Fabio Magno Falcão b , Luis Arthur Moreira Ferreira b , Lucas Corrêa Modesto b , Pablo Rodrigues Nunes de Souza b , Rafael Malcher Meira Rocha b , Arnaldo Jorge Martins Filho a , Jorge Rodrigues de Sousa a,b,c# , Mayumi Duarte Wakimoto d# , Pedro Fernando da Costa Vasconcelos a,b# , Carla Pagliari f , Hellen Thais Fuzii c , Juarez Antônio Simões Quaresma b,e# a. Evandro Chagas Institute, Ministry of Health, Ananindeua, PA, Brazil. b. Center for Biological and Health Sciences, Pará State University, Belem, PA, Brazil. c. Federal University of Para, Belem, PA, Brazil. d. Evandro Chagas National Institute of Infectious Diseases (INI-FIOCRUZ), Rio de Janeiro, RJ, Brazil. e. Department of Pathology, Paulista School of Medicine, Federal University of Sao Paulo, Sao Paulo-SP, Brazil.* f. University of Sao Paulo, Sao Paulo-SP, Brazil. # These authors contributed equally to this work. CORRESPONDING AUTHOR: Juarez Antônio Simões Quaresma Institution: Federal University Of São Paulo Address: Avenida Hiléia, s/n, Amapá, Marabá, Pará CEP: 68.502-100. E-mail: [email protected] Declarations Ethics approval and consent to participate Patient samples were secured and processed as part of the emergency response activities for the surveillance of the Yellow Fever Virus (YFV) epidemic in Brazil, as defined by the Brazilian Ministry of Health. This investigation received formal approval (No. 2.824.592) from the Research Ethics Committee (CEP) of the Evandro Chagas Institute (IEC) in Ananindeua, Pará, Brazil. All procedures were conducted in full accordance with the relevant guidelines and regulations sanctioned by the CEP/IEC and the rules of the Brazilian Ministry of Health for studies that involve biological samples. Consent for Publication Not applicable. Availability of Data and Materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding sources This research did not receive any specific grant from funding agencies in the public, commercial or not for profit sectors. Authors’ contributions VSCM, AJMF, JRS, MDW, PFCV, HLF, CP and JASQ contributed to the conceptualization of the project, acquisition, analysis and interpretation of the data. LCM, LAMF, PRNS, RMMR and LFMF contributed significantly to the draft, writing and review of the manuscript. All authors read and approved the final manuscript, as well as agreed both to be personally accountable for author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which they were not personally involved, are properly investigated, resolved and the resolution documented in the literature. Acknowledgments Not applicable. Abstract: Background: Yellow fever virus (YFV) infection causes severe liver damage, yet the specific immunopathological mechanisms remain partially unclear. This study investigated the contribution of the inflammasome complex to the pathogenesis of fatal yellow fever (YF) in human liver tissue. Methods: We analyzed liver tissue samples from 21 patients with fatal YFV infection and five flavivirus-negative controls. Immunohistochemical analysis was performed to detect receptors (NLRP1, NLRP3, AIM2), cytokines (IL-1β, IL-18, IL-33), and enzymes (caspase 1, iNOS, arginase 1). Results: Upregulation of NLRP1, NLRP3, and AIM2 receptors, along with increased expression of IL-1β, IL-18, IL-33, caspase 1, iNOS, and arginase 1, was observed in infected tissues compared to controls. These markers were predominantly expressed in the midzonal region. Statistical analysis revealed strong correlations among these markers across hepatic zones. Conclusion: The findings demonstrate that inflammasome activation is associated with a robust inflammatory response in fatal human YF cases. The midzonal zone acts as a critical area for the production of inflammatory markers, suggesting a key role for the inflammasome in YFV-induced liver pathology. Keywords: Yellow Fever; Inflammasome; Liver parenchyma; Arbovirus; Pathogenesis. Introduction YFV is a member of the genus Flavivirus (family Flaviviridae), possesses a positive-sense single-stranded RNA genome, and is recognized as the etiological agent of yellow fever (YF), transmitted by mosquitoes such as Aedes and Haemagogus [1]. In most cases, yellow fever does not require specific treatment; however, up to 47% of individuals infected with YFV may develop severe or fulminant forms due to an imbalance between immune and inflammatory responses [2,3]. The lethality of severe cases is higher in Brazil, where it ranges from 40% to 60% [4,5].Despite the availability of a highly effective vaccine, YFV remains a public health problem in several countries. Its transmission can occur through urban, sylvatic, or savannah cycles (the latter in Africa); however, the connection between the wild and urban cycles is particularly concerning due to the widespread adaptation of the Aedes aegypti mosquito to urban centers. The latest YFV outbreak in Brazil, which involved the spread of the virus in parks within urban areas, highlighted the high risk of establishing an urban transmission cycle in the country [6]. YFV infection can affect several organs, such as the spleen, kidneys, and heart, but the predilection for hepatocytes is a hallmark of this virus [7].The damage to hepatocytes caused by yellow fever is mainly attributed to apoptosis, necrosis, and steatosis, especially in the midzonal region of the liver [8]. Studies with other flaviviruses, such as Zika virus (ZIKV) [9,10], dengue virus (DENV) [11,12], and hepatitis C virus (HCV) [13], have shown that the inflammasome complex can exacerbate tissue damage and induce programmed cell death known as pyroptosis, possibly contributing to fatal cases of these diseases [14,15].Inflammasomes are multiprotein complexes that induce inflammation by increasing the expression of NLRP1, NLRP3, AIM2, and caspase 1, which cleave pro-IL-1β, pro-IL-18, and pro-IL-33 cytokines, ultimately causing cell death by pyroptosis [16–19]. In Zika virus infection, the inflammasome response is directly linked to neuroinflammation through the expression of