Caspase-1/11 Controls Zika Virus Replication in Astrocytes by Regulating Glycolytic Metabolism

doi:10.21203/rs.3.rs-4320553/v1

Download PDF

Research Article

Caspase-1/11 Controls Zika Virus Replication in Astrocytes by Regulating Glycolytic Metabolism

https://doi.org/10.21203/rs.3.rs-4320553/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

The Zika virus (ZIKV) poses a significant threat due to its association with severe neurological complications, particularly during pregnancy. Although viruses exhibit tropism for neural cells, including astrocytes, the role of these cells in controlling ZIKV replication remains unclear. In this study, we demonstrated that ZIKV induces caspase-1 activation in primary astrocytes despite the absence of classical signs of inflammasome activation. Caspase-1/11^−/− astrocytes exhibit heightened permissiveness to viral replication, accompanied by overactivation of glycolytic metabolism. Inhibition of glycolysis reversed the susceptibility of caspase-1/11^−/− astrocytes to ZIKV infection. Protein network analysis revealed mTORC as a link between proteins involved in glycolysis and caspase-1, and mTORC inhibition also suppressed viral replication. Furthermore, we found that the impact of caspase-1/11 on astrocytes is dependent on pyruvate transport to mitochondria for viral replication, emphasizing the role of the mTORC/glycolytic pathway/pyruvate axis in the caspase-1/11-mediated control of ZIKV. Overall, our findings elucidate a caspase-1/11-dependent microbicidal mechanism in astrocytes, providing insights into potential therapeutic targets for ZIKV infection.

Caspase-1/11

Astrocytes

Zika virus

Metabolism

Glycolysis

mTORC

Scientific category: Immunobiology

Zika virus (ZIKV) is a mosquito-borne flavivirus that is primarily transmitted through the bite of Aedes mosquitoes, but it can also be transmitted through sexual contact and from mother to fetus during pregnancy. Although most of the clinical manifestations of ZIKV infection range from asymptomatic to mild flu-like symptoms, in 2016, the virus gained global attention due to its association with severe neurological complications, such as Guillain–Barré syndrome, encephalopathies, and fetal microcephaly (1–3). This led to the declaration of a state of emergency and international concern by the WHO due to hundreds of cases of fetal malformation and neonatal microcephaly. Although the situation is now under control, with only approximately 500 cases of ZIKV infection in Brazil, the lack of available vaccines or treatments urges a deeper investigation of its neuropathogenesis.

Although ZIKV exhibits greater tropism for neural cells, specifically neuroprogenitor cells, it has already been demonstrated to infect multiple cell types in the brain, including oligodendrocytes (1), microglia (2), endothelial cells (3) and astrocytes (4, 5). Astrocytes are highly abundant in the central nervous system (CNS), and they play a role in maintaining blood brain barrier (BBB) functions and regulating brain energy metabolism (6, 7). Interestingly, astrocytes can support persistent and productive infection of ZIKV for a minimum of one month and are regarded as the first brain cell type targeted by ZIKV upon infection (8, 9).

To eradicate invading viruses quickly and efficiently, the host has evolved highly conserved sensors called pattern recognition receptors (PRRs), which recognize viral infections and subsequently trigger antiviral immune responses (10, 11). PRRs include Toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), DNA sensors such as cyclic GMP-AMP synthase (cGAS), nucleotide-binding oligomerization domain leucine-rich repeats (LRR)-containing receptors (NLRs), and members of the Pyrin and HIN domain-containing protein (PYHIN) family (12). Upon activation, members of the NLR and PYHIN families oligomerize, forming inflammasome complexes—multiprotein platforms present in the cytosol capable of activating the protease caspase-1. Active caspase-1 leads to the maturation and release of IL-1β and IL-18, along with cell death through pyroptosis via gasdermin-D (GSDMD) activation (13, 14).

While it has been demonstrated that ZIKV induces inflammasome activation in human and murine monocytes, leading to inflammatory responses through caspase-1 cleavage and IL-1β secretion (15–20), the impact of this activation on the control of infection remains controversial. The genetic or pharmacological inhibition of caspase-1 and NLRP3 in human and murine monocytes results in an increased IFN-I-dependent response, leading to better control of viral replication (16, 18). However, caspase-1 inhibition with Ac-YVAD-cmk increased viral dissemination and mortality in an in vivo ZIKV infection model, demonstrating a protective role of this protease (17).

The activation of inflammasomes in glial cells by ZIKV appears to be even more controversial, as ZIKV infection in a human microglial cell line (U87-MG) resulted in IL-1β secretion (21), while in mixed glial cells derived from the mouse brain, ZIKV not only failed to induce inflammasome activation but also inhibited inflammasome activation in response to classical NLRP3 agonists (20). Since astrocytes harbor ZIKV (8), understanding the autonomous effector mechanisms by which these cells control infection is crucial. In this study, we revealed that ZIKV infection activates caspase-1 in primary astrocytes, yet classical signs of inflammasome activation (IL-1β and pyroptosis) are absent. Caspase-1/11-deficient astrocytes exhibit increased permissiveness to viral replication, despite elevated cytokine secretion (e.g., IFN-I and TNF-α). Moreover, caspase-1/11^−/− astrocytes exhibit dysregulated glycolytic metabolism, and inhibiting glycolytic flux reverses their susceptibility to ZIKV infection. Notably, inhibiting the transport of pyruvate to mitochondria, but not its conversion to lactate, reversed caspase-1/11^−/− astrocyte susceptibility, while pyruvate addition enhanced viral replication. These findings collectively underscore caspase-1/11-dependent control of ZIKV replication in astrocytes through the regulation of the glycolytic pathway.

