Interleukin-17A signaling promotes CD8+ T cell cytotoxicity against West Nile virus infection through enhancing PI3K-mTOR-mediated metabolism

Farzana Nazneen; Biswas Neupane; Yao Chen; Shazeed-Ul Karim; Zongbing You; Weiguo Cui; Fengwei Bai

doi:10.1371/journal.ppat.1013218

Abstract

West Nile Virus (WNV), a mosquito-borne neurotropic flavivirus, is a major cause of viral encephalitis in the United States, posing a continuous threat to public health. Unfortunately, no vaccine or specific therapeutic intervention is available against WNV infection. Previous studies, including ours, demonstrated that interleukin-17A (IL-17A) signaling promotes the cytotoxicity of CD8⁺ T cells to facilitate WNV and parasite clearance; however, the molecular mechanism is not understood. IL-17 receptor C (IL-17RC) is an obligatory co-receptor with IL-17 receptor A (IL-17RA) for signaling induced by IL-17A, IL-17A/F, and IL-17F. In this study, we found that IL-17RC deficient (Il17rc^-/-) mice were more susceptible to WNV infection with a higher viral load in the brain than wild-type (WT) control mice. The number of infiltrating WNV-specific CD8⁺ T cells and the expression levels of cytotoxicity mediators, such as perforin, in the T cells in the brain of Il17rc^-/- mice were reduced. In addition, WNV-specific CD8⁺ T cells from IL-17RA deficient (Il17ra^-/-) mice and CD8⁺ cell-specific Il17ra conditional knockout (cre-KO) mice expressed lower levels of perforin than their counterpart controls. Moreover, supplementing mouse recombinant IL-17A ex vivo increased the perforin production in WNV-specific CD8⁺ T cells from the WT mice but not Il17rc^-/- or cre-KO mice. Interestingly, we found that IL-17A signaling activated the phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K-mTOR) signaling pathway in CD8⁺ T cells, leading to increased metabolism of CD8⁺ T cells to cope with the higher energy demand for WNV clearance in the brain. In summary, our findings reveal a novel IL-17A-PI3K-mTOR signaling axis in promoting the effector functions of CD8⁺ T cells, suggesting potential broader implications in stimulating immune responses to combat WNV and other intracellular infections.

Author summary

West Nile encephalitis is the leading viral neurological disease in the United States and North America. No approved vaccine or specific therapeutics are available to prevent or treat human West Nile virus (WNV) infection. Interleukin 17A (IL-17A) is a pleiotropic cytokine that regulates inflammatory diseases and cancers. One of our previous studies has demonstrated that IL-17A signaling promotes the cytotoxicity of CD8⁺ T cells and facilitates WNV clearance from the brain in the mouse model, as well as the therapeutic potential of this cytokine. However, the molecular mechanism is not understood. This study uncovered a new role of IL-17A signaling in activating a metabolic pathway in CD8⁺ T cells to cope with the higher energy demand for WNV clearance from the brain. This finding may have broader therapeutic implications in stimulating IL-17A-mediated metabolic pathways in CD8⁺ T cells to combat other viral and intracellular infections.

Citation: Nazneen F, Neupane B, Chen Y, Karim S-U, You Z, Cui W, et al. (2025) Interleukin-17A signaling promotes CD8⁺ T cell cytotoxicity against West Nile virus infection through enhancing PI3K-mTOR-mediated metabolism. PLoS Pathog 21(7): e1013218. https://doi.org/10.1371/journal.ppat.1013218

Editor: Alexander Ploss, Princeton University, UNITED STATES OF AMERICA

Received: October 23, 2024; Accepted: May 15, 2025; Published: July 9, 2025

Copyright: © 2025 Nazneen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: RNA-Seq data deposited into Dryad with the Reviewer URL: http://datadryad.org/stash/share/6SwKf1cOtKadBrKfBhwfAqQSJ-Urxngbyfc7iS-LdkE. A unique digital object identifier (DOI): https://doi.org/10.5061/dryad.2bvq83c14.

Funding: This work was partly supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health and the American Heart Association (1R15AI178654 and 23AIREA1051360 to FB). ZY was partially supported by a Lavin Bernick Grant and the Tulane University Office of Vice President for Research Bridge Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

West Nile virus (WNV) is a mosquito-transmitted, single-stranded, positive-sense RNA virus belonging to the Flaviviridae family [1]. Human WNV infection is typically subclinical, but in symptomatic individuals, the manifestations range from fever and myalgias to meningoencephalitis [2–5]. The WNV-associated neuroinvasive disease affects all ages; however, elderly and immunocompromised individuals are particularly at risk [6], with 4–24% mortality rates among the elderly with encephalitis [7]. WNV was initially identified in the West Nile province of Uganda in 1937 [8], and since it arrived in the United States in 1999, WNV has claimed the lives of over 2,900 people and sickened more than 60,600 individuals as of January 14, 2025, as reported by the Centers for Disease Control and Prevention (CDC) [9,10]. Furthermore, up to 50% of WNV survivors experience significant morbidity for at least a year following the infection [11]. WNV has become the primary viral agent responsible for human neurological diseases in North America [12]. Despite the intensive research of over 20 years, vaccines or specific treatments are still unavailable for human WNV infection, and treatment remains supportive [1].

After an infected mosquito bite, WNV replicates in various cells, including keratinocytes, neutrophils, and monocytes, and disseminates via the bloodstream into peripheral organs, such as the liver, kidney, and spleen [1,13]. Although WNV induces a lower level of viremia that usually clears up within a few days in humans, it may invade the central nervous system (CNS) in some individuals, causing various neuroinvasive complications [14]. While innate immune responses serve as the first line of host defense in controlling the viral replication in the peripheral cells and organs, the B and T cell-mediated adaptive immunity plays a key role in clearing the WNV infection in the CNS. Particularly, CD8⁺ T cells are essential in protecting from WNV infection, and they control viral infections by triggering apoptosis of infected cells through perforin- or Fas ligand-dependent pathways or producing antiviral cytokines (e.g., IFN-γ and TNF-α). Our previous study demonstrated that interleukin-17A (IL-17A) enhanced CD8⁺ T cell cytotoxicity, and a supplement of IL-17A as late as day 6 post-WNV infection significantly increased the survival of the infected mice [15], suggesting a therapeutic role of IL-17A against WNV infection in the CNS.

IL-17A is the founding member of IL-17 family that also includes IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F [15,16]. With 50% structural homology to IL-17F, IL-17A acts as both a homodimer and an IL-17A/IL-17F heterodimer [15]. IL-17A is produced primarily by T helper 17 (Th17) cells, cytotoxic T 17 cells (Tc17), γδT cells, natural killer T (NKT) cells, group 3 innate lymphoid (ILC3) cells, neutrophils, and mast cells [15,17,18]. The biological functions of IL-17A and IL-17F are mediated through binding to IL-17 receptor A (IL-17RA) and IL-17 receptor C (IL-17RC) complex [19]. While IL-17RA participates in the formation of the receptor complexes for other members of the IL-17 family, IL-17RC is specific to IL-17A signaling [20]. IL-17RA is widely expressed in the bone marrow, thymus, spleen, and hematopoietic cells, whereas IL-17RC is more abundant in epithelial cells of prostate, kidney, and joints, with lower levels in hematopoietic cells [21–23].