NLRP3, caspase 1, and IL-1β [16]. In hepatitis C, hepatocyte damage is associated with IL-1β-dependent NLRP3 activation and the production of reactive oxygen species (ROS) [13].Some studies have highlighted the mechanisms of the immune response against YFV in the liver parenchyma, but there are no in situ descriptions of cytosolic inflammasome complex participation in human fatal cases resulting from viral infection [8,20,21]. In light of the above, we analyzed liver samples from fatal cases of yellow fever in humans and hypothesized that the inflammasome plays a crucial role in the development of tissue injury in response to YFV infection. Material and Methods 2.1. Patient Cohort, Specimen Acquisition, and Yellow Fever (YF) Diagnosis This investigation was performed using a cohort of 26 human liver biopsy specimens. Among these, 21 samples originated from fatal Yellow Fever (YF) cases. Diagnosis confirmation was achieved by detecting the virus via RT−PCR and/or immunohistochemistry, as per the protocol established by Olímpio et al. [22]. The control group consisted of five samples. These were sourced from patients who exhibited preserved liver architecture and tested negative for YF and other flaviviruses circulating in Brazil. The verification of death and diagnosis for the control specimens was carried out by the Renato Chaves Scientific Expertise Center in Belém city, Pará state, Brazil. Histopathological diagnosis for both positive and control biopsies utilized the Hematoxylin−Eosin(H&E) method. Tissue samples were initially fixed in 10% neutral buffered formalin, followed by routine processing and paraffin embedding. Subsequently, sections were cut at 5μm and stained with H&E for histological examination. Patient characteristics are detailed in Table 1. [Table 1 ] 2.2. Immunostaining Protocol for IHC The inflammasome profile within the samples was analyzed by Immunohistochemistry(IHC) using the streptavidin-biotin peroxidase (SABC) method. The procedure was adapted from the method described by Hsu et al. (1981) [23], specifically following the adaptations outlined by Olímpio et al. (2022) [22]. The primary antibodies selected for each inflammasome marker are listed in Table 2.The initial steps involved deparaffinizing the tissue samples in xylene and then rehydrating them through a graded ethanol series (90%, 80%, and 70%). Endogenous peroxidase activity was blocked by incubating the liver sections in 3% hydrogen peroxide for 45 minutes. Antigen retrieval was conducted by incubating the sections in citrate buffer (pH6.0) for 20 minutes at 90∘C. Nonspecific protein binding was minimized via a 30-minute incubation with 10% skim milk.Subsequently, the histological sections were incubated overnight with the primary antibodies, which were diluted in 1% bovine serum albumin. Following a wash in 1×PBS, the slides were incubated with the biotinylated secondary antibody (LSAB; DakoCytomation, Glostrup, Denmark) in an oven for 30 minutes at 37∘C. After a second wash in 1× PBS, the streptavidin peroxidase (LSAB; DakoCytomation) was applied for 30 minutes at 37∘C. Visualization of the reaction was achieved by treating the specimens with a chromogenic solution (0.03% diaminobenzidine and 3% hydrogen peroxide). The final steps included washing the sections in distilled water, counterstaining with Harris hematoxylin for 1 minute, dehydrating (ethanol series), clearing in xylene, and mounting with Entellan®. [Table 2] 2.3. Quantitative Assessment and Photo-Documentation The markers used to define the in situ inflammasome profile were observed and visualized using a Zeiss Axio Imager Z1 microscope. Quantitative evaluation of the immunostaining results was performed by the random selection of ten fields within the hepatic parenchyma. This assessment covered each liver zone evaluated in the fatal YF cases: Z3 (centrolobular zone), Z2 (midzonal zone), Z1 (periportal zone), and PT (portal tract). For the negative control cases, quantification was carried out specifically in the midzonal (Z2) region, as this zone is the most representative site for alterations caused by YF infection. Each field was subdivided into 10×10 areas, delineated by a 0.0625mm 2 grid. 2.4. Statistical Procedures The compiled data was stored in a Microsoft Excel 2016 spreadsheet and analyzed utilizing GraphPad Prism 9.0. Numerical variables were presented as mean, median, standard deviation, and variance. Statistical tests applied included One-way ANOVA, Tukey’s test, and Pearson correlation. Specifically, the Pearson’s correlation coefficient (r) was employed to evaluate correlations between immunostaining for NLRP3, NLRP1, AIM2, IL−1β, IL−18, IL−33, iNOS, caspase 1, and arginase 1 across the different liver areas (Z3, Z2, Z1, and PT). Values of r>0.7 indicated strong correlations, and r>0.9 represented very strong correlations. Results were designated as statistically significant when the p value was <0.05. 2.5. Ethical Compliance Patient samples were secured and processed as part of the emergency response activities for the surveillance of the YFV epidemic in Brazil, as defined by the Ministry of Health. This investigation received approval (No. 2.824.592) from the Research Ethics Committee (CEP) of the Evandro Chagas Institute (IEC) in Ananindeua, Pará, Brazil. All procedures were conducted in full accordance with the relevant guidelines and regulations sanctioned by the CEP/IEC and the rules of the Brazilian Ministry of Health for studies that involve biological samples. Results 3.1. Expression of NLRP1, NLRP3, AIM2, IL-1β, IL-18, IL-33, caspase 1, iNOS, and arginase 1 in the hepatic parenchyma in fatal Yellow Fever cases To investigate the levels of protein expression related to the inflammasome pathway, we performed immunohistochemistry (IHC) for NLRP1, NLRP3, AIM2, IL-1β, IL-18, IL-33, iNOS, arginase 1, and caspase 1 on a total of 26 samples—21 fatal YF cases and 5 negative controls. All zones (Z3, Z2, and Z1) and the portal tract were analyzed to quantify protein expression in