Animals

This work was submitted and accepted by the Ethics Committee on Animal Use of the Federal University of São Paulo (UNIFESP) and is registered under number 1368130918. Wild-type C57BL/6 mice (WT) and Caspase-1/11 knockout (Caspase-1/11^−/−) mice (kindly provided by Dr. Richard Flavell, Yale University, USA) were provided by the Center for the Development of Experimental Models for Medicine and Biology (CEDEME) at UNIFESP. All animals were kept under specific germ-free (SPF) conditions in microisolators with free access to water and feed.

Primary cell culture of astrocytes

Primary microglia and astrocytes were obtained according to a previously published protocol (22). In summary, animals that were up to 3 days postnatal were used. The animals were anesthetized by inhalation of isoflurane anesthetic (Cristália) and then quickly euthanized by decapitation, after which the brain was removed. The tissue was processed and placed in culture in a greenhouse at 37°C. After 14 days, microglia and astrocytes were cultured. To separate the cell populations, the culture was subjected to circular shaking in an orbital shaker at 200 rpm and 37°C overnight. After shaking, the detached microglia are located in the supernatant, and the astrocytes adhere to the supernatant. After separation, the cells were counted and incubated at the planned density for the experiments.

Virus

The ZIKVBR strain (isolated, purified and titrated) was kindly donated by Prof. Dr. Ricardo Weinlich and Dr. Rafaela da Rosa Ribeiro of the Albert Einstein Israeli Teaching and Research Institute (IIEPAE) São Paulo, Brazil (which was obtained in collaboration with the laboratory of Prof. Dr. Luís Carlos Ferreira of the Institute of Biomedical Sciences – ICB of USP).

Treatment with agonists and inhibitors

To induce the transcription of inflammasome components, cells were treated with LPS (200 ng/ml) (InvivoGen, San Diego, CA, USA) for 3 h. Nigericin (10 nM/ml, 1 h) (InvivoGen) was added directly to the cultures. To inhibit glycolytic pathway enzymes, cells were treated 1 h before infection with the following inhibitors: 5 mM 2DG (cat. Number 14325), 40 mM oxamate (Cayman Chemical – cat. Number 19057), 10 µM UK 5099 (Cayman Chemical – cat. Number 16980) and 10 µM rapamycin (InvivoGen).

Glial cell infection

Astrocytes from WT and Caspase-1/11^−/− mice were plated in 48-well plates (Costar) at a concentration of 3x10⁵ cells per well (for quantitative real-time PCR) or 3x10⁴ cells per well in a 96-well plate (Costar) (for immunofluorescence). Then, the cells were infected with ZIKVBR at MOIs of 0.1, 1 and 10 for a period of 2 h in DMEM/F12 without fetal bovine serum (FBS). After this period, the plate was washed to remove extracellular viruses and incubated at 37°C for 24, 48 and 72 h for subsequent analysis.

Quantitative real-time PCR

To determine the viral load of the infected cells, real-time PCR was used. Astrocytes were incubated and treated as described above, and total cell RNA was isolated by the TRIzol method (Thermo Fisher Scientific, 15596026) following the manufacturer's instructions. The concentration and purity of the mRNA were analyzed by a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Inc.). The absorbance of the samples was evaluated at 260/280 nm and 260/230 nm, where only ratios above 1.8 were used, indicating the absence of contaminants. cDNA was generated from 500 ng of total RNA with a High-Capacity cDNA Reverse Transcription Kit (catalog number: 4368814) following the manufacturer's instructions. cDNA was homogenized with TaqMan Universal PCR Master Mix (Applied Biosystems, 4369016). The primer sequences for ZIKABR were as follows: primer 1, 5'-CCGCTGCCCACACAAG-3'; primer 2, 5'-CCACTAACGTTCTTTTGCAGACAT-3'; and the TaqMan FAM probe, AGCCTACCTTGACAAGCAGTCAGACACTCAA. Reactions were conducted in a 7500 Real-Time PCR system (Applied Biosystems). Cycle threshold values (Ct) were converted to PFU/mL using a quantitative RNA curve constructed with each point of the serial dilution of viral stock used to determine the PFU/mL titer.

Measurement of cytokines and NO

IL-1β cytokine quantification was performed by collecting the supernatant, and a sandwich ELISA was performed according to the manufacturer's instructions (Invitrogen). The absorbance of the plate was read at 450 nm on a 165 SpectraMax instrument. The detection of the cytokines TNF-α, IL-6, IL-27, IL-10, and IFN-β in the cell supernatant was performed using the Cytometry Beads Array (CBA) kit LEGENDplex Mouse Inflammation Panel (BioLegend). The assay was performed following the manufacturer's instructions. Samples were acquired with a FACSCanto II instrument (BD Bioscience) and analyzed using LEGENDplex software (BioLegend). NO production was assessed indirectly by measuring the nitrite concentration via the Griess reaction immediately after the end of each time point. Briefly, 50 µL of Griess reagent (1% sulfanilamide, 0.1% naphthalene diamine dihydrochloride (NEED), and 45% CH₃COOH) was added to 50 µL of each supernatant sample and to 50 µL of a serially diluted standard curve. The absorbance of the samples was read at 540 nm on a SpectraMax instrument.