IL-17A is a pro-inflammatory cytokine with prominent roles in allergic and autoimmune diseases, such as multiple sclerosis [24–28], rheumatoid arthritis [29–33], psoriasis [33–36], asthma [31,37,38], Crohn’s disease [39,40], and cancers [41–43]. IL-17A signaling mediates protective immunity against fungi and bacteria by promoting neutrophil recruitment, inducing antimicrobial peptides, and enhancing barrier functions [19]. In terms of viral infections, IL-17A plays both protective and pathogenic roles. It promotes infections in influenza virus [44], respiratory syncytial virus [45], murine encephalitis virus [46], hepatitis virus [47], and chikungunya virus (CHIKV) [48]. IL-17A can also promote the adaptive immune responses to lymphocytic choriomeningitis virus infection [49]. Research from our lab indicates that IL-17A enhances the cytotoxicity of CD8⁺ T cells, thereby promoting WNV clearance from the brain in a mouse model [50]. Consistent with our findings, IL-17A signaling has also been observed to sustain CD8⁺ T cell immunity against Trypanosoma cruzi infection [51] to enhance CD8⁺ T cell effector function [52]. During other viral infections, IL-17A was found to be crucial for priming hepatic CD8⁺ T cells in adenovirus-infected mice [53] and partly responsible for the clustering and activation of CD8⁺ T cells in the spleen following vaccinia virus infection [54]. However, the detailed mechanism by which IL-17A signaling enhances the cytotoxicity of CD8⁺ T cells is not understood. In this study, we unveiled a novel function of IL-17A, which shows that it activates the PI3K-mTOR signaling pathway-mediated metabolism in CD8⁺ T cells to cope with a higher energy demand and protein synthesis of the cytotoxicity mediators, such as perforin, for WNV clearance in the brain.

Results

IL-17RC is essential to protect mice from WNV infection

It has been reported that IL-17A initiates its signaling via the IL-17RA/IL-17RC receptor complex [20]. While some other members of the IL-17 cytokine family use IL-17RA as one of their receptor subunits, IL-17RC is an obligate co-receptor with IL-17RA for signaling induced by IL-17A [20]. Therefore, we employed the Il17rc^-/-mice [55] to study the signaling of IL-17A in the pathogenesis of WNV. Eight to nine weeks old Il17rc^-/- and WT (C57BL/6J) mice were infected with 100 plaque-forming units (PFU) of WNV via the footpad (f.p.) route, which partially mimicked mosquito inoculation, and monitored the survival for 21 days. The infected animals showed a wide range of disease symptoms, such as ruffled fur, hunched back, increased body temperature, weight loss, some form of paralysis, mostly hind limb weakness, decreased movement, huddled together, and increased mortality [56]. The survival results showed a lower survival rate in Il17rc^-/- (28%) than in WT (66%) mice (Fig 1A), suggesting that the absence of the IL-17RC results in Il17rc^-/- mice being more susceptible to WNV infection. After WNV infection in mice, the viral burden in the blood and peripheral organs typically peaks between days 3 and 4 post-infection (p.i.). Subsequently, the virus enters the central nervous system (CNS) and clears from the peripheral organs after about a week [57–59]. To monitor the virological profiles in Il17rc^-/- mice, blood, liver, and spleen samples were collected on days (D) 2, 4, and 6 p.i., and the viral load was measured by quantifying the WNV envelope gene (WNV-E) by RT-qPCR. There was no apparent difference in the blood, liver, and spleen samples between Il17rc^-/- and WT mice (S1A-C Fig). However, there was a significantly higher viral load in the brain tissues of Il17rc^-/- mice collected on D7 p.i. than in the WT controls by both RT-qPCR (Fig 1B) and plaque-forming assay (Fig 1C). To visualize the impact of WNV infection in the brains of Il17rc^-/- mice, the expression of WNV-E antigen was detected using an immunofluorescence assay (IFA). A higher expression of WNV-E antigen was observed in Il17rc^-/- mice (Fig 1D). In addition, TUNEL assay results show that there are more apoptotic bodies in the brains of Il17rc^-/- mice (Fig 1E).

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Fig 1. Il17rc^-/^- mice are susceptible to WNV infection.

Eight to nine weeks old Il17rc^-/- (n = 21) and WT mice (n = 30) were infected with 100 PFU of WNV via f.p. inoculation and monitored daily for mortality for 21 days. (A) The survival curves of Il17rc^-/- and WT mice after WNV infection. The survival was compared with a Log-rank test with * denoting p <0.05. Brain tissue was collected on D7 p.i., and the viral load was measured by qRT-PCR (B, WT, n =12; Il17rc^-/-, n = 22), and plaque-forming assay (C, WT, n = 14; Il17rc^-/-, n = 13). Data were presented as mean ± s.e.m. and compared with two-tailed student’s t-tests with * denoting p <0.05. The data represent at least three independent experiments. (D) IFA images of WNV-E antigens in mouse brains. The WNV-E antigen and nuclei were stained with Alexa fluor 568 (red) and DAPI (blue). (E) TUNEL assay images of WNV-infected mouse brain. The TUNEL-positive cells and nuclei were stained with FITC (green) and DAPI (blue). Images in (D) and (E) were taken by Nikon A1R confocal microscope at 40x magnification (2.5x zoomed in, scale bar = 20 µm).

https://doi.org/10.1371/journal.ppat.1013218.g001

Next, we evaluated the expression of inflammatory and antiviral cytokines and chemokines in the blood and brain tissues collected from Il17rc^-/- mice on D2 and D7 p.i., respectively. The RT-qPCR results showed no change in the transcripts of IFN-α (Fig 2A), IL-1β (Fig 2D), and TNF-α (Fig 2H). But interestingly, there was significant down-regulation in the transcripts of IFN-β (Fig 2B), IFN-γ (Fig 2C), CXCL-10 (Fig 2F), and IL-6 (Fig 2G) in the brain of Il17rc^-/- mice compared to WT controls. On the contrary, the expression of CXCL-2 was increased in the brain of Il17rc^-/- mice compared to WT mice (Fig 2E). Compared to the brain samples, the changes of expression of these genes in the blood were mostly insignificant except for IL-1β, which was reduced in Il17rc^-/- mice (S2D Fig). Collectively, these findings suggest that Il17rc^-/- mice are more susceptible to WNV infection and IL-17A signaling via IL-17RC may play a more vital role in promoting the anti-viral function in brain than in the periphery to inhibit WNV infection.

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Fig 2. The antiviral and inflammatory responses in the brain of Il17rc^-/^- mice.

Eight to nine weeks old Il17rc^-/- (n = 8) and WT mice (n = 6) were infected with 100 PFU of WNV via f.p. inoculation, and brain tissues were collected on D7 p.i. and the expression of anti-viral and inflammatory cytokine genes were measured by RT-qPCR and normalized to mouse β-actin. The relative fold changes (RFC) of gene expressions of IFN-α (A), IFN-β (B), IFN-γ (C), IL-1β (D), CXCL-2 (E), CXCL-10 (F), IL-6 (G), and TNF-α (H). Data were obtained from two independent experiments and presented as mean ± s.e.m. and analyzed by two-tailed student’s t-tests with *, and ** denoting p <0.05, and p <0.01, respectively.

https://doi.org/10.1371/journal.ppat.1013218.g002

IL-17A-IL-17RC signaling promotes infiltration and cytotoxicity of CD8⁺ T cells in the brain

IL-17A signaling has been reported to facilitate the migration and activation of innate immune cells, such as neutrophils, during infections and autoimmune disorders [21,60]. Therefore, we asked if the absence of IL-17A signaling is linked to a deficiency of immune cell infiltration in responding to WNV infection in the brain. To address this question, Il17rc^-/- and WT mice were infected with WNV and sacrificed on D7 p.i. to collect the brain. Total leukocytes in the brain were isolated and characterized using flow cytometry. The results showed no change in the total infiltrating leukocytes in the brain of Il17rc^-/- mice compared to WT controls (Fig 3A). While no apparent changes were detected in the cell populations of residential microglia, infiltrating neutrophils, monocytes, or CD4⁺ T lymphocytes, a significant decrease in CD8⁺ T cell population was noted in Il17rc^-/- mice (Fig 3B-F), suggesting that IL-17A-IL-17RC signaling may promote the infiltration of CD8⁺ T cells into the brain during WNV infection. To exclude the possibility that IL-17RC deficiency might negatively affect the development of CD8⁺ T cells in mice, we quantified CD8⁺ T cells as well as neutrophils, monocytes, B cells, and CD4⁺ T cells in spleen samples collected from Il17rc^-/- and WT mice and analyzed by flow cytometry. The results showed no difference in any of these cell populations in the spleen and blood from Il17rc^-/- and WT mice (S3 Fig), further confirming that the decrease of CD8⁺ T cell infiltration in the brain of Il17rc^-/- mice are due to the deficiency of the signaling of IL-17A through IL-17RC.