each area. The expression levels of NLRP1, NLRP3, and AIM2 receptors in samples from fatal YF cases showed significant differences compared to the respective controls (Table 3, Fig. 1A). Figure 2B and Table 3 show that the cytokines cleaved in response to inflammasome activation (IL-1β, IL-18, and IL-33) were significantly upregulated compared to the control group in all zones. In addition, YFV infection induced high levels of caspase 1, iNOS, and arginase 1 expression in hepatocytes (Table 3, Fig. 3B). Among all expressions, the midzonal zone showed the highest levels for all proteins (Fig. 1A, 2A, 3A). To verify the relationships among these protein expression levels in the Z3, Z2, Z1, and PT areas, we used Pearson’s correlation, which demonstrated strong to very strong correlations among the expressions of receptors (NLRP1, NLRP3, and AIM2), cytokines (IL-1β, IL-18, and IL-33), and enzymes (iNOS, arginase 1, and caspase 1) across the liver parenchyma zones of fatal yellow fever cases (Table 4, Fig. 4). [Table 3] [Figure 1] [Figure 2] [Figure 3] [Table 4] [Figure 4] [Figure 4 . (Continuation)] Discussion Yellow fever in humans is an acute infection of variable severity, with a predilection for liver tissue, causing severe damage to hepatocytes in fatal cases, especially in the midzonal area, as a result of cell death processes such as apoptosis, necrosis, and steatosis [24–26]. The extent of these injuries and hepatic parenchymal involvement is directly related to a disproportionate host immune response [27–29]. However, the mechanisms by which the immune system interacts with YFV are not yet fully elucidated. After YFV infects hepatocytes, resident innate immune cells in the liver parenchyma, such as Kupffer cells, neutrophils, CD4+, CD8+, CD20+, CD68+ cells, and NK cells, interact with the virus to limit and inhibit viral replication. This interaction contributes to the generation of an inflammatory response at sites of injury [30,31]. Our results indicate that the inflammatory response triggered by YFV infection is initiated by in situ inflammasome activation in human liver tissue. The primary promoter proteins of the inflammasome complex—NLRP3, NLRP1, and AIM2—differed significantly from those in the negative controls (Table 3, Fig. 1A). Furthermore, we found that these receptors, as well as inflammatory response markers such as the cytokines IL-1β, IL-18, and IL-33 and the enzymes iNOS, caspase 1, and arginase 1, interact across the liver zones to promote inflammasome activity in this organ (Table 4, Fig. 4). Among all evaluated markers, the highest expression levels were observed in the midzonal zone (Table 3, Fig. 1A, 2A, 3A). Our findings underscore the importance of the midzonal zone in YFV infection and corroborate previous in situ studies that associated this region with greater YFV activity, which consequently leads to more extensive tissue injury, including apoptosis, necrosis, and steatosis [24,27,28]. Recently, several groups have evaluated the immune responses generated by flavivirus infections; however, none of these studies has clarified which inflammasome complex markers are present in situ in fatal YF cases. Flaviviruses such as Zika virus (ZIKV) [32,33], dengue virus (DENV) [34,35], Japanese encephalitis virus (JEV) [36], and West Nile virus (WNV) [37] trigger cascades of mediators associated with inflammasome activation as a response mechanism to infection. In DENV infection in murine models, the NLRP3 complex activates caspase-1 and cleaves IL-1β and IL-18 to induce the inflammatory response [38]. Additionally, WNV and JEV infections also activate NLRP3 and produce IL-1β in murine neural tissue [36,39]. Sousa et al. [33], studying ZIKV in human brain tissue, demonstrated the relationship among the inflammasomes NLRP3, NLRP1, and AIM2 and the cytokines IL-1β, IL-18, and IL-33 in aggravating the in situ neuroinflammatory response. Our results identified, for the first time, the occurrence of markers related to the in situ inflammasome pathway in the human liver parenchyma, highlighting the importance of in vivo studies in fatal cases of YFV infection. Few studies have analyzed NLRP1 activation in YFV infection. A cohort study of individuals who received the YF vaccine showed upregulation of caspases-1 and -5, which are part of the NLRP1 inflammasome complex; these data suggest that the NLRP1 inflammasome can be activated by YFV [40]. Our results corroborate these findings by showing that NLRP1 inflammasome markers were significantly higher than those in the negative control across all liver zones (Table 3, Fig. 1A). Furthermore, a strong correlation was identified between NLRP1 and caspase 1, indicating the activation of the canonical pathway of this inflammasome in YF (Table 4, Fig. 4). Additionally, we found significant expression of AIM2 compared to the negative control. The AIM2 complex is widely associated with the detection of viral DNA, and the AIM2 pathway can form an inflammasome activated by intracytoplasmic DNA [41–46]. To date, no studies have investigated the occurrence of AIM2 in YFV infection. However, Hamel et al. [47] found that the Zika virus, an ssRNA virus similar to YFV, can stimulate AIM2 expression and IL-1β secretion in primary human skin fibroblasts. Moreover, a study by Sousa et al. [33] also found AIM2 in brain tissue, aggravating the neuroinflammatory response in cases of microcephaly due to ZIKV infection. These findings, together with ours, indicate that AIM2 signaling may also be involved in the recognition of viral RNA from these flaviviruses. In the liver parenchyma, all three cytokines—IL-1β, IL-18, and IL-33—were significantly higher than in the negative controls (Table 3, Fig. 2B). IL-18 and IL-33 immunostaining was intense in liver cells from fatal YF cases (Fig. 2B Z2-b,c). IL-1β and IL-18 secretion in the human liver occurs via Kupffer cells during viral infections and has been associated with higher degrees of inflammation in liver diseases [48,49]. The activated form of IL-18 