Detection of caspase-1 (p10 and p20 domains)

Mature caspase-1 in mice was quantified by collecting the supernatant, and a sandwich ELISA was performed according to the manufacturer's instructions (AdipoGen Life Sciences). The absorbance of the plate was read at 450 nm on a 165 SpectraMax instrument.

Measurement of lactate and glutamate

To assess lactate release from ZIKV-infected cells, lactate levels in the supernatant were measured using an enzymatic lactate kit (Lab test). For this, 2 µL of sample and 100 µL of kit reagent were utilized. Subsequently, the plate was incubated in an oven for 5 minutes before being read on a spectrophotometer at 500 nm. For the glutamate assay, the commercial Amplex™ Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen) was used following the manufacturer's instructions.

Immunofluorescence

The cells were plated for 72 hours in a 96-well black plate (Greiner) with a clear bottom for microscopy. After infection, the supernatant was removed, and the cells were fixed with 3% paraformaldehyde (Sigma Aldrich) diluted in PBS for at least 30 min. Then, the wells were washed twice with warm PBS, followed by blocking/permeabilization buffer [10% BSA (Sigma Aldrich), 1% FBS (LGC), 0.5% Triton-X 100 (Sigma Aldrich), diluted in PBS] for 30 min at room temperature. The wells were carefully washed twice with warm PBS and incubated overnight at 4°C with 1:200 anti-GFAP (ab4674) and 1:750 anti-flavivirus (envelope protein 4G2) antibodies. The next day, the wells were washed again with warm PBS and incubated with Alexa Fluor 488 (green) (Abcam) or Alexa Fluor 647 (red) (Invitrogen) secondary antibodies (1:1000) for 1 h at room temperature. The wells were washed again and incubated with 5 µg/mL DAPI (blue) (Sigma Aldrich), and images were acquired on an IN Cell 200 Analyzer 2200 instrument.

Real-Time Metabolic Assays

An XFe24 extracellular flux analyzer (Agilent) was used to determine the bioenergetic profile of the astrocytes. A total of 3x10^{^4} cells were seeded in XFe24 plates and maintained for 18 hours at 37°C and 5% CO2. The following day, the cells were treated with or without LPS for 3 hours and infected with ZIKV as described above for 72 hours. Glycolytic stress tests (ECARs) and mitochondrial stress tests (OCRs) were performed on an XFe96 Bioanalyzer 72 hours after infection. One hour prior to the assay, the cell medium was replaced with Seahorse XF RPMI Medium (pH 7.4), and the cells were incubated at 37°C without CO₂. For ECAR measurements, Seahorse XF RPMI medium supplemented with glutamine (2 mM) was used, and all parameters were determined by subsequent injections of glucose (10 mM), oligomycin (2.5 µM), and 2-DG (50 mM). For OCR measurements, Seahorse XF RPMI medium supplemented with glucose (10 mM), pyruvate (1 mM), and glutamine (2 mM) was used, and all parameters were determined by subsequent injections of oligomycin (3 µM), FCCP (1 µM), and rotenone (0.5 µM) + antimycin (5 µM). The results were normalized to total protein.

Cell lysis analysis

Astrocyte lysis was evaluated after cell treatments, as described above, using a lactate dehydrogenase (LDH) activity assay kit according to the manufacturer's instructions (Sigma‒Aldrich).

Statistical analysis

All the statistical analyses were performed using the computer software GraphPad Prism 9.3.0 from the company GraphPad Software Incorporation, following the most appropriate test for each dataset.

1 - Caspase-1/11 controls ZIKV replication in murine astrocytes

Primary astrocytes obtained from newborn (E0-E3) cortices, primed with or without LPS, were infected at different multiplicities of infection (MOIs) to determine the proper MOI for subsequent experiments. Astrocytes infected at an MOI of 1 had a greater viral load, as determined by the number of viral genome copies, than those infected at an MOI of 0.1 and an MOI of 10 (Fig. 1A). Based on these observations, subsequent experiments were performed using a MOI of 1. Moreover, at 72 h post infection, astrocytes presented a robust increase in viral load (Fig. 1B). The presence of the envelope viral protein was demonstrated by immunofluorescence with an anti-pan flavivirus 4G2 antibody, confirming that astrocytes are permissive to ZIKV (Fig. 1C).

We then evaluated the inflammatory response induced by ZIKV infection in astrocytes and detected marked secretion of TNF-α and IL-6 but not of IL-27 or IL-10 (Supplemental Fig. 1), demonstrating that ZIKV infection in astrocytes triggers the induction of a specific set of cytokines. Additionally, an increase in the secretion of mature caspase-1 was observed upon ZIKV infection, which was potentiated after exposure to nigericin, a classical NLRP3 agonist (Fig. 1D). Notably, caspase-1/11^−/− astrocytes primed with or without LPS exhibited a significantly greater viral load (Fig. 1E and Supplemental Fig. 2A), followed by an increase in the presence of intracellular virus compared to their wild-type (WT) counterparts (Fig. 1F), underscoring the role of caspase-1/11 in controlling virus replication in astrocytes.