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Fig 3. The cytotoxicity of infiltrated CD8⁺ T cells in the brain of Il17rc^-/^- mice is reduced.

Eight to nine weeks old Il17rc^-/- (n = 9) and WT mice (n = 4 to 10) were infected with 100 PFU of WNV via f.p. inoculation, and brain tissue was collected on D7 p.i. The brain leukocytes were characterized by flow cytometry after probing with antibodies against CD45, CD11b, Ly6G, CD3, CD4, CD8, and perforin. (A) The infiltrating leukocytes (CD45^hi) in the brain of Il17rc^-/- and WT mice; (B) microglia (CD45^lowCD11b⁺); (C) neutrophils (CD45^hiCD11b⁺Ly6G⁺); (D) monocytes (CD45^hiCD11b⁺); (E) CD4⁺ T cells (CD45^hiCD3⁺CD4⁺); (F) CD8⁺ T cells (CD45^hiCD3⁺CD8⁺); (G) percentages of perforin expression in CD8⁺ T cells (CD45^hiCD3⁺CD8⁺perforin⁺); (H) MFI of perforin expression in CD8⁺ T cells (CD45^hiCD3⁺CD8⁺perforin⁺); and (I) CD8⁺ T cells isolated from leukocytes infiltrated into brain of Il17rc^-/- (n = 10) and WT mice (n = 10) were co-cultured with target (MC57GL_WNV-E) or control (MC57GL_vector) cells at a 10:1 effector/target ratio for 8 h, and cytotoxicity was evaluated by quantifying by the release of intracellular lactate dehydrogenase in culture supernatants. (J) percentages of perforin expression in splenic CD8⁺ T cells (CD3⁺CD8⁺perforin⁺); (K) MFI of perforin expression in splenic CD8⁺ T cells (CD3⁺CD8⁺perforin⁺); All experiments were performed at least twice. Data were presented as mean ± s.e.m. and analyzed by Mann-Whitney U tests (A-H) or two-tailed student’s t-tests (I-K) with * and ** denoting p < 0.05 and p < 0.01, respectively.

https://doi.org/10.1371/journal.ppat.1013218.g003

Our previous study has demonstrated that IL-17A promotes cytotoxicity of CD8⁺ T cells in combating WNV infection in mice [50]; therefore, we examined the expression of cytotoxicity gene mediators, such as perforin and granzyme B, in CD8⁺ T cells from Il17rc^-/- mice by flow cytometry. The results indicated no changes in the cell percentages and the MFI of the granzyme B expressing CD8⁺ T cells in Il17rc^-/- mice compared to WT mice (S4 Fig). However, a significant decrease in the MFI, but not the cell percentages, of the expression of perforin (Fig 3G and 3H) in CD8⁺ T cells was observed in Il17rc^-/- mice, suggesting that CD8⁺ T cells from Il17rc^-/- mice may have defects in cytotoxic effector function. To confirm this phenotype in the brain, we isolated CD8⁺ T cells from the brain-infiltrated leukocytes from Il17rc^-/- and WT mice infected with WNV. These CD8⁺ T cells (effector cells) were co-cultured with either WNV-E ectodomain expressing MC57GL cells (target cells) or control cells containing the empty vectors for 8 hours (h). The cytotoxicity of the effector CD8⁺ T cells against WNV-E-expressing target cells was determined by measuring the level of released lactate dehydrogenase (LDH) in the cell culture medium. The results showed that the cytotoxicity of the CD8⁺ T cells from Il17rc^-/- mice was significantly lower compared to that of WT mice (Fig 3I), suggesting that the CD8⁺ T cells of Il17rc^-/- mice are less cytotoxic in WNV-infected cells in the brain. To test whether this phenotype is not specific to brain-infiltrated CD8⁺ T cells, we also quantified the cell percentages and MFI of the perforin expression in splenic CD8⁺ T cells from WNV-infected WT and Il17rc^-/- mice. The results indicate that both splenic and brain-infiltrated CD8⁺ T cells of Il17rc^-/- mice produce less perforin compared to their counterparts from WT mice (Fig 3J and 3K). Collectively, these results indicate that IL-17A signaling through its receptor, IL-17RC, promotes the infiltration and cytotoxicity of CD8⁺ T cells in the brain.

IL-17A-IL-17RA signaling increases the cytotoxicity of WNV-specific CD8⁺ T cells

In our previous study, we found that an ex vivo supplement of mouse rIL-17A augmented the gene expression of perforin and granzymes in the splenic CD8⁺ T cells isolated from WNV-infected mice deficient in IL-17A (Il17a^-/-) and WT mice [50]. In the current study, we further investigated if IL-17A signaling via its receptors promotes cytotoxicity of WNV-specific CD8⁺ T cells from WNV-infected IL-17RA deficient (Il17ra^-/-) mice. The mice were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. We used a lower dose to infect the animals so that the animals remained alive for more days, which allowed us to collect the spleen at D8 p.i. when the WNV-specific CD8⁺ T cells become mature [50]. Splenocytes were stimulated with WNV ex vivo, and WNV-specific CD8⁺ T cells were identified using WNV-NS4B tetramer [50]. Perforin and granzyme A production in WNV-specific CD8⁺ T cells (NS4B⁺CD8⁺) was quantified with flow cytometry. There was a significant increase in the percentage of NS4B⁺CD8⁺ T cells in Il17ra^-/- mice (Fig 4A); however, the production perforin was lower in the NS4B⁺CD8⁺ T cells from Il17ra^-/- mice than in the WT controls (Fig 4B). There was no change in the level of granzyme A (Fig 4C). To confirm the phenotype, we infected Il17ra conditional knockout mice, which lack Il17ra expression on CD8⁺ cells (cre-KO mice) and their WT littermates’ control (cre-WT) with WNV as above. The flow cytometric results showed no changes in the splenic NS4B⁺CD8⁺ T cell numbers (Fig 4D) or granzyme A expression levels (Fig 4F), while a significant decrease was noted in perforin expression levels in the NS4B⁺CD8⁺ T cells from the cre-KO mice compared to the cre-WT controls (Fig 4E). These results suggest that the IL-17A-IL-17RA signaling axis is essential to the cytotoxicity of WNV-specific CD8⁺ T cells. To determine if a supplement of rIL-17A could promote the cytotoxicity of WNV-specific CD8⁺ T cells, we treated the splenocytes collected from WNV-infected cre-WT and cre-KO mice ex vivo and measured the expression of perforin in these cells by flow cytometry. Results showed that rIL-17A treatment increased the MFI of perforin⁺NS4B⁺CD8⁺ T cells from the WNV-infected cre-WT mice but not the cells from WNV-infected cre-KO mice (Fig 4G and 4H). Collectively, these results suggest that IL-17A signaling via the IL-17A-IL-17RA signaling axis can increase the cytotoxicity of WNV-specific CD8⁺ T cells both in vivo and ex vivo.

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Fig 4. The effects of IL-17A-IL-17RA signaling on the cytotoxicity of WNV-specific CD8⁺ T cells.