can modulate both innate and adaptive immunity through IFN-γ production by T cells [50,51], Th1 polarization [52], cytotoxicity of T cells and natural killer (NK) cells, and maturation of T, NK, and CD cells [53,54]. IL-1β can also induce the development of various types of T cells that participate in inflammatory pathways and neutrophil recruitment to the site of infection [55,56]. An in vitro study using macrophages derived from human monocytes (GM-Mϕ) infected with DENV showed transcriptional upregulation of pro-IL-1β, pro-IL-18, and the NLRP3 inflammasome, as well as increased caspase-1 activity [57]. In ZIKV infection, IL-1β and IL-18 can contribute to worsening the neuronal response, cell death, and the production of free radicals [33]. The strong correlation between IL-1β, IL-18, and NLRP3 in our results further demonstrates the activity of this inflammasome pathway in YF in situ in the human liver (Table 4, Fig. 4). The increased expression of IL-33 in liver areas characterized by necrosis indicates that IL-33 may be a key factor in the induction of necrosis in the liver parenchyma (Fig. 2B.c), as this cytokine directly participates in necroptosis through RIPK1/RIPK3 regulation [58,59]. In ZIKV infection in human microcephaly cases, IL-33 and inflammasome activation appear to trigger Th2 cytokine signaling and a neuroinflammatory response, contributing to the mechanisms of cell damage in the CNS [32]. It is important to emphasize that IL-33 signaling involves the production of arginase 1 and iNOS, factors that promote cellular stress and directly contribute to cell death through necrosis and apoptosis [32,60]. In our results, we detected high expression of iNOS and arginase 1, mainly in the midzonal area of the hepatic parenchyma (Table 3, Fig. 3). Previous studies have shown that iNOS and arginase 1 are antagonistic enzymes that compete for L-arginine [61,62]. Arginase 1 is a constitutively expressed enzyme in the liver that converts L-arginine to L-ornithine and urea, while iNOS production and L-arginine metabolism result in nitric oxide synthesis and tissue-damaging free radical production [63]. These results indicate that YFV-infected liver cells play an important role in creating an inflammatory environment rich in reactive oxygen and nitrogen intermediates, leading to cell damage and cell death. Further studies, including experimental models, are needed to elucidate in detail the pathogenic mechanisms and immunological factors involved in the host response against YFV infection in hepatocytes. Finally, it is important to mention the limitations of the present study, such as difficulties in evaluating medical records with complete patient information for positive samples. The difficulty in finding positive samples with all patient information to complete the clinical dataset (Table 1) was due to differences in the standardization of medical records between states in the country, which are usually handwritten [64], especially at the time when most samples were collected for this study (Table 1). However, we selected patients with the most complete information available and with confirmatory laboratory results from RT-PCR and/or IHC for YFV. Thus, the positive samples were considered adequate for this methodology and resulted in significant differences in expression levels between the markers used, which were sufficient for comparison with the controls. Conclusion Our findings indicate that lesions caused by YFV infection may be mediated by the modulation of NLRP1, NLRP3, and AIM2 expression in liver parenchyma cells. These molecular receptors associated with YFV appear to induce caspase 1 production, promoting the cleavage of pro-IL-1β, pro-IL-18, and pro-IL-33 and converting these cytokines into their bioactive forms. The results obtained in this study suggest that in situ inflammasome activation significantly contributes to the development of the inflammatory response as well as oxidative stress in fatal cases of YF. Additional in vitro and in vivo studies are necessary to better establish the relationship between inflammasome activation and YFV-related liver pathology. Bibliographic References 1. 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. Memorias do Instituto Oswaldo Cruz 2020, 115, 1–8, doi:10.1590/0074-02760190501. 2. Servadio, J.L.; Muñoz-Zanzi, C.; Convertino, M. Estimating Case Fatality Risk of Severe Yellow Fever Cases: Systematic Literature Review and Meta-Analysis. BMC Infectious Diseases 2021, 21, doi:10.1186/s12879-021-06535-4. 3. Gardner, C.L.; Ryman, K.D. Yellow Fever: A Reemerging Threat. Clinics in Laboratory Medicine 2010, 30, 237–260. 4. Vasconcelos, P.F.C.; Costa, Z.G.; Travassos Da Rosa, E.S.; Luna, E.; Rodrigues, S.G.; Barros, V.L.R.S.; Dias, J.P.; Monteiro, H.A.O.; Oliva, O.F.P.; Vasconcelos, H.B.; et al. Epidemic of Jungle Yellow Fever in Brazil, 2000: Implications of Climatic Alterations in Disease Spread; 2001; Vol. 65;. 5. Vasconcelos, P.F.; Luna, E.J.; Galler, R.; Silva, L.J.; Coimbra, T.L.; Barros, V.L.; Monath, T.P.; Rodigues, S.G.; Laval, C.; Costa, Z.G.; et al. Serious Adverse Events Associated with Yellow Fever 17DD Vaccine in Brazil: A Report of Two Cases. The Lancet 2001, 358, 91–97, doi:10.1016/S0140-6736(01)05326-0. 6. Fernandes, C.F.; Uesugi, J.H.E.; Silva, J.C.C. da; Martins, H.H.S.; Nascimento, B.R. de S.; Trindade, E.L. da; Ferreira, L.A. Prevalence and Evolution of Clinical Cases of Yellow Fever in Brazil between the Years 2007 to 2016. Research, Society and Development 2022, 11, e28611124852, doi:10.33448/rsd-v11i1.24852. 7. Junior, S.; Keyler, A.; Santos, T.; Taís, M.; Souza, O.; Daher, E.D.F. Kidney Involvement in Yellow Fever: A Review. 2019, 1–11. 8. Quaresma, J.A.S.; Barros, V.L.R.S.; Pagliari, C.; Fernandes, E.R.; Guedes, F.; Takakura, C.F.H.; Andrade, H.F.; Vasconcelos, P.F.C.; Duarte, M.I.S. 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, doi:10.1016/j.virol.2005.09.058. 