2 - ZIKV does not elicit a canonical inflammasome response in murine astrocytes

To investigate the mechanism by which caspase-1/11 controls ZIKV infection in astrocytes, we first assessed the secretion of IL-1β and the induction of pyroptosis, which are canonical responses mediated by this protease (13). ZIKV failed to induce IL-1β secretion (Fig. 2A) or lactate dehydrogenase (LDH) release (Fig. 2B), a signal of lytic cell death, in astrocytes. This finding contrasts with the robust response observed with nigericin, which served as a positive control for inflammasome activation. Furthermore, unlike treatment with recombinant IFN-g, ZIKV did not elicit nitric oxide (NO) release by astrocytes (Fig. 2C).

Next, we investigated the impact of caspase-1/11 on the induction of IFN-β, a pivotal mediator for controlling viral infections (23). As described for monocytes (16, 18), astrocytes lacking caspase-1/11 exhibited increased secretion of IFN-β in response to ZIKV compared to their WT counterparts (Fig. 2D). Notably, compared with WT astrocytes, caspase-1/11^−/− astrocytes secreted more TNF-α (Fig. 2E), while no differences were observed in the secretion of IL-6 (Fig. 2F), IL-27 (Fig. 2G) or IL-10 (Fig. 2H). These findings suggest that the susceptibility observed in caspase-1/11^−/− astrocytes may not be directly linked to classical inflammasome-related effector responses or antiviral cytokine secretion.

3 - Caspase-1/11 regulate glycolytic metabolism in astrocytes

Given that cellular metabolism profoundly influences immune responses and the capacity to control infections (24–30), our subsequent investigation focused on elucidating the potential role of caspase-1/11 in the metabolic reprogramming of astrocytes during ZIKV infection. To this end, we initially quantified the secretion of lactate and glutamate, products of the glycolytic pathway, and an amino acid produced from glutamine conversion, respectively (31). ZIKV infection did not alter lactate (Fig. 3A) or glutamate (Fig. 3B) production by astrocytes. Remarkably, both infected and noninfected caspase-1/11^−/− astrocytes displayed increased levels of lactate (Fig. 3A) but not glutamate (Fig. 3B) compared to those in WT cells, suggesting the influence of caspase-1/11 on the glycolytic pathway in astrocytes.

In fact, extracellular acidification rate (ECAR) analysis confirmed that astrocytes deficient in caspase-1/11 had increased levels of glycolysis (Fig. 3C and 3D), increased glycolytic reserve (Fig. 3C and 3E), and increased glycolytic capacity (Fig. 3C and 3F) under basal conditions compared to their wild-type counterparts, even in the absence of LPS priming (Supplemental Fig. 2B-E). Additionally, all these parameters remained elevated in astrocytes lacking caspase-1/11, despite ZIKV infection (Fig. 3C-F).

4 – Glycolytic metabolism is critical for ZIKV replication by astrocytes

To understand how caspase-1/11 regulate glycolysis in astrocytes, we initially examined the expression of glycolysis-related genes (Fig. 4A). We observed elevated expression of hexokinase-1 (HK-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH), and hypoxia-inducible factor 1-alpha (HIF-1α) in caspase-1/11^−/− astrocytes compared to that in WT astrocytes, both in the absence (Fig. 4B) and presence of ZIKV (Fig. 4C). Among these factors, HK-1, the first enzyme in the glycolytic pathway, was particularly upregulated in the absence of caspase-1/11.

To investigate the importance of glycolysis for the permissiveness of caspase-1/11^−/− astrocytes to ZIKV infection, we used 2-deoxi-D-glucose (2-DG) to pharmacologically inhibit glycolytic flux. The inhibition of glycolytic flux dramatically reduced viral replication in caspase-1/11^−/− astrocytes and mimicked the outcome observed in WT astrocytes (Fig. 4D and 4E). These findings suggest that enhanced glycolytic flux due to caspase-1/11 deficiency reduces the ability of astrocytes to control ZIKV infection.

To further explore how caspase-1/11 modulates glycolytic flux, we performed an interactome analysis of caspase-1 and HK-1 proteins using the STRING platform, which indicated an association and revealed two distinct clusters (Fig. 4F). A possible link between these clusters was identified as the metabolic sensor mammalian target of rapamycin (mTOR), a crucial regulator of metabolism and mammalian physiology (32). Consistent with this finding, mTORC inhibition with rapamycin mimicked the effect of 2-DG, resulting in the suppression of viral replication in caspase-1/11^−/− astrocytes (Fig. 4D and 4E). This finding underscores the connection between glycolytic flux and mTORC in the caspase-1/11-mediated control of ZIKV by astrocytes.