(A-F) Eight to nine weeks old Il17ra^-/- (n = 4), WT (n = 4 to 11), cre-WT (n = 8), and cre-KO mice (n = 4) were infected with 20 PFU of WNV via f.p. inoculation. Spleens were collected on D8 p.i. Splenocytes were stimulated with 0.01 MOI of WNV in vitro for 24 h with the addition of Brefeldin-A solution for the last 6 h of stimulation. Splenocytes were then characterized by flow cytometry after probing with WNV NS4B-tetramer, and antibody against CD8 followed by re-probing intracellularly with antibodies against perforin, and granzyme A. (A) The percentages of NS4B⁺CD8⁺ T cells of Il17ra^-/- and WT mice; (B) the expression of perforin⁺NS4B⁺CD8⁺ T cells; and (C) granzyme A⁺NS4B⁺CD8⁺ T cells from Il17ra^-/-, and WT mice. (D) The percentages of NS4B⁺CD8⁺ T cells; (E) the expression of perforin⁺NS4B⁺CD8⁺ T cells; and (F) granzyme A⁺NS4B⁺CD8⁺ T cells from cre-KO, and cre-WT mice. (G-H) Eight to nine weeks old cre-WT (n = 7), and cre-KO mice (n = 4) were infected with 20 PFU of WNV via f.p. inoculation, and splenocytes were collected on D8 p.i. then treated with either rIL-17A (100 ng/ml) or RPMI (control) for 24 h and characterization by flow cytometry. (G) MFI of perforin⁺NS4B⁺CD8⁺ T cells in cre-WT mice. (H) MFI of perforin⁺NS4B⁺CD8⁺ T cells in cre-KO mice. Data were analyzed by two-tailed student’s t-tests (A-G), and Mann Whitney U tests (H) and presented as mean ± s.e.m. with *, and ** denoting p <0.05, and p <0.01, respectively.

https://doi.org/10.1371/journal.ppat.1013218.g004

IL-17A-IL-17RC signaling increases the cytotoxicity of WNV-specific CD8⁺ T cells

Both IL-17RA and IL-17RC receptors are required for initiating IL-17A signaling, while IL-17RC is the IL-17A-specific receptor. Hence, we investigated if IL-17A signaling via IL-17RC promotes cytotoxicity of WNV-specific CD8⁺ T cells utilizing Il17a^-/- and Il17rc^-/- mice, which are deficient in IL-17A ligand and the receptor C, respectively. Eight to nine weeks old Il17a^-/-, Il17rc^-/-, and WT mice were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. Splenocytes were stimulated with 0.01 MOI of WNV ex vivo, and WNV-specific CD8⁺ T cells were identified using WNV-NS4B tetramer [50]. Perforin and granzyme A production in WNV-specific CD8⁺ T cells (NS4B⁺CD8⁺) was quantified with flow cytometry. There was no significant change in the percentage of NS4B⁺CD8⁺ T cells (Fig 5A); however, the production of both perforin and granzyme A was dramatically reduced in the NS4B⁺CD8⁺ T cells from Il17a^-/- mice than in the WT controls (Fig 5B and 5C). Although Il17rc^-/- mice exhibited a higher percentage of NS4B⁺CD8⁺ T cells in the spleen (Fig 5D), the production of perforin was lower in these cells compared to WT controls (Fig 5E), and a similar trend was also detected for granzyme A, but it was not statistically significant (Fig 5F). To determine if a supplement of rIL-17A could also promote the cytotoxicity of WNV-specific CD8⁺ T cells, we treated the splenocytes collected from WNV-infected WT and Il17rc^-/- mice ex vivo and measured the expression of perforin in these cells by flow cytometry. Consistent with our previous findings, rIL-17A treatment enhanced the percentage of perforin⁺NS4B⁺CD8⁺ T cell populations from the WNV-infected WT mice at both 6 h and 24 h time points (Fig 5G and 5J). In addition, there was also an increase in the level of MFI in the WT NS4B⁺CD8⁺ T cell populations after rIL-17A treatment (Fig 5H, 5I, 5K, and 5L). In contrast, no change of perforin expression was detected in these cells isolated from WNV-infected Il17rc^-/- mice after rIL-17A treatment for 24 h (Fig 5M and 5N). These results suggest that the ex vivo treatment of IL-17A can increase the cytotoxicity of WNV-specific CD8⁺ T cells from WT mice but not from Il17rc^-/- mice, reinforcing the notion that IL-17A signals through IL-17RC to promote the cytotoxicity of CD8⁺ T cells during WNV infection.

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Fig 5. The effects of IL-17A signaling on the cytotoxicity of WNV-specific CD8⁺ T cells.

Eight to nine weeks old Il17a^-/- (n = 5), Il17rc^-/- (n = 3 to 8), and WT (n = 4 to 11) mice were infected with 20 PFU of WNV via f.p. inoculation. Spleens were collected on D8 p.i., and splenocytes were stimulated with 0.01 MOI of WNV in vitro for 24 h, with the addition of Brefeldin-A solution for the last 6 h of stimulation. Splenocytes were then characterized by flow cytometry after probing with WNV NS4B-tetramer and antibody against CD8 followed by re-probing intracellularly with antibodies against perforin and granzyme A. (A) The percentages of NS4B⁺CD8⁺ T cells of Il17a^-/- and WT mice; (B) the expression of perforin⁺; and (C) granzyme A⁺NS4B⁺CD8⁺ T cells from Il17a^-/- and WT mice. (D) The percentages of NS4B⁺CD8⁺ T cells; (E) the expression of perforin⁺; and (F) granzyme A⁺NS4B⁺CD8⁺ T cells from Il17rc^-/- and WT mice. Eight to nine weeks old WT (n = 8) and Il17rc^-/- mice (n = 4 to 6) were infected with 20 PFU of WNV via f.p. inoculation, and splenocytes were collected on D8 p.i. then treated with either rIL-17A (100 ng/ml) or RPMI (control) for 6 or 24 h, and characterization by flow cytometry. (G) Perforin⁺NS4B⁺CD8⁺ T cell percentages; (H-I) histograms and values of MFI of perforin expression in 6 h treatment in WT mice. (J) Perforin⁺NS4B⁺CD8⁺ T cell percentages; (K-L) histograms and values of MFI of perforin expression in 24 h treatment in WT mice. (M) Perforin⁺NS4B⁺CD8⁺ T cell percentages; and (N) MFI of perforin expression in 24 h treatment in Il17rc^-/- mice. Data were analyzed by Mann Whitney U tests (A-C, F, and M), and two-tailed student’s t-tests (D-E, G, I-J, L, and N) and presented as mean ± s.e.m. with *, and ** denoting p <0.05, and p <0.01, respectively. The data represent at least two independent experiments.

https://doi.org/10.1371/journal.ppat.1013218.g005

IL-17A signaling activates PI3K-mTOR signaling in CD8⁺ T cells

To understand the mechanism by which IL-17A signaling promotes cytotoxicity of CD8⁺ T cells, we analyzed the profiles of transcription in splenic CD8⁺ T cells collected from WNV-infected WT and Il17ra^-/- mice by RNA sequencing. IL-17RA is widely expressed in hematopoietic tissue, epithelial, endothelial, fibroblast cells, and lymphocytes, including CD8⁺ T cells [61]. The RNA sequencing data [62] showed that the PI3K-mTOR signaling may be activated by IL-17A signaling (Fig 6A). To verify if IL-17A signaling activates the PI3K-mTOR pathway, we treated CD8⁺ T cells isolated from splenocytes of WNV-infected Il17a^-/-, Il17rc^-/-, and WT mice with rIL-17A and measured the gene expression of PIK3ca, mTOR, and S6K1 by RT-qPCR. The results indicate that rIL-17A treatment increases the expression of PIK3ca, mTOR, and S6K1 in CD8⁺ T cells from WT mice but not from Il17rc^-/- mice (Fig 6B-D). In addition, treatment with rIL-17A also increased the expression of S6K1 in CD8⁺ T cells from Il17a^-/- mice, albeit the expression of PIK3ca and mTOR showed a slight trend of increase after the treatment, but the change was not statistically significant (Fig 6B-D). Immunoblots were conducted to confirm that IL-17A signaling can activate the PI3K-mTOR pathway at the protein level. The splenic CD8⁺ T cells isolated from WNV-infected WT mice were treated with rIL-17A and simultaneously co-treated with either PI3K inhibitor (LY294002) [63] or mTOR inhibitor (Rapamycin) [64], or RPMI (control) ex vivo. Data showed that rIL-17A treatment slightly increases the production of phosphorated AKT, p70S6K (pS6K), and 4EBP1 at 15- or 60-minute (min) time points (Fig 6E, 6G, and 6I-J). Moreover, the presence of the inhibitors of PI3K and mTOR decreases the phosphorylation of pS6K, and 4EBP1, but not PI3K and mTOR (Fig 6E-J).

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Fig 6. IL-17A enhances PI3K-mTOR signaling in CD8⁺ T cells.