9. Wang, H.; Mao, L.; Meng, G. The NLRP3 Inflammasome Activation in Human or Mouse Cells, Sensitivity Causes Puzzle. Protein and Cell 2013, 4, 565–568, doi:10.1007/s13238-013-3905-0. 10. Wang, W.; Li, G.; De, W.; Luo, Z.; Pan, P.; Tian, M.; Wang, Y.; Xiao, F.; Li, A.; Wu, K.; et al. Zika Virus Infection Induces Host Inflammatory Responses by Facilitating NLRP3 Inflammasome Assembly and Interleukin-1β Secretion. Nature Communications 2018, 9, doi:10.1038/s41467-017-02645-3. 11. Hottz, E.D.; Lopes, J.F.; Freitas, C.; Valls-De-Souza, R.; Oliveira, M.F.; Bozza, M.T.; Da Poian, A.T.; Weyrich, A.S.; Zimmerman, G.A.; Bozza, F.A.; et al. Platelets Mediate Increased Endothelium Permeability in Dengue through NLRP3-Inflammasome Activation. Blood 2013, 122, 3405–3414, doi:10.1182/blood-2013-05-504449. 12. Wu, M.; Chen, S.; Yang, A.; Lin, W.; Lin, Y.; Chen, N.; Tsai, I.; Li, L. CLEC5A Is Critical for Dengue Virus – Induced Inflammasome Activation in Human Macrophages. Immunobiology 2013, 121, 95–106, doi:10.1182/blood-2012-05-430090.The. 13. Negash, A.A.; Ramos, H.J.; Crochet, N.; Lau, D.T.Y.; Doehle, B.; Papic, N.; Delker, D.A.; Jo, J.; Bertoletti, A.; Hagedorn, C.H.; et al. IL-1β Production through the NLRP3 Inflammasome by Hepatic Macrophages Links Hepatitis C Virus Infection with Liver Inflammation and Disease. PLoS Pathogens 2013, 9, 1–13, doi:10.1371/journal.ppat.1003330. 14. de Sousa, J.R.; Azevedo, R. do S. da S.; Martins Filho, A.J.; de Araujo, M.T.F.; Cruz, E. do R.M.; Vasconcelos, B.C.B.; Cruz, A.C.R.; de Oliveira, C.S.; Martins, L.C.; Vasconcelos, B.H.B.; et al. In Situ Inflammasome Activation Results in Severe Damage to the Central Nervous System in Fatal Zika Virus Microcephaly Cases. Cytokine 2018, 111, 255–264, doi:10.1016/j.cyto.2018.08.008. 15. Shrivastava, G.; Valenzuela Leon, P.C.; Calvo, E. Inflammasome Fuels Dengue Severity. Frontiers in Cellular and Infection Microbiology 2020, 10, 1–13, doi:10.3389/fcimb.2020.00489. 16. Gardner, C.L.; Ryman, K.D. Yellow Fever: A Reemerging Threat. (Special Issue: Emerging Pathogens.). Clinics in Laboratory Medicine 2010, 30, 237–260, doi:10.1016/j.cll.2010.01.001.Yellow. 17. Tricarico, P.M.; Caracciolo, I.; Crovella, S.; D’Agaro, P. Zika Virus Induces Inflammasome Activation in the Glial Cell Line U87-MG. Biochemical and Biophysical Research Communications 2017, 492, 597–602, doi:10.1016/j.bbrc.2017.01.158. 18. Wu, M.F.; Chen, S.T.; Yang, A.H.; Lin, W.W.; Lin, Y.L.; Chen, N.J.; Tsai, I.S.; Li, L.; Hsieh, S.L. CLEC5A Is Critical for Dengue Virus-Induced Inflammasome Activation in Human Macrophages. Blood 2013, 121, 95–106, doi:10.1182/blood-2012-05-430090. 19. Liston, A.; Masters, S.L. Homeostasis-Altering Molecular Processes as Mechanisms of Inflammasome Activation. Nature Reviews Immunology 2017, 17, 208–214, doi:10.1038/nri.2016.151. 20. Quaresma, J.A.S.; Barros, V.L.R.S.; Pagliari, C.; Fernandes, E.R.; Andrade, H.F.; Vasconcelos, P.F.C.; Duarte, M.I.S. Hepatocyte Lesions and Cellular Immune Response in Yellow Fever Infection. Trans R Soc Trop Med Hyg 2007, 101, 161–168, doi:10.1016/j.trstmh.2006.02.019. 21. 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, doi:10.1002/rmv.1752. 22. Olímpio, F.A.; Falcão, L.F.M.; Carvalho, M.L.G.; Lopes, J. da C.; Mendes, C.C.H.; Filho, A.J.M.; da Silva, C.A.M.; Miranda, V.D.S.C.; dos Santos, L.C.; Vilacoert, F.S. da S.; et al. Endothelium Activation during Severe Yellow Fever Triggers an Intense Cytokine-Mediated Inflammatory Response in the Liver Parenchyma. Pathogens 2022, 11, doi:10.3390/pathogens11010101. 23. Hsu, S.M.; Raine, L.; Fanger, H. Use of Avidin-Biotin-Peroxidase Complex (ABC) in Immunoperoxidase Techniques: A Comparison between ABC and Unlabeled Antibody (PAP) Procedures. Journal of Histochemistry & Cytochemistry 1981, 29, 577–580, doi:10.1177/29.4.6166661. 24. 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. Reviews in Medical Virology 2013, 23, 305–318. 25. Quaresma, J.A.S.; Barros, V.L.R.S.; Pagliari, C.; Fernandes, E.R.; Andrade, H.F.; Vasconcelos, P.F.C.; Duarte, M.I.S. Hepatocyte Lesions and Cellular Immune Response in Yellow Fever Infection. Trans R Soc Trop Med Hyg 2007, 101, 161–168, doi:10.1016/j.trstmh.2006.02.019. 26. Simões Quaresma, J.A.; Souza Barros, V.L.R.; Rainero Fernandes, E.; Pagliari, C.; Takakura, C.; da Costa Vasconcelos, P.F.; de Andrade, H.F.; Seixas Duarte, M.I. Reconsideration of Histopathology and Ultrastructural Aspects of the Human Liver in Yellow Fever. Acta Tropica 2005, 94, 116–127, doi:10.1016/j.actatropica.2005.03.003. 27. Quaresma, J.A.S.; Barros, V.L.R.S.; Pagliari, C.; Fernandes, E.R.; Andrade, H.F.; Vasconcelos, P.F.C.; Duarte, M.I.S. Hepatocyte Lesions and Cellular Immune Response in Yellow Fever Infection. Trans R Soc Trop Med Hyg 2007, 101, 161–168, doi:10.1016/j.trstmh.2006.02.019. 28. Quaresma, J.A.S.; Duarte, M.I.S.; Vasconcelos, P.F.C. Midzonal Lesions in Yellow Fever: A Specific Pattern of Liver Injury Caused by Direct Virus Action and in Situ Inflammatory Response. Medical Hypotheses 2006, 67, 618–621, doi:10.1016/j.mehy.2006.01.060. 29. 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. Reviews in Medical Virology 2013, 23, 305–318. 30. Quaresma, J.A.S.; Barros, V.L.R.S.; Pagliari, C.; Fernandes, E.R.; Guedes, F.; Takakura, C.F.H.; Andrade, H.F.; Vasconcelos, P.F.C.; Duarte, M.I.S. 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, doi:10.1016/j.virol.2005.09.058. 31. Quaresma, J.A.S.; Barros, V.L.R.S.; Fernandes, E.R.; Pagliari, C.; Guedes, F.; Vasconcelos, P.F.D.C.; Andrade, H.F. de; Duarte, M.I.S. Immunohistochemical Examination of the Role of Fas Ligand and Lymphocytes in the Pathogenesis of Human Liver Yellow Fever. Virus Research 2006, 116, 91–97, doi:10.1016/j.virusres.2005.08.019. 32. Azevedo, 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.; et al. In Situ Immune Response and Mechanisms of Cell Damage in Central Nervous System of Fatal Cases Microcephaly by Zika Virus. Scientific Reports 2018, 8, doi:10.1038/s41598-017-17765-5. 