5 - The impact of caspase-1/11 on ZIKV control by astrocytes is contingent on pyruvate, with no observable effect on lactate conversion

The glycolytic pathway generates pyruvate, which can be converted to lactate by LDH or can enter the mitochondria to participate in the tricarboxylic acid (TCA) cycle (33). To gain a comprehensive understanding of how caspase-1/11-mediated glycolysis regulation affects ZIKV replication in astrocytes, the conversion of pyruvate to lactate was inhibited by the addition of oxamate, and the mitochondrial pyruvate carrier was blocked with UK-5099 (Fig. 5A). Our results revealed that treatment with UK-5099 but not oxamate (Fig. 5B and 5C) effectively reduced viral replication, indicating the necessity of pyruvate transport to mitochondria for ZIKV replication in astrocytes.

To confirm the role of pyruvate in this pathway, WT and caspase-1/11^−/− astrocytes were incubated with exogenous pyruvate, which increased viral replication in WT cells (Fig. 5D), whereas no significant effect was observed in caspase-1/11^−/− astrocytes (Fig. 5E). These results underscore the role of pyruvate in facilitating ZIKV replication in astrocytes and implicate caspase-1/11 in the regulation of this process.

ZIKV, a neurotropic virus, poses significant threats to both the adult and developing brain. While initial investigations predominantly focused on ZIKV's effects on neural progenitors and stem cells, recent evidence points to the virus's preference for glial cells (5, 8, 34–36). The ability of astrocytes to fight ZIKV infection (8, 9, 37) and the role of inflammasome activation in astrocytes during viral infections remain poorly understood (20, 21).

Inflammasome activation is pivotal for infection control, yet excessive responses, particularly within the CNS, can contribute to severe neurological disorders (38). Previous studies have associated inflammasome activation in astrocytes with various neurodegenerative and neuropsychiatric conditions (38, 39). However, reports also highlight the protective role of inflammasomes in the CNS, such as in normal brain neurodevelopment (40) and in preventing experimental autoimmune encephalomyelitis (EAE) (41–43). Although the specific molecular pathways driving caspase-1/11 activation by ZIKV in astrocytes await further elucidation, our findings reinforce the cited protective effects, underscoring the pivotal involvement of caspase-1/11 in regulating virus replication within astrocytes. Notably, this regulatory role persists even in the absence of typical indicators of inflammasome activation, such as IL-1β secretion and pyroptosis, as evidenced in previous studies during EAE (41).

The absence of caspase-1/11 increased IFN-β secretion in response to ZIKV, as previously demonstrated (16, 18). However, the increase in IFN-β was insufficient to rescue ZIKV replication in caspase-1/11^−/− astrocytes. Additionally, caspase-1/11-deficient astrocytes displayed elevated glycolysis and glycolytic reserve, even without ZIKV infection. Pharmacological inhibition of the glycolytic pathway reversed the susceptibility of caspase-1/11^−/− astrocytes, highlighting the role of caspase-1/11 in astrocytic control of ZIKV infection through metabolic regulation rather than cytokine secretion.

Like proliferating cells, viruses require sufficient nutrients for their metabolic needs during replication, often by manipulating host cell metabolism to facilitate this process (25, 28–30, 44–46). Metabolic reprogramming by ZIKV is correlated with impaired fetal development and microcephaly (24, 26, 44, 46). Our demonstration that glycolysis inhibition with 2-DG reversed susceptibility in caspase-1/11-deficient astrocytes aligns with broader observations that inhibiting glycolysis is crucial for controlling viral infections, including SARS-CoV-2 infection (30, 47), underscoring the importance of caspase-1/11 in regulating this metabolic pathway.

Although the involvement of metabolic pathways in inflammasome activation is well documented, particularly in inflammatory cells such as M1-like proinflammatory macrophages (M1) (48, 49), the influence of inflammasomes on the metabolic regulation of CNS cells remains largely unexplored. Our protein association network analysis identified mTORC1 as a potential link between HK-1 and caspase-1. Inhibiting mTOR with rapamycin effectively reversed the susceptibility of caspase-1/11-deficient astrocytes to rapamycin, which mirrored the effects of HK-1 inhibition. These findings underscore the potential involvement of caspase-1 in orchestrating the regulation of the mTOR-glycolysis axis, although the precise molecular mechanism awaits further elucidation.

Intriguingly, our exploration of pyruvate generated by the glycolytic pathway revealed that mitochondrial pyruvate transport is crucial for controlling ZIKV infection in caspase-1/11^−/− astrocytes. Its inhibition successfully reversed the susceptibility observed in the absence of caspase-1/11, while the inhibition of the conversion of pyruvate to lactate had no effect on ZIKV replication in astrocytes. These findings, distinct from those observed in SARS-CoV-2 infection in human monocytes (30), emphasize a specific requirement for ZIKV replication in astrocytes. In addition, pyruvate supplementation increased viral replication in WT astrocytes but had no significant effect on caspase-1/11^−/− cells, highlighting the requirement of pyruvate for facilitating ZIKV replication in astrocytes and the role of caspase-1/11 in the regulation of this process.

In summary, our findings present compelling evidence of the role of caspase-1/11 in regulating astrocyte glycolytic metabolism. The intricate interplay between caspase-1/11, mTOR, glycolysis, and pyruvate yields a beneficial effect, effectively hindering ZIKV replication. These findings identify potential targets for anti-ZIKV therapeutic development, contributing to our understanding of inflammasome interactions in CNS cells during infections.