(A) Eight weeks old Il17ra^-/-, and WT (C57BL/6J) mice (n = 3) were infected with 100 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i., followed by purification of CD8⁺ T cells. The transcription profile was analyzed by RNA sequencing and the representative data were shown as a heatmap. (B-D) Eight to nine weeks old Il17a^-/- (n = 5), Il17rc^-/- (n = 4), and WT (n = 8) mice were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. CD8⁺ T cells were isolated from the splenocytes, cultured ex vivo with mouse rIL-17A (50 ng/ml) or RPMI (control) for 24 h and measured the gene expression of PIK3ca (B), mTOR (C), and S6K1 (D) by RT-qPCR, expressed as the relative fold changes (RFC). Data were analyzed by two-tailed student’s t-tests (B-D) and presented as mean ± s.e.m. with *, and ** denoting p <0.05, and p <0.01, respectively. (E-J) Eight to nine weeks-old WT mice were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. CD8⁺ T cells were isolated from the splenocytes, stimulated ex vivo with mouse rIL-17A (100 ng/ml) or RPMI (control), and co-treated with either rapamycin (20 nM), LY294002 (12.5 µM) for 15- or 60- minutes. Cell lysates were resolved with SDS-PAGE. The protein expressions of PI3K, p-PI3K^Tyr458, AKT, p-AKT^Thr308, mTOR, p-mTOR^Ser2481, pS6K, p-pS6K^Thr389, 4EBP1, and p-4EBP1^Thr37/46 were analyzed by immunoblot. GAPDH was used as the loading control. (E) The protein bands of PI3K, p-PI3K^Tyr458, AKT, p-AKT^Thr308, mTOR, and p-mTOR^Ser2481 were from the 15-min treatment, and the remaining bands were from the 60-min treatment. (F-J) Densitometric quantification of the phosphorylated protein normalized to corresponding total protein and analyzed by Image Lab software.

https://doi.org/10.1371/journal.ppat.1013218.g006

Once the PI3K-mTOR pathway gets activated, there is phosphorylation of effector molecules such as pS6K, and 4EBP1. The phosphorylated pS6K (p-S6K) activates the downstream signal transduction by phosphorylating its substrate, the ribosomal S6 protein, thereby increasing the production of phosphorylated S6 (p-S6) [65]. To further confirm that IL-17A activates the PI3K-mTOR pathway, we assessed the level of p-S6 in splenic CD8⁺ T cells from WNV-infected WT, Il17a^-/-, and Il17rc^-/- mice by flow cytometry. The splenocytes were treated with rIL-17A and simultaneously co-treated with either PI3K inhibitor (LY294002) or mTOR inhibitor (Rapamycin), or RPMI (control) ex vivo for 1 or 6 h. The results demonstrated that adding PI3K and mTOR inhibitors in the presence of rIL-17A reduced the expression of p-S6 in CD8⁺ T cells from WT, and Il17a^-/- but not from Il17rc^-/- mice (Fig 7A-I). The flow cytometry results confirm that IL-17A signaling activates the PI3K-mTOR pathway at the substate level. Thus, these findings collectively suggest that IL-17A activates PI3K-mTOR signaling in CD8⁺ T cells during WNV infection.

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Fig 7. IL-17A activates PI3K-mTOR signaling at the substrate level in CD8⁺ T cells.

(A-E) Splenocytes from WNV-infected WT mice (n=8) were stimulated with mouse rIL-17A (100 ng/ml) and treated with either rapamycin (20 nM), LY294002 (12.5 µM), or RPMI (control) for 1 or 6 h. (A) The representative pseudocolor plots and MFI of p-S6 of p-S6⁺CD3⁺CD8⁺ T cells by flow cytometry. (B-E) The flow cytometry results of p-S6 expression in p-S6⁺CD3⁺CD8⁺ T cells post 1 h or 6 h of treatment. (F-I) Splenocytes from WNV-infected Il17a^-/- (n=6) and Il17rc^-/- mice (n=5) were stimulated with mouse rIL-17A (100 ng/ml) and treated with either rapamycin (20 nM), LY294002 (12.5 µM), or RPMI (control) for 1 h. (F) Percentages of p-S6⁺CD3⁺CD8⁺ T cells and (G) MFI of p-S6⁺CD3⁺CD8⁺ T cells in Il17a^-/- mice. (H) Percentages p-S6⁺CD3⁺CD8⁺ T cells and (I) MFI of p-S6⁺CD3⁺CD8⁺ T cells in Il17rc^-/- mice. Data were analyzed by two-tailed student’s t-tests (B-G), and Mann Whitney U tests (H-I) and presented as mean ± s.e.m. with *, **, and **** denoting p <0.05, p <0.01, and p <0.0001, respectively. The data represent at least two independent experiments.

https://doi.org/10.1371/journal.ppat.1013218.g007

IL-17A signaling stimulates metabolism in CD8⁺ T cells during WNV infection

It has been reported that the activation of the PI3K-mTOR signaling pathway leads to increased metabolism in CD8⁺ T cells to cope with the higher energy demand in the inflammatory microenvironment and promotes CD8⁺ T cell effector function [61,66–68]. Therefore, we evaluated whether IL-17A signaling promotes metabolism in CD8⁺ T cells during WNV infection. Global profiling of glucose, amino acids, and nucleotides metabolism was performed on CD8⁺ T cells purified from WNV-infected WT and Il17rc^-/- mice with or without rIL-17A treatment by liquid chromatography-mass spectrometry (LC-MS/MS). The metabolomic results showed that the expression levels of fructose 1,6 bisphosphate (FBP), galactose, and lactose in CD8⁺ T cells from Il17rc^-/- mice were lower than those in WT mice (Fig 8A). FBP is generated from phosphorylation of fructose-6-phosphate (F6P) by phosphofructokinase-1 (PFK-1). FBP directly increases aerobic glycolysis by stimulating pyruvate kinase, a key rate-limiting glycolytic enzyme [69]. During FBP production, a fraction of F6P bypasses glycolysis to be converted into fructose 2,6-bisphosphate (F2,6-BP) by PFK-2. F2,6-BP acts as an allosteric activator of PFK-1, increasing aerobic glycolysis [69,70]. Consequently, with the help of PFK-1, FBP indirectly increases glycolytic flux to support higher proliferation. Thus, the reduced level of FBP suggests a decrease in aerobic glycolysis within CD8⁺ T cells of Il17rc^-/- mice, implying that IL-17A signaling increases aerobic glycolysis.

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Fig 8. IL-17A signaling promotes metabolism of CD8⁺ T cells.

Eight to nine weeks old Il17rc^-/- (n = 3) and WT (n = 3) mice were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. Levels of metabolites of glucose (A), amino acid (C), nucleotide (E), and redox homeostasis (G) in the purified CD8⁺ T cells were measured by mass spectrometry. The CD8⁺ T cells from the WT mice were treated with mouse rIL-17A (100 ng/ml) or RPMI (control) for 24 h, and the levels of metabolites of glucose (B), amino acid (D), and nucleotide metabolism (F) were measured by mass spectrometry. Data were analyzed by one-tailed student’s t-tests and presented as mean ± s.e.m. with * and ** denoting p <0.05, and p <0.01, respectively.

https://doi.org/10.1371/journal.ppat.1013218.g008

During anaerobic glycolysis, a newly formed pyruvate undergoes reduction to lactate by LDH in the cytosol, along with the generation of two adenosine triphosphate (ATP) molecules per glucose. Therefore, a lower lactate level indicates a decline in anaerobic glycolysis and gluconeogenesis in CD8⁺ T cells of Il17rc^-/- mice (Fig 8A). In addition, decreased galactose, a substrate for lactose, also results in a reduction in anaerobic glycolysis. Thus, the results suggest that IL-17A-IL-17RC signaling is required for both aerobic and anaerobic glycolysis in CD8⁺ T cells. This hypothesis is supported by the ex vivo rIL-17A treatment results, showing that the rIL-17A treatment decreases the production of adenosine diphosphate (ADP), an inhibitor of glycolytic enzymes [69], which in turn increases aerobic glycolysis in CD8⁺ T cells of WT mice (Fig 8B).