33. de Sousa, J.R.; Azevedo, R. do S. da S.; Martins Filho, A.J.; de Araujo, M.T.F.; Cruz, E. do R.M.; Vasconcelos, B.C.B.; Cruz, A.C.R.; de Oliveira, C.S.; Martins, L.C.; Vasconcelos, B.H.B.; et al. In Situ Inflammasome Activation Results in Severe Damage to the Central Nervous System in Fatal Zika Virus Microcephaly Cases. Cytokine 2018, 111, 255–264, doi:10.1016/j.cyto.2018.08.008. 34. Srikiatkhachorn, A.; Mathew, A.; Rothman, A.L. Immune-Mediated Cytokine Storm and Its Role in Severe Dengue. Seminars in Immunopathology 2017, 39, 563–574, doi:10.1007/s00281-017-0625-1. 35. Shrivastava, G.; Valenzuela Leon, P.C.; Calvo, E. Inflammasome Fuels Dengue Severity. Frontiers in Cellular and Infection Microbiology 2020, 10. 36. Kaushik, D.K.; Gupta, M.; Kumawat, K.L.; Basu, A. Nlrp3 Inflammasome: Key Mediator of Neuroinflammation in Murine Japanese Encephalitis. PLoS ONE 2012, 7, doi:10.1371/journal.pone.0032270. 37. Kumar, M.; Roe, K.; Orillo, B.; Muruve, D.A.; Nerurkar, V.R.; Gale, M.; Verma, S. Inflammasome Adaptor Protein Apoptosis-Associated Speck-Like Protein Containing CARD (ASC) Is Critical for the Immune Response and Survival in West Nile Virus Encephalitis. Journal of Virology 2013, 87, 3655–3667, doi:10.1128/jvi.02667-12. 38. Lien, T.S.; Sun, D.S.; Chang, C.M.; Wu, C.Y.; Dai, M.S.; Chan, H.; Wu, W.S.; Su, S.H.; Lin, Y.Y.; Chang, H.H. Dengue Virus and Antiplatelet Autoantibodies Synergistically Induce Haemorrhage through Nlrp3-Inflammasome and FcγRIII. Thrombosis and Haemostasis 2015, 113, 1060–1070, doi:10.1160/TH14-07-0637. 39. Ramos, H.J.; Lanteri, M.C.; Blahnik, G.; Negash, A.; Suthar, M.S.; Brassil, M.M.; Sodhi, K.; Treuting, P.M.; Busch, M.P.; Norris, P.J.; et al. IL-1β Signaling Promotes CNS-Intrinsic Immune Control of West Nile Virus Infection. PLoS Pathogens 2012, 8, doi:10.1371/journal.ppat.1003039. 40. Sakhon, O.S.; Severo, M.S.; Kotsyfakis, M.; Pedra, J.H.F. A Nod to Disease Vectors: Mitigation of Pathogen Sensing by Arthropod Saliva. Frontiers in Microbiology 2013, 4, doi:10.3389/fmicb.2013.00308. 41. Lugrin, J.; Martinon, F. The AIM2 Inflammasome: Sensor of Pathogens and Cellular Perturbations. Immunological Reviews 2018, 281, 99–114. 42. Schattgen, S.A.; Gao, G.; Kurt-Jones, E.A.; Fitzgerald, K.A. Cutting Edge: DNA in the Lung Microenvironment during Influenza Virus Infection Tempers Inflammation by Engaging the DNA Sensor AIM2. The Journal of Immunology 2016, 196, 29–33, doi:10.4049/jimmunol.1501048. 43. Zhang, H.; Luo, J.; Alcorn, J.F.; Chen, K.; Fan, S.; Pilewski, J.; Liu, A.; Chen, W.; Kolls, J.K.; Wang, J. AIM2 Inflammasome Is Critical for Influenza-Induced Lung Injury and Mortality. The Journal of Immunology 2017, 198, 4383–4393, doi:10.4049/jimmunol.1600714. 44. Reinholz, M.; Kawakami, Y.; Salzer, S.; Kreuter, A.; Dombrowski, Y.; Koglin, S.; Kresse, S.; Ruzicka, T.; Schauber, J. HPV16 Activates the AIM2 Inflammasome in Keratinocytes. Archives of Dermatological Research 2013, 305, 723–732, doi:10.1007/s00403-013-1375-0. 45. Torii, Y.; Kawada, J.I.; Murata, T.; Yoshiyama, H.; Kimura, H.; Ito, Y. Epstein-Barr Virus Infection-Induced Inflammasome Activation in Human Monocytes. PLoS ONE 2017, 12, doi:10.1371/journal.pone.0175053. 46. van de Veerdonk, F.L.; Netea, M.G.; Dinarello, C.A.; Joosten, L.A.B. Inflammasome Activation and IL-1β and IL-18 Processing during Infection. Trends in Immunology 2011, 32, 110–116. 47. Hamel, 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. Journal of Virology 2015, 89, 8880–8896, doi:10.1128/jvi.00354-15. 48. Chattergoon, M.A.; Levine, J.S.; Latanich, R.; Osburn, W.O.; Thomas, D.L.; Cox, A.L. High Plasma Interleukin-18 Levels Mark the Acute Phase of Hepatitis C Virus Infection. Journal of Infectious Diseases 2011, 204, 1730–1740, doi:10.1093/infdis/jir642. 49. Sharma, A.; Chakraborti, A.; Das, A.; Dhiman, R.K.; Chawla, Y. Elevation of Interleukin-18 in Chronic Hepatitis C: Implications for Hepatitis C Virus Pathogenesis. Immunology 2009, 128, doi:10.1111/j.1365-2567.2008.03021.x. 50. A Yoshimoto T, Takeda K, Tanaka T, et al. IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN-gamma production. J Immunol. 1998;161(7):3400-3407.; 51. Chirathaworn, C.; Rianthavorn, P.; Wuttirattanakowit, N.; Poovorawan, Y. Serum IL-18 and IL-18BP Levels in Patients with Chikungunya Virus Infection. Viral Immunology 2010, 23, 113–117, doi:10.1089/vim.2009.0077. 52. Fujikura, D.; Muramatsu, D.; Toyomane, K.; Chiba, S.; Daito, T.; Iwai, A.; Kouwaki, T.; Okamoto, M.; Higashi, H.; Kida, H.; et al. Aureobasidium Pullulans-Cultured Fluid Induces IL-18 Production, Leading to Th1-Polarization during Influenza A Virus Infection. Journal of Biochemistry 2018, 163, 31–38, doi:10.1093/jb/mvx062. 53. Dinarello, C.A.; Fantuzzi, G. Interleukin-18 and Host Defense against Infection; 54. Li, J.; Mbow, M.L.; Sun, L.; Li, L.; Yang, G.; Griswold, D.E.; Schantz, A.; Shealy, D.J.; Goletz, T.J.; Wan, J.; et al. Induction of Dendritic Cell Maturation by IL-18. Cellular Immunology 2004, 227, 103–108, doi:10.1016/j.cellimm.2004.02.002. 55. Santarlasci, V.; Cosmi, L.; Maggi, L.; Liotta, F.; Annunziato, F. IL-1 and T Helper Immune Responses. Frontiers in Immunology 2013, 4. 56. Niu, J.; Wu, S.; Chen, M.; Xu, K.; Guo, Q.; Lu, A.; Zhao, L.; Sun, B.; Meng, G. Hyperactivation of the NLRP3 Inflammasome Protects Mice against Influenza A Virus Infection via IL-1β Mediated Neutrophil Recruitment. Cytokine 2019, 120, 115–124, doi:10.1016/j.cyto.2019.04.019. 57. Wu, M.F.; Chen, S.T.; Yang, A.H.; Lin, W.W.; Lin, Y.L.; Chen, N.J.; Tsai, I.S.; Li, L.; Hsieh, S.L. CLEC5A Is Critical for Dengue Virus-Induced Inflammasome Activation in Human Macrophages. Blood 2013, 121, 95–106, doi:10.1182/blood-2012-05-430090. 58. Liu, Y.; Liu, T.; Lei, T.; Zhang, D.; Du, S.; Girani, L.; Qi, D.; Lin, C.; Tong, R.; Wang, Y. RIP1/RIP3-Regulated Necroptosis as a Target for Multifaceted Disease Therapy (Review). International Journal of Molecular Medicine 2019, 44, 771–786, doi:10.3892/ijmm.2019.4244. 59. Zhou, H.; Liu, L.; Ma, X.; Wang, J.; Yang, J.; Zhou, X.; Yang, Y.; Liu, H. RIP1/RIP3/MLKL-Mediated Necroptosis Contributes to Vinblastine-Induced Myocardial Damage. Molecular and Cellular Biochemistry 2021, 476, 1233–1243, doi:10.1007/s11010-020-03985-3. 