Collectively, our findings elucidate a pivotal role for caspase-1/11 in regulating astrocytic glycolytic metabolism, thereby influencing their autonomous capacity to control ZIKV infection.

DATA AVAILABILITY

All data in this study are included in this published article and its additional files.

2-DG

2-deoxi-D-glucose

ANOVA

Analysis of variance

ASC

Apoptosis-associated speck

BBB

Blood brain barrier

BSA

Bovine serum albumin

CBA

Cytometry Beads Array

cGAS

Cyclic GMP-AMP synthase

CNS

Central nervous system

DAPI

4′,6-Diamidino-2-phenylindole

EAE

Experimental autoimmune encephalomyelitis

ECAR

Extracellular acidification rate

ELISA

Enzyme-linked immunosorbent assay

FBS

Fetal bovine serum

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

GFAP

Glial fibrillary acidic protein

GSDMD

Gasdermin-D

HIF-1α

hypoxia-inducible factor 1-alpha

HK-1

Hexokinase-1

IFN-β

Interferon beta

IFN-I

Type I interferon

IL-10

Interleukin 10

IL-18

Interleukin 18

IL-1β

Interleukin 1beta

IL-6

Interleukin 6

LDH

Lactate dehydrogenase

LPS

Lipopolysaccharide

LRR

Leucine-rich repeats

MOI

Multiplicities of infection

mRNA

Messenger RNA

mTOR

Mammalian target of rapamycin

NLR

nucleotide-binding oligomerization domain leucine-rich repeats-containing receptors

NLRP3

NACHT-LRR and pyrin (PYD) domain-containing protein 3

Nitric Oxide

PBS

Phosphate-buffered saline

PCR

Polymerase chain reaction

PFA

Paraformaldehyde

PFU

Plaque forming unit

PRR

Pattern recognition receptors

PYHIN

Pyrin and HIN domain-containing protein

RIG-I

Retinoic acid-inducible gene-I

RLR

Retinoic acid-inducible gene-I-like receptors

SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2

TCA

Tricarboxylic acid

TLR

Toll-like receptors

TNF-α

Tumor necrosis factor alpha

TNF-α

Tumor necrosis factor-alpha

Wildtype

ZIKV

Zika virus

ETHICS STATEMENT

All experimental procedures involving mice were carried out in accordance to the Brazilian National Law (11.794/2008), the Guide for the Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimentation (CONCEA) and the ARRIVE guidelines. The animal experimentation research was also approved by the Institutional Animal Care and Use Committees (IACUC) from the Federal University of São Paulo (UNIFESP) under protocol number: 8178110121.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

FUNDING

This research was supported by São Paulo Research Foundation [FAPESP, grant numbers: 2018/19411-4; 2020/13493-9; 2021/03371-6; 2023/16013-6]; the Brazilian National Research Council (CNPq); the Higher Education Improvement Coordination [CAPES, Finance code 001] and the National Institute of Science and Technology in Vaccines (INCTV). ISF received fellowships from FAPESP.

Author Contribution

ISF, JCAF, and KRB: conception and design of the work; ISF, GR, and IN: data acquisition; ISF, GR, IN, JPSP, PMMV, JCAF, and KRB: Analysis and interpretation of data; ISF, GR, IN, JPSP, PMMV, JCAF, and KRB: validation; ISF, and KRB: writing - original draft; ISF, GR, IN, JPSP, PMMV, JCAF, and KRB: writing –review & editing; JPSP, PMMV, and KRB: Resources; KRB: Project supervision.

Acknowledgement

We thank Dr. Ricardo Weinlich and Dr. Rafaela da Rosa Ribeiro for providing the Zika virus for this work and Elisabeth de Souza, for the technical assistance in the laboratory. Cartoons in Figures 4A and 5A were created with BioRender.com.