Activated CD8⁺ T cells also require an augmented influx of various amino acids to support the effector functions. Next, we examined if IL-17A signaling alters the amino acid metabolism in CD8⁺ T cells from WNV-infected Il17rc^-/- and WT mice. Compared with those from WT mice, the levels of histidine, leucine, lysine, arginine (Arg), glutamine, and tyrosine (Tyr) in CD8⁺ T cells from Il17rc^-/- mice are significantly decreased (Fig 8C). The increase of amino acid metabolism augmented by IL-17A signaling was also confirmed by the ex vivo rIL-17A treatment data showing significant increases in methionine, Tyr, aspartate, glutamate, Arg, tryptophan, phenylalanine, and cysteine in CD8⁺ T cells from WT mice (Fig 8D). Thus, these data suggest that IL-17A signaling increases amino acid metabolism.

To evaluate nucleotide metabolism in CD8⁺ T cells, we analyzed the metabolites related to purine and pyrimidine metabolism. The data showed a significant reduction of the levels of adenosine diphosphate/deoxyguanosine diphosphate (ADP/dGDP), and allantoin in CD8⁺ T cells from Il17rc^-/- mice compared to those from WT mice (Fig 8E). ADP/dGDP decreases both de novo and salvage purine synthesis via feedback inhibition of 5-phosphoribosyl-α-1-pyrophosphate (PRPP), the rate-limiting substrate for both pathways [71]. Allantoin is a metabolic intermediate of purine catabolism produced from the oxidation of uric acid [72]. Thus, decreases in ADP/dGDP and allantoin in CD8⁺ T cells from Il17rc^-/- mice suggest more purine synthesis and less purine breakdown, respectively. The data from pyrimidine metabolism showed a significant increase in uridine 5′ monophosphate (UMP) level in CD8⁺ T cells of Il17rc^-/- mice compared to WT mice (Fig 8E). High levels of UMP decrease de novo pyrimidine biosynthesis by feedback inhibition of carbamoyl phosphate synthase (CPS)- II, the primary regulatory enzyme of pyrimidine synthesis [73,74]. Therefore, decreased de novo pyrimidine synthesis in Il17rc^-/- mice implies that IL-17A signaling may increase pyrimidine metabolism. We checked if ex vivo rIL-17A treatment can affect nucleotide metabolism to confirm the results. Data on the purine metabolism showed increased inosine and hypoxanthine levels in the rIL-17A treatment group in WT mice (Fig 8F). Hypoxanthine, derived from inosine, is an important metabolic intermediate, required for salvage purine synthesis [71]. Interestingly, rIL-17A treatment also significantly inhibits the production of ADP/dGDP (Fig 8F), which is the feedback inhibitor of both de novo and salvage purine synthesis [75]. These results suggest that IL-17A signaling enhances the nucleotide metabolism in CD8⁺ T cells during WNV infection.

During T-cell activation, higher metabolic demand initiates an excess production of reactive oxygen species (ROS), resulting in the toxic accumulation of ROS and triggering oxidative stress in the cells [76]. In responding to oxidative stress, antioxidants, particularly reduced glutathione (GSH), are produced to neutralize ROS [76]. GSH neutralizes the ROS by continuously supplying reducing equivalents and undergoes conversion into oxidized glutathione (GSSG). Sustenance of the reduced state of other antioxidants is also maintained via GSH through a redox cycling reaction [77]. To assess the oxidative stress in the purified CD8⁺ T cells from WNV-infected Il17rc^-/- and WT mice, we measured the levels of GSH, GSSG, and pyroglutamate, a substrate for GSH synthesis. The results indicate a reduction in GSH levels and an increase in GSSG in Il17rc^-/- mice (Fig 8G). This suggests that oxidative stress is elevated in these mice, leading to an increase in ROS production. The ROS are neutralized by GSH but are simultaneously oxidized into GSSG. Consequently, there is a decrease in GSH levels and an increase in GSSG. Additionally, the lower pyroglutamate levels in Il17rc^-/- mice are associated with reduced GSH production, which further contributes to oxidative stress. Hence, these results collectively indicate that the CD8⁺ T cells from Il17rc^-/- mice exhibit a higher ratio of GSSG to GSH and lower pyroglutamate levels, suggesting increased oxidative stress in these cells compared to those from WT mice during WNV infection (Fig 8G). Therefore, it is reasonable to conclude that IL-17A signaling reduces oxidative stress by maintaining redox homeostasis and increasing glutathione metabolism in CD8⁺ T cells.

IL-17A-mediated PI3K-mTOR signaling increases the cytotoxicity of CD8⁺ T cells

Next, we evaluated the effect of IL-17A-mediated PI3K/mTOR signaling activation on the cytotoxicity of WNV-specific CD8⁺ T cells. The splenocytes were isolated from WNV-infected WT and Il17rc^-/- mice on D8 p.i. and treated with rIL-17A in the presence or absence of rapamycin and LY294002 for 6 or 24 h ex vivo. The production of perforin in the NS4B⁺CD8⁺ T cells was quantified by flow cytometry. The results showed a decrease in perforin production in the WT cells with the rapamycin treatment at 6 h (Fig 9A and 9B). A similar reduction trend was also detected at 24 h treatment, although the difference is not statistically significant (Fig 9C and 9D). The treatment of LY294002 decreased the percentage of perforin-expressing NS4B⁺CD8⁺ T cells compared to the rIL-17A treatment control group at both 6 and 24 h time points (Fig 9A and 9C). As expected, no changes in the perforin expression were observed in either rapamycin or LY294002 treatment groups in NS4B⁺CD8⁺ T cells from Il17rc^-/- mice (Fig 9E and 9F), indicating that IL-17RC was required for the activation of IL-17A-mediated PI3K-mTOR signaling. Hence, these results collectively suggest that IL-17A signaling-mediated PI3K-mTOR pathway can promote cytotoxicity in CD8⁺ T cells during WNV infection.

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Fig 9. IL-17A signaling promotes perforin expression by activating the PI3K-mTOR pathway in WNV-specific CD8+ T cells.

Eight to nine weeks old WT (n = 7 to 8) and Il17rc^-/- mice (n = 4) were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. The collected splenocytes were treated with mouse rIL-17A (100 ng/ml) with or without rapamycin (20 nM) and LY294002 (12.5 µM) for 6 or 24 h and characterized by flow cytometry after probing with WNV NS4B tetramer and antibodies against CD8 and perforin. The perforin⁺NS4B⁺CD8⁺ T cell percentages (A), MFI (B) of 6 h treatment; the percentages (C), and MFI (D) of 24 h treatment in WT mice. The data represent at least two independent experiments. The percentages of perforin⁺NS4B⁺CD8⁺ T cells (E) and MFI (F) for 24 h treatment in Il17rc^-/- mice. Data were analyzed by two-tailed student’s t-tests and presented as mean ± s.e.m. with * denoting p <0.05.