60. Liang, Y.; Yi, P.; Yuan, D.M.K.; Jie, Z.; Kwota, Z.; Soong, L.; Cong, Y.; Sun, J. IL-33 Induces Immunosuppressive Neutrophils via a Type 2 Innate Lymphoid Cell/IL-13/STAT6 Axis and Protects the Liver against Injury in LCMV Infection-Induced Viral Hepatitis. Cellular and Molecular Immunology 2019, 16, 126–137, doi:10.1038/cmi.2017.147. 61. Cao, W.; Sun, B.; Feitelson, M.A.; Wu, T.; Tur-Kaspa, R.; Fan, Q. Hepatitis C Virus Targets Over-Expression of Arginase I in Hepatocarcinogenesis. International Journal of Cancer 2009, 124, 2886–2892, doi:10.1002/ijc.24265. 62. Wagener, J.; Maccallum, D.M.; Brown, G.D.; Gow, N.A.R. Candida Albicans Chitin Increases Arginase-1 Activity in Human Macrophages, with an Impact on Macrophage Antimicrobial Functions. mBio 2017, 8, doi:10.1128/mBio.01820-16. 63. Wheatley, D.N. Controlling Cancer by Restricting Arginine Availability–Arginine-Catabolizing Enzymes as Anticancer Agents. Anti-Cancer Drugs 2004, 15, 825–833, doi:10.1097/00001813-200410000-00002. 64. Timóteo, M. do S.T.B.A.; Dantas, R.A.N.; Costa, I.C.S.; Silva, T.T.M. da; Santos, K.V.G. dos; Oliveira, E.D.S.; Dantas, D.V. Implementation of Improvement Cycle in Health Records of Mobile Emergency Prehospital Care. Rev Bras Enferm 2020, 73, e20190049, doi:10.1590/0034-7167-2019-0049. Figures Figure 1. Quantitative and immunohistochemical analysis for NLRP1, NLRP3, and AIM2. (A) Quantitative analysis of NLRP1, NLRP3, and AIM2 in zones Z3, Z2, Z1, and PT of the hepatic parenchyma in fatal YF cases and YF-negative controls. Tukey test: *p ≤ 0.05; **p ≤ 0.001; ***p ≤ 0.0001. (B) Immunohistochemical analysis showing positive staining for NLRP3, NLRP1, and AIM2 in zones Z3, Z2, Z1, and PT of the hepatic parenchyma in fatal YF cases and controls. Immunostaining for NLRP1 (a) in hepatocytes (arrows) (a–Z3, b–Z2, b–Z1) and in the inflammatory infiltrate (a–PT). Immunolabeling for NLRP3 (b) in hepatocytes (arrows) (b–Z3, b–Z2, b–Z1) and in the inflammatory infiltrate (b–PT). Immunolabeling for AIM2 (c) in hepatocytes (arrows) (c–Z3, c–Z2, c–Z1) and in the inflammatory infiltrate (c–PT). In the negative control, hepatic parenchyma is preserved and there is minimal expression of NLRP3, NLRP1, and AIM2. Z3: centrolobular zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract; (NC): negative controls. Magnification: 400X (20 µm). Figure 2. Figure 2. Quantitative and immunohistochemical analysis for IL-1β, IL-18, and IL-33. (A) Quantitative analysis of IL-1β, IL-18, and IL-33 in zones Z3, Z2, Z1, and PT of the hepatic parenchyma in fatal YF cases and YF-negative controls. Tukey test: *p ≤ 0.05; **p ≤ 0.001; ***p ≤ 0.0001. (B) Immunohistochemical analysis showing positive staining for IL-1β, IL-18, and IL-33 in zones Z3, Z2, Z1, and PT of the hepatic parenchyma in fatal YF cases and controls. Immunostaining for IL-1β (a) in hepatocytes (arrows) (a–Z3, b–Z2, b–Z1) and in the inflammatory infiltrate (a–PT). Immunolabeling for IL-18 (b) in hepatocytes (arrows) (b–Z3, b–Z2, b–Z1) and in the inflammatory infiltrate (b–PT). Immunolabeling for IL-33 (c) in hepatocytes (circled) (c–Z3, c–Z2, c–Z1) and in the inflammatory infiltrate (c–PT). In the negative control, hepatic parenchyma is preserved and minimal expression of IL-1β, IL-18, and IL-33 is observed. Z3: centrolobular zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract; (NC): negative controls. Magnification: 400X (20 µm). Figure 3. Quantitative and immunohistochemical analysis for iNOS, caspase 1, and arginase 1. (A) Quantitative analysis of iNOS, caspase 1, and arginase 1 in zones Z3, Z2, Z1, and PT of the hepatic parenchyma in fatal YF cases and YF-negative controls. Tukey test: *p ≤ 0.05; **p ≤ 0.001; ***p ≤ 0.0001. (B) Immunohistochemical analysis showing positive staining for iNOS, caspase 1, and arginase 1 in zones Z3, Z2, Z1, and PT of the hepatic parenchyma in fatal YF cases and controls. Immunostaining for iNOS (a) in hepatocytes (arrows) (a–Z3, b–Z2, b–Z1) and in the inflammatory infiltrate (a–PT). Immunolabeling for caspase 1 (b) in hepatocytes (arrows) (b–Z3, b–Z2, b–Z1) and in the inflammatory infiltrate (b–PT). Immunolabeling for arginase 1 (c) in hepatocytes (arrows) (c–Z3, c–Z2, c–Z1) and in the inflammatory infiltrate (c–PT). In the negative control, hepatic parenchyma is preserved and minimal expression of iNOS, caspase 1, and arginase 1 is observed. Z3: centrolobular zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract; (NC): negative controls. Magnification: 400X (20 µm). Figure 4. Linear correlation between inflammasome markers and liver parenchyma areas in fatal cases of yellow fever, showing strong (r > 0.7) to very strong (r > 0.9) correlation coefficients. Pearson correlation: ***p < 0.0001; **p < 0.001; *p 0.7) to very strong (r > 0.9) correlation coefficients. Pearson correlation: **p < 0.001; ***p < 0.0001. Tables Table 1 - Characterization of patients with YF according to gender, age, state, year and duration of disease. 1 001/00 M 25 Tocantins 2000 8 2 106/00 M 75 Goiás 2000 NR** 3 108/00 M 49 Goiás 2000 7 4 494/00 M NR** Distrito Federal 2000 NR** 5 251/00 M 16 Mato Grosso do Sul 2000 6 6 252/00 M 49 Goiás 2000 NR** 7 253/00 M 23 Goiás 2000 NR** 8 255/00 M NR** Goiás 2000 NR** 9 291/00 M NR** Goiás 2000 NR** 10 158/00 M 33 Goiás 2000 NR** 11 063/03 M NR** Minas Gerais 2003 NR** 12 339/04 M 36 Amazonas 2004 11 13 019/08 M 64 Goiás 2008 7 14 273/08 M 57 Goiás 2008 7 15 068/08 F 65 Goiás 2008 2 16 095/08 M 42 Goiás 2008 3 17 143/08 M 37 Distrito Federal 2008 NR** 18 361/15 F 53 Rio grande do Norte 2015 4 19 062/16 M 35 Goiás 2016 NR** 20 346/16 M 15 Goiás 2016 7 21 369/16 M 27 Goiás 2016 1 *DD= duration of disease / **NR= No registry Table 2- Antibodies used in the analysis of the inflammasome profile in the liver of fatal YFV-induced cases NLRP1 Abcam/ab98181 1/50 NLRP3 Abcam/ab214185 1/50 AIM2 Abcam/ab93015 1/50 iNOS Abcam/ab15323 1/200 IL-1β Abcam/ab9722 1/100 IL-18 Abcam/ab68435 1/100 IL-33 