Li C, Wang Q, Jiang Y, Ye Q, Xu D, Gao F et al. Disruption of glial cell development by Zika virus contributes to severe microcephalic newborn mice. Cell Discov. 2018;4(1).
Olagnier D, Muscolini M, Coyne CB, Diamond MS, Hiscott J. Mech Zika Virus Infect Neuropathogenesis. 2016;35(8):367–72.
Komarasamy TV, Adnan NAA, James W, Balasubramaniam VRMT. Zika Virus Neuropathogenesis: The Different Brain Cells, Host Factors and Mechanisms Involved. Front Immunol. 2022;13:773191.
Miner JJ, Diamond MS. Zika Virus Pathogenesis and Tissue Tropism. 21, Cell Host Microbe. 2017.
Retallack H, Di Lullo E, Arias C, Knopp KA, Laurie MT, Sandoval-Espinosa C et al. Zika virus cell tropism in the developing human brain and inhibition by azithromycin. Proceedings of the National Academy of Sciences. 2016;113(50):14408–13.
Chen Z, Yuan Z, Yang S, Zhu Y, Xue M, Zhang J, et al. Brain Energy Metabolism: Astrocytes in Neurodegenerative Diseases. CNS Neurosci Ther. 2023;29(1):24–36.
Beard E, Lengacher S, Dias S, Magistretti PJ, Finsterwald C. Astrocytes as Key Regulators of Brain Energy Metabolism: New Therapeutic Perspectives. Front Physiol. 2021;12:825816.
Jorgačevski J, Korva M, Potokar M, Lisjak M, Avšič-Županc T, Zorec R. ZIKV Strains Differentially Affect Survival of Human Fetal Astrocytes versus Neurons and Traffic of ZIKV-Laden Endocytotic Compartments. Sci Rep. 2019;9(1):8069.
Limonta D, Jovel J, Kumar A, Airo A, Hou S, Saito L, et al. Human Fetal Astrocytes Infected with Zika Virus Exhibit Delayed Apoptosis and Resistance to Interferon: Implications for Persistence. Viruses. 2018;10(11):646.
Thompson MR, Kaminski JJ, Kurt-Jones EA, Fitzgerald KA. Pattern Recognition Receptors and the Innate Immune Response to Viral Infection. Viruses. 2011;3(6):920–40.
Tan X, Sun L, Chen J, Chen ZJ. Detection of Microbial Infections Through Innate Immune Sensing of Nucleic Acids. Annu Rev Microbiol. 2018;72(1):447–78.
Pradeu T, Thomma BPHJ, Girardin SE, Lemaitre B. The conceptual foundations of innate immunity: Taking stock 30 years later. Immunity. 2024;57(4):613–31.
Broz P, Dixit VM. Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol. 2016;16(7):407–20.
Barnett KC, Li S, Liang K, Ting JPY. A 360° view of the inflammasome: Mechanisms of activation, cell death, and diseases. Cell. 2023;186(11):2288–312.
He Z, Chen J, Zhu X, An S, Dong X, Yu J, et al. NLRP3 Inflammasome Activation Mediates Zika Virus–Associated Inflammation. J Infect Dis. 2018;217(12):1942–51.
Zheng Y, Liu Q, Wu Y, Ma L, Zhang Z, Liu T, et al. Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS 1-caspase‐1 axis. EMBO J. 2018;37:18.
Wang W, Li G, De W, Luo Z, Pan P, Tian M et al. Zika virus infection induces host inflammatory responses by facilitating NLRP3 inflammasome assembly and interleukin-1β secretion. Nat Commun. 2018;9(1).
Khaiboullina SF, Uppal T, Sarkar R, Gorzalski A, St Jeor S, Verma SC. ZIKV infection regulates inflammasomes pathway for replication in monocytes. Sci Rep. 2017;7(1).
Wen C, Yu Y, Gao C, Qi X, Cardona CJ, Xing Z. Concomitant pyroptotic and apoptotic cell death triggered in macrophages infected by Zika virus. PLoS ONE. 2022;17(4 April).
Gim E, Shim DW, Hwang I, Shin OS, Yu JW. Zika virus impairs host NLRP3-mediated inflammasome activation in an NS3-dependent manner. Immune Netw. 2019;19(6).
Tricarico PM, Caracciolo I, Crovella S, D’Agaro P. Zika virus induces inflammasome activation in the glial cell line U87-MG. Biochem Biophys Res Commun. 2017;492(4):597–602.
Pacheco A, de OL, Amaral MP, de Farias IS, Bottino LZMF, Bortoluci KR. Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection. J Visualized Experiments. 2020;(157).
Mesev EV, LeDesma RA, Ploss A. Decoding type I and III interferon signalling during viral infection. 4, Nat Microbiol. 2019.
Pang H, Jiang Y, Li J, Wang Y, Nie M, Xiao N, et al. Aberrant NAD + metabolism underlies Zika virus–induced microcephaly. Nat Metab. 2021;3(8):1109–24.
Thai M, Graham NA, Braas D, Nehil M, Komisopoulou E, Kurdistani SK, et al. Adenovirus E4ORF1-Induced MYC Activation Promotes Host Cell Anabolic Glucose Metabolism and Virus Replication. Cell Metab. 2014;19(4):694–701.
Yau C, Low JZH, Gan ES, Kwek S, Sen, Cui L, Tan HC, et al. Dysregulated metabolism underpins Zika-virus-infection-associated impairment in fetal development. Cell Rep. 2021;37(11):110118.
González Plaza JJ, Hulak N, Kausova G, Zhumadilov Z, Akilzhanova A. Role of metabolism during viral infections, and crosstalk with the innate immune system. Intractable Rare Dis Res. 2016;5(2):90–6.