https://doi.org/10.1371/journal.ppat.1013218.g009

Discussion

CD8⁺ T cells recognize peptide-class I major histocompatibility complexes (MHC) and play a key role in both clearing acute viral infection and generating long-lived memory cells primed to provide a rapid response to repeated infections [78]. CD8⁺ T cells control viral infections by triggering apoptosis of infected cells via perforin or Fas ligand-dependent pathways or producing antiviral cytokines (e.g., IFN-γ and TNF-α) [78]. Infection with WNV can lead to life-threatening encephalitis and meningitis, and CD8⁺ T cells are essential in clearing WNV infection from the CNS of humans and experimental mice. Mice deficient in CD8⁺ T cells or class I MHC antigens had higher viral burdens in the CNS and increased mortality rates. The absence of CD8⁺ T cells did not affect the kinetics or magnitude of viremia nor the production of anti-WNV antibodies [79]. In addition, WNV infection of mice deficient in perforin, Fas ligand, and TNF-related apoptosis-inducing ligand (TRAIL) resulted in an increased viral burden in the CNS and mortality following infection [80–82]. In contrast, anti-viral antibodies and B cells are essential in protecting against WNV infection by controlling viremia and viral loads in the peripheral organs, thus limiting viral dissemination into the CNS. Prophylactic and therapeutic transfer of human IgG or mouse serum delayed or protected mice from WNV infection. However, antibodies alone cannot eliminate viral reservoirs in host tissues, further suggesting CD8⁺ T cell-mediated cellular immune responses are required for WNV clearance [79,83]. Although some evidence suggests that CD8⁺ T cells might contribute to neuronal damage in several inflammatory, neurodegenerative, and paraneoplastic neurological disorders [84], in the case of WNV infection, CD8⁺ T lymphocytes play a crucial protective role in eliminating virus-infected cells [79] by inducing robust anti-viral adaptive immunity [85]. Considering the risk vs benefit of the cytotoxic potential of CD8⁺ T cells in the CNS, it is crucial to balance their ability to target and eliminate virus-infected cells with unintended damage to the healthy neuronal tissue. Careful modulation and control of CD8⁺ T cells are important to optimize their effector function. However, the regulation of their effector functions remains poorly understood. Through investigating WNV pathogenesis, we have uncovered a novel function of IL-17A signaling in promoting the cytotoxicity of CD8⁺ T cells. Importantly, a single injection of 2.5 μg of rIL-17A into WT mice on day 6 after WNV infection significantly increased expression of the cytotoxic mediator genes in CD8⁺ T cells and profoundly reduced viral burden in the brain and increased animal survival, suggesting a great therapeutic potential of IL-17A against WNV infection in the CNS [50]. Therefore, understanding how IL-17A signaling promotes the cytotoxicity of CD8⁺ T cells paves the way for applying IL-17A as a potential therapeutic agent to treat WNV infection in the CNS.

IL-17A induces signaling transduction through a heterodimer of IL-17RA and IL-17RC complex. While IL-17RA participates in the signaling pathways of other members of the IL-17 family, such as IL-17B, C, and E, IL-17RC is specific to the signaling of IL-17A, IL-17F, and IL-17A/F. IL-17F has been reported to have similar biological functions as IL-17A [86]. Thus, we utilized Il17rc^-/- mice to study how IL-17A signaling regulates the cytotoxicity of CD8⁺ T cells against WNV infection. After WNV infection, Il17rc^-/- mice bear a higher viral burden in the brain than the WT mice, although the viral load in the blood, liver, and spleen samples showed no apparent difference. In addition, the TUNEL assay results showed more apoptotic bodies in the brains of Il17rc^-/- mice. These findings suggest that IL-17A signaling is crucial to protect mice against WNV infection in the brain. During WNV neuroinvasion, monocytes, neutrophils, NK cells, DC cells, and effector CD4⁺ and CD8⁺ T cells were found to infiltrate the brain to respond to the infection [87]. Of these, monocytes have been reported as the most predominant immune cells infiltrated into the brains of WNV-infected mice [87,88]. Neutrophils exhibit a dual role in the pathogenesis of WNV, serving as viral reservoirs, facilitating the early viral spread to the CNS, and subsequently inhibiting viral replication [89]. Our flow cytometry results showed that the numbers of infiltrated CD8⁺ T cells were reduced compared to the WT control mice, suggesting that the higher susceptibility of Il17rc^-/- mice to WNV infection may be due to the lower magnitude of the functions of CD8⁺ T cells. The flow cytometry results also excluded the possibility that the absence of IL-17RC might negatively affect CD8⁺ T cell development, thus further supporting the notion that IL-17A signaling is essential to the effector functions of CD8⁺ T cells. Effector CD8⁺ T cells destroy target cells by releasing several cytotoxic mediators, such as perforin and granzymes. While perforin binds to the target cell membrane to form pores, damaging the integrity of the target cell membrane [90], granzymes activate apoptosis pathways after entering the target cells through the perforin pores [78]. In addition, effector CD8⁺ T cells can initiate the Fas-FasL interaction, triggering the caspase cascade and inducing apoptosis of the target cells [91,92]. The flow cytometry results showed that the expression of perforin in the brain-infiltrated CD8⁺ T cells from Il17rc^-/- mice was significantly lower than that of the WT mice, suggesting that IL-17A signaling is indispensable to maintaining the cytotoxicity of CD8⁺ T cells. A previous report indicated that the absence of CD8⁺ T cells in mice did not affect the kinetics or magnitude of WNV viremia but did increase the viral burden in the brain and mortality [79]. This aligns with our findings that Il17rc^-/- mice exhibited a higher viral burden in the brain compared to WT mice while maintaining similar viral loads in the blood and peripheral organs. One possible explanation is that WNV burdens in the blood and peripheral organs of mice typically peak between D3 and D4 p.i. before WNV-specific CD8⁺ T cells have matured and become functional [57–59].

It is worth noting that we used 100 PFU of infection via footpad inoculation to assess the susceptibility of IL17rc^-/- mice, measuring viral load, cytokine profiles, and immune cell brain infiltration as described in our previous publications [50,57,93]. This optimal dose was chosen because it results in approximately 50% mortality in WT C57BL/6 mice aged 7–8 weeks. Due to the variability among animals, even within the same group, some may exhibit sickness sooner and die, while others survive the infection. In this study, we collected brain and spleen samples on day 7 post-infection (p.i.), when WNV typically infects the brain, and all the animals in both groups are still alive, as death usually occurs between days 8 and 15 post-infection. Although some animals may start showing signs of sickness on day 7, most appear healthy. By collecting and analyzing samples from each surviving animal in both groups, we ensured unbiased sample collection and data presentation regardless of their health status. Any observed differences between WT and the knockout groups should reflect their immune responses to WNV infection. However, we cannot exclude the possibility that clinical heterogeneity within the same group may affect the measurements and data interpretation.

To further investigate whether IL-17A signaling promotes the cytotoxicity of WNV-specific CD8⁺ T cells via the IL-17A-IL-17RC axis, we evaluated the expression of perforin and granzyme A and the percentages of WNV NS4B tetramer and CD8 double-positive (NS4B⁺CD8⁺) cells from WNV-infected Il17a^-/- and Il17rc^-/- mice. The results of both mouse models suggest that IL-17A signaling promotes the cytotoxicity of WNV-specific CD8⁺ T cells. Interestingly, we noticed a significant increase in the percentage of NS4B⁺CD8⁺ T cells in Il17rc^-/- compared to WT mice. The higher number of NS4B⁺CD8⁺ T cells but lower cytotoxicity in Il17rc^-/- mice further implied that these WNV-specific CD8⁺ T cells were functionally defective. The ex vivo rIL-17A treatment experiment results confirmed that rIL-17A increased the perforin expression in the WNV-specific CD8⁺ T cells isolated from WT mice but not Il17rc^-/- mice. Thus, these findings further demonstrate the essential role of the IL-17A-IL-17RC axis on the cytotoxicity in WNV-specific CD8⁺ T cells. In addition, we did not notice any noticeable change in the expression of granzyme A, suggesting that the IL-17A signaling may be more effective in inducing perforin expression than granzyme A. Consistent with our findings on the roles of perforin, previous studies have demonstrated that CD8⁺ T cells require perforin to clear viral infections, including WNV from infected neurons [82,94,95]. Mice deficient in perforin resulted in a higher viral burden in the CNS and increased mortality after WNV infection [82]. Although perforin and granzyme A are key components of the cytotoxic machinery in CD8⁺ T cells, literature suggests their expression can be regulated independently under different conditions. For instance, IL-2 has been found to increase the expression of both perforin and granzyme A in CD8⁺ T cells, but the regulation of these molecules can occur independently [96]. Additionally, most CD8⁺ T cells in rectal tissue, including HIV (human immunodeficiency virus)-1-specific cells, fail to express perforin but granzyme A, indicating differential expression patterns of perforin and granzyme A [97]. The detailed molecular mechanisms of why IL-17A-IL17RC signaling may be more effective in inducing perforin expression than granzyme A are warranted for further investigation.