Abcam/ab118503 1/100 Caspase 1 Abcam/ab1872 1/100 Arginase 1 Novus/NBP1-87455 1/300 Table 3 - Quantitative analysis of inflammasome markers in the hepatic parenchyma of fatal YFV cases comparing to control cases. NLRP1 105.90 ± 29.00 * 212.60 ± 48.16 *** 87.62 ± 20.73 * 81.52 ± 20.89 * *** Control 51.20 ± 11.97 76.00 ± 13.27 41.60 ± 12.80 35.20 ± 11.97 NLRP3 136.38 ± 25.00 *** 285.71 ± 87.12 *** 101.33 ± 19.96 * 80.76 ± 24.43 * *** Control 57.60 ± 16.32 80.00 ± 11.31 51.20±7.155 38.40 ± 12.80 AIM2 140.95 ± 25.95 *** 356.57 ± 48.92 *** 119.62 ± 23.48 *** 80.00 ± 20.36 * *** Control 38.40 ± 12.80 48.00 ± 10.12 48.00 ± 10.12 51.20 ± 18.66 Caspase 1 102.86 ± 21.84 *** 147.0 ± 22.96 *** 89.90 ± 17.44 * 70.10 ± 15.98 * *** Control 51.20 ± 11.87 60.80 ± 18.66 54.40 ± 12.80 35.20 ± 11.79 iNOS 100.57 ± 19.84 *** 150.1 ± 21.79 *** 89.90 ± 19.42 *** 76.19 ± 15.54 ** *** Control 54.40 ± 16.32 54.40 ± 16.32 44.80 ± 11.97 48.00 ± 10.12 Arginase 1 139.43 ± 19.21 *** 323.81 ± 46.56 *** 115.81 ± 15.54 *** 75.43 ± 14.93 * *** Control 38.40 ± 16.32 38.40 ± 7.84 44.80 ± 11.97 48.00 ± 17.53 IL-1β 109.0 ± 23.48 * 190.48 ± 73.22 *** 86.10 ± 15.98 * 70.10 ± 15.98 * *** Control 64.00 ± 22.63 83.20 ± 11.97 54.40 ± 16.32 48.00 ± 10.12 IL-18 154.7 ± 20.56 *** 364.95 ± 43.78 *** 123.43 ± 16.48 *** 90.67 ± 21.14 * *** Control 83.20 ± 11.97 92.80 ± 27.53 70.40 ± 21.07 60.80 ± 6.49 IL-33 146.3 ± 19.93 * 351.24 ± 42.04 *** 177.2 ± 50.39 *** 89.14 ± 20.70 * *** Control 92.80 ± 21.23 89.60 ± 19.20 37.60± 6.97 41.60 ± 16.32 Z3: Centrolobular zone; Z2: Midzonal zone; Z1: Periportal zone; PT: Portal tract. Tukey-test;***p < 0.0001; ** p < 0.001; * p < 0.05. Table 4. Linear correlation among receptors, cytokines, and enzymes that characterize the in situ inflammasome response and hepatic parenchyma areas in fatal YFV cases. Pearson correlation; *p < 0.05; **p < 0.001; ***p < 0.0001. NLRP1 Z3 x IL-18 PT -0.4707 0.0313* NLRP1 Z3 x IL-33 Z1 0.8230 <0.0001*** NLRP1 Z3 x Caspase 1 Z3 -0.7991 0.0004*** NLRP1 Z2 x IL-33 Z1 0.9143 <0.0001*** NLRP1 Z1 x IL-33 PT -0.5034 0.0200* NLRP3 Z3 x iNOS Z3 -0.8149 0.0002*** NLRP3 Z2 x Caspase 1 Z2 0.8549 <0.0001*** NLRP3 Z2 x Arginase 1 Z1 0.7830 0.0003*** NLRP3 Z2 x IL-1β Z3 0.7097 0.0021** NLRP3 Z1 x IL-18 Z2 0.8598 0.0002*** NLRP3 PT x Arginase 1 Z2 0.4369 0.0477* NLRP3 PT x IL-1β Z1 0.3380 0.0057** IL-1β Z2 x Caspase 1 Z2 0.8569 <0.0001*** IL-1β Z1 x Arginase 1 Z2 0.4931 0.0231* IL-1β PT x iNOS Z1 0.7120 0.0004*** IL-1β PT x iNOS PT 0.5843 0.0054** IL-18 Z3 x IL-33 Z3 0.8430 <0.0001*** IL-18 Z3 x Arginase 1 Z3 0.8411 <0.0001*** IL-18 Z2 x AIM2 Z2 0.8465 <0.0001*** IL-18 Z2 x Caspase 1 Z3 -0.8042 0.0002*** IL-18 Z2 x iNOS Z3 -0.7750 0.0004*** IL-18 Z1 x Arginase 1 Z1 0.4487 0.0413* IL-18 PT x iNOS Z2 -0.5380 0.0119* IL-33 Z3 x IL-18 PT 0.4630 0.0346* IL-33 Z3 x NLRP3 Z1 0.7817 0.0010** IL-33 PT x AIM2 PT 0.6075 0.0035** iNOS Z3 x IL-1β Z3 0.7824 0.0003*** iNOS Z3 x Caspase 1 Z3 0.7987 0.0004*** iNOS Z2 x Caspase 1 Z3 0.8099 0.0004*** iNOS Z2 x IL-33 PT -0.4479 0.0417* iNOS Z2 x NLRP3 Z2 0.8123 <0.0001*** iNOS Z1 x AIM2 PT 0.4624 0.0348* iNOS PT x IL-18 Z2 -0.5546 0.0091** iNOS PT x IL-1β PT 0.5843 0.0054** Caspase 1 Z3 x IL-18 PT -0.8021 <0.0001*** Caspase 1 Z2 x Arginase 1 PT 0.5030 0.0201* Caspase 1 Z2 x NLRP3 Z3 0.7883 0.0003 Caspase 1 Z2 x NLRP3 Z2 0.8429 <0.0001*** Caspase 1 Z1 x IL-33 PT -0.5210 0.0154* AIM2 Z3 x Arginase 1 Z3 0.6811 0.0007*** AIM2 Z2 x Caspase 1 Z3 -0.8302 0.0001*** AIM2 Z2 x Arginase 1 Z2 0.8396 <0.0001** * AIM2 Z2 x IL-18 Z2 0.8306 <0.0001** * AIM2 Z1 x Arginase 1 Z1 0.8764 <0.0001*** AIM2 Z1 x Caspase 1 Z3 0.7809 0.0006*** AIM2 Z1 x NLRP3 Z2 0.8056 0.0005*** Arginase 1 Z2 x Caspase 1 Z3 -0.7932 0.0007*** Arginase 1 PT x AIM2 PT 0.5615 0.0081** Supplementary Material File (table 1 - characterization of patients with yf.docx) Download 17.21 KB File (table 2- antibodies used in the analysis.docx) Download 15.45 KB File (table 3 - quantitative analysis of inflammasome.docx) Download 17.09 KB File (table 4 - linear correlation among receptors.docx) Download 17.21 KB Information & Authors Information Version history V1 Version 1 04 December 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cytokines disease control flavirus immune responses innate immunity virus classification yellow fever virus Authors Affiliations Vanessa do Socorro Cabral Miranda Instituto Evandro Chagas View all articles by this author Luiz Fábio Magno Falcão Universidade do Estado do Para Centro de Ciencias Biologicas e da Saude View all articles by this author Luis Arthur Moreira Ferreira Universidade do Estado do Para Centro de Ciencias Biologicas e da Saude View all articles by this author Lucas Corrêa Modesto Universidade do Estado do Para Centro de Ciencias Biologicas e da Saude View all articles by this author Pablo Rodrigues Nunes de Souza Universidade do Estado do Para Centro de Ciencias Biologicas e da Saude View all articles by this author Rafael Malcher Meira Rocha Universidade do Estado do Para Centro de Ciencias Biologicas e da Saude View all articles by this author Arnaldo Jorge Martins Filho Instituto Evandro Chagas View all articles by this author Jorge Rodrigues de Sousa Instituto Evandro Chagas View all articles by this author Mayumi Duarte Wakimoto Instituto Nacional de Infectologia Evandro Chagas View all articles by this author Pedro Fernando da Costa Vasconcelos Instituto Evandro Chagas View all articles by this author Carla Pagliari 0000-0001-6210-6917 Universidade de Sao Paulo Departamento de Patologia e Medicina Legal View all articles by this author Hellen Fuzii Universidade Federal do Para View all articles by this author Juarez Antônio Simões Quaresma [email protected] Universidade do Estado do Para Centro de Ciencias Biologicas e da Saude View all articles by this author Metrics & Citations Metrics Article Usage 196 views 138 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Vanessa do Socorro Cabral Miranda, Luiz Fábio Magno Falcão, Luis Arthur Moreira Ferreira, et al. 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