de Oliveira LG, de Souza Angelo Y, Yamamoto P, Carregari VC, Crunfli F, Reis-de‐Oliveira G, et al. <scp > SARS‐CoV ‐2 infection impacts carbon metabolism and depends on glutamine for replication in Syrian hamster astrocytes</scp >. J Neurochem. 2022;163(2):113–32.
Thaker SK, Chapa T, Garcia G, Gong D, Schmid EW, Arumugaswami V, et al. Differential Metabolic Reprogramming by Zika Virus Promotes Cell Death in Human versus Mosquito Cells. Cell Metab. 2019;29(5):1206–e12164.
Codo AC, Davanzo GG, Monteiro L, de B GF, Muraro SP, Virgilio-da-Silva JV, et al. Elevated Glucose Levels Favor SARS-CoV-2 Infection and Monocyte Response through a HIF-1α/Glycolysis-Dependent Axis. Cell Metab. 2020;32(3):437–e4465.
Yoo HC, Yu YC, Sung Y, Han JM. Glutamine reliance in cell metabolism. Exp Mol Med. 2020;52(9):1496–516.
Panwar V, Singh A, Bhatt M, Tonk RK, Azizov S, Raza AS, et al. Multifaceted role of mTOR (mammalian target of rapamycin) signaling pathway in human health and disease. Signal Transduct Target Ther. 2023;8(1):375.
Gray LR, Tompkins SC, Taylor EB. Regulation of pyruvate metabolism and human disease. Cell Mol Life Sci. 2014;71(14):2577–604.
Miner JJ, Diamond MS. Understanding How Zika Virus Enters and Infects Neural Target Cells. Cell Stem Cell. 2016;18(5):559–60.
van den Pol AN, Mao G, Yang Y, Ornaghi S, Davis JN. Zika Virus Targeting in the Developing Brain. J Neurosci. 2017;37(8):2161–75.
Simonin Y, Loustalot F, Desmetz C, Foulongne V, Constant O, Fournier-Wirth C, et al. Zika Virus Strains Potentially Display Different Infectious Profiles in Human Neural Cells. EBioMedicine. 2016;12:161–9.
Stefanik M, Formanova P, Bily T, Vancova M, Eyer L, Palus M, et al. Characterisation of Zika virus infection in primary human astrocytes. BMC Neurosci. 2018;19(1):5.
Ravichandran KA, Heneka MT. Inflammasomes in neurological disorders — mechanisms and therapeutic potential. Nat Rev Neurol. 2024;20(2):67–83.
Mata-Martínez E, Díaz-Muñoz M, Vázquez-Cuevas FG. Glial Cells and Brain Diseases: Inflammasomes as Relevant Pathological Entities. Front Cell Neurosci. 2022;16.
Lammert CR, Frost EL, Bellinger CE, Bolte AC, McKee CA, Hurt ME, et al. AIM2 inflammasome surveillance of DNA damage shapes neurodevelopment. Nature. 2020;580(7805):647–52.
Barclay WE, Aggarwal N, Deerhake ME, Inoue M, Nonaka T, Nozaki K et al. The AIM2 inflammasome is activated in astrocytes during the late phase of EAE. JCI Insight. 2022;7(8).
Ma C, Li S, Hu Y, Ma Y, Wu Y, Wu C et al. AIM2 controls microglial inflammation to prevent experimental autoimmune encephalomyelitis. J Exp Med. 2021;218(5).
Chou WC, Guo Z, Guo H, Chen L, Zhang G, Liang K, et al. AIM2 in regulatory T cells restrains autoimmune diseases. Nature. 2021;591(7849):300–5.
Chen Q, Gouilly J, Ferrat YJ, Espino A, Glaziou Q, Cartron G, et al. Metabolic reprogramming by Zika virus provokes inflammation in human placenta. Nat Commun. 2020;11(1):2967.
Munger J, Bennett BD, Parikh A, Feng XJ, McArdle J, Rabitz HA, et al. Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy. Nat Biotechnol. 2008;26(10):1179–86.
Giovannoni F, Bosch I, Polonio CM, Torti MF, Wheeler MA, Li Z, et al. AHR is a Zika virus host factor and a candidate target for antiviral therapy. Nat Neurosci. 2020;23(8):939–51.
Bhatt AN, Kumar A, Rai Y, Kumari N, Vedagiri D, Harshan KH, et al. Glycolytic inhibitor 2-deoxy-d-glucose attenuates SARS-CoV-2 multiplication in host cells and weakens the infective potential of progeny virions. Life Sci. 2022;295:120411.
Olona A, Leishman S, Anand PK. The NLRP3 inflammasome: regulation by metabolic signals. Trends Immunol. 2022;43(12):978–89.
Hughes MM, O’Neill LAJ. Metabolic regulation of < scp > NLRP 3. Immunol Rev. 2018;281(1):88–98.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Caspase-1/11 Controls Zika Virus Replication in Astrocytes by Regulating Glycolytic Metabolism

Status:

Version 1

Abstract

Figures

BACKGROUND

MATERIALS AND METHODS

Animals

Primary cell culture of astrocytes

Virus

Treatment with agonists and inhibitors

Glial cell infection

Quantitative real-time PCR

Measurement of cytokines and NO

Detection of caspase-1 (p10 and p20 domains)

Measurement of lactate and glutamate

Immunofluorescence

Real-Time Metabolic Assays

Cell lysis analysis

Statistical analysis

RESULTS

1 - Caspase-1/11 controls ZIKV replication in murine astrocytes

2 - ZIKV does not elicit a canonical inflammasome response in murine astrocytes

3 - Caspase-1/11 regulate glycolytic metabolism in astrocytes

4 – Glycolytic metabolism is critical for ZIKV replication by astrocytes

DISCUSSION

CONCLUSION

DATA AVAILABILITY

Abbreviations

Declarations

ETHICS STATEMENT

CONFLICTS OF INTEREST

FUNDING

Author Contribution

Acknowledgement

References

Additional Declarations

Status:

Version 1