During a viral infection, effector T cells experience an increased influx of nutrients due to higher energy demands [66]. mTOR serves as a molecular sensor of metabolism that integrates signals associated with both immune activation and metabolic requirements [66]. Additionally, mTOR signaling via the mTORC1 complex not only induces differentiation of the effector over the memory lineage of CD8⁺ T cells but also prompts their cytotoxic effector function by inducing the expression of T-bet [98]. The CD28-mediated costimulatory signal required for activating CD8⁺ T cells during antigenic stimulation also serves as a classic signal for activating the PI3K-AKT-mTOR signaling pathway [61,66,67]. The engagement of TCR-MHC class I complex also activates PI3K in the activated CD8⁺ T cells [67]. Similar to the mTOR pathway, PI3K-AKT signaling also promotes the activation and differentiation of effector CD8⁺ T cells [67]. It has been reported that IL-17A via the IL-17RA axis activates the PI3K-mTOR pathway in mouse alveolar epithelial cells and promotes fibrosis [99]. Using a bleomycin-induced IL-17A activation model, Cong et al. also reported that IL-17A-IL-17RA signaling could activate the PI3K-AKT-mTOR signaling and increase pulmonary inflammation and fibrosis in mouse bronchial epithelial cells [61,66,67]. In the current study, our results suggest that IL-17A signaling upregulates the expression of PI3K3ca, mTOR, and S6K1, suggesting that IL-17A signaling may activate the PI3K-mTOR signaling pathway and increase the activity of p-pS6K. This notion is supported by evidence that rIL-17A treatment increases, while rapamycin and LY294002 inhibit the phosphorylation of pS6K in CD8⁺ T cells from WNV-infected WT mice in the immuno-blotting analysis. Moreover, IL-17A treatment also increases the phosphorylation of 4EBP1 at the protein level. Thus, IL-17A may affect the PI3K/mTOR signaling pathway via mediating phosphorylation of both downstream effectors, pS6K and 4EBP1.

Upon viral antigen activation, naïve CD8⁺ T cells undergo rapid differentiation into activated CD8⁺ T cells, subsequently engaging in swift proliferation to generate a pool of effector CD8⁺ T cells. This process requires higher energy and a continuous supply of different macromolecules as building blocks to generate robust antiviral responses. This increased energy demand is met by a radical shift in metabolism, transitioning from a resting catabolic state to a highly proliferative anabolic state [68]. While the naïve CD8⁺ T cells mainly rely on fatty acid oxidation and oxidative phosphorylation (OXPHOS), the short-lived effector CD8⁺ T cells differentiated due to any type of cellular insult, depend on both aerobic and anaerobic glycolysis [100], glutaminolysis, protein, and nucleotide synthesis [68]. It has been reported that PI3K-mTOR signaling acts as a metabolic switch for the differentiation and proliferation of CD8⁺ T cells to promote the effector functions [66]. However, the impact of IL-17A signaling on the metabolism of CD8⁺ T cells has not been studied. To address this question, we measured metabolites related to glucose, amino acids, nucleotides, and redox metabolism with LC-MS. Since fatty acid oxidation is primarily utilized by naïve CD8⁺ T cells for energy production [101], lipid metabolism is not included in the analysis. Data from the metabolomic study demonstrated that IL-17A signaling elevated both aerobic and anaerobic glycolysis in CD8⁺ T cells, which were further confirmed by the ex vivo rIL-17A treatment study on the cells from WNV-infected WT mice. T cells have been documented to metabolize glucose through both aerobic and anaerobic glycolysis during proliferation [100]. It has been reported that PI3K-AKT-mTORC1 signaling increases glycolysis in metabolically demanding conditions in actively proliferating immune cells [102,103]. Thus, our results suggest that IL-17A signaling activates the PI3K-mTOR pathway, leading to the increase of glycolysis in CD8⁺ T cells during WNV infection. Similarly, it also increases the metabolism of amino acids and nucleotides within CD8⁺ T cells during WNV infection. T-cell activation promotes the uptake of different amino acids, activating mTOR signaling, thereby promoting protein synthesis and metabolic reprogramming [104]. In addition to upholding structural integrity, amino acids are also required to participate in nucleotide metabolism, redox homeostasis, and epigenetic modifications of histones within T cells [104,105]. While purine and pyrimidine regulate the cell cycle differently, there is evidence suggesting that both nucleotides contribute to T cell proliferation and survival [106]. In highly proliferative T cells, mTOR, as a master regulator, promotes amino acid influx required for purinosomes formation and upregulates purine synthesis through the tetrahydrofolate cycle [106,107]. Similarly, activation of PI3K-mTOR signaling phosphorylates the trifunctional enzymes, i.e., CPS, aspartate carbamoyltransferase, and dihydroorotase, resulting in the initiation of pyrimidine synthesis [74]. Therefore, our results suggest that IL-17A signaling activates PI3K-mTOR signaling and subsequently increases the metabolism of glycolysis, amino acids, and nucleotides, enhancing the effector functions of CD8⁺ T cells.

Finally, we confirm if IL-17A signaling augments the cytotoxicity of CD8⁺ T cells via activating the PI3K-mTOR signaling pathway with their inhibitors, rapamycin and LY294002. The results demonstrated that IL-17A-mediated increase of the perforin expression was suppressed by both rapamycin and LY294002 in CD8⁺ T cells from WT but not in Il17rc^-/- mice. Thus, collectively, these results suggest that IL-17A signaling activates PI3K-mTOR-mediated metabolism and increases the cytotoxicity of CD8⁺ T cells for WNV clearance in the CNS. This study contributes by elucidating a novel mechanism of IL-17A signaling in WNV pathogenesis, adding valuable insights to the current understanding of the complex interplay between IL-17A and WNV infection.

Methods

Ethical statement and biosafety

The animal experiments in this study were reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) at the University of Southern Mississippi (USM) under the IACUC protocols # 15101601 and 17110903. The experiments involving live WNV were performed by certified personnel in the Biosafety Level-2 (BSL-2, cell culture) and BSL-3 (animal) laboratories, following the biosafety protocols approved by the USM Institutional Biosafety Committee.

Cells and viruses

Vero cells (ATCC CCL-81) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies) supplemented with 1% Penicillin/Streptomycin (P/S, Gibco) and 10% Fetal Bovine Serum (FBS, Atlanta Biologicals). The splenic CD8⁺ T cells were maintained in RPMI 1640 medium (Gibco) supplemented with 1% P/S and 10% FBS. The cells were kept in an incubator at 37°C with 5% CO₂ and a relative humidity of 95%. The WNV isolate CT2741 [108] was kindly provided by John F. Anderson at the Connecticut Agricultural Experiment Station, propagated in Vero cells, and quantified by a plaque-forming assay as previously described [109,110].

Mice and animal studies

The breeding pair of C57BL/6J (WT) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The breeding pair of Il17a^-/- mice were kindly provided by Richard A. Flavell at the Yale University School of Medicine, and Il17rc^-/- mice by Dr. Zongbing You at Tulane University School of Medicine [55], and Il17ra^-/- mice by Dr. Sarah Gaffen at the University of Pittsburgh. All the knockout strains are in the C57BL/6J background. The breeding pairs and pups were kept in a clean room until they were ready to be used. The infection experiments were performed in an animal BSL-3 lab at USM. Seven to nine weeks old Il17a^-/-, Il17rc^-/-, and WT mice were subcutaneously injected on the ventral side of the right hind footpad with either 20 or 100 PFU of WNV in 50 µl of phosphate buffer saline (PBS) containing 5% gelatin after 30% isoflurane anesthesia [93]. In the survival experiments, the infected animals were monitored daily for 21 days for morbidity and mortality. Blood samples were collected from the retro-orbital sinus on D2 and 4 p.i. in 0.5M EDTA.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

The total RNA was extracted from cells and tissue samples using TRI Reagent (Molecular Research Center, Inc) and converted into the first-strand complementary DNA (cDNA) using the iSCRIPT cDNA synthesis kit (Bio-Rad). The RT-qPCR assays were performed in a CFX Connect Real-Time System (Bio-Rad) using iTaq Universal SYBR Green Supermix (Bio-Rad). The WNV-E RNA copies were measured by probe-based RT-qPCR, and data were normalized by mouse β-actin as a housekeeping gene [50,109]. Genes of various cytokines, CD8⁺ T cell cytotoxicity mediators, and PI3K-mTOR signaling mediators were quantified by SYBR Green-based RT-qPCR, and data were normalized by the ΔΔC_T method using β-actin as the house-keeping gene to determine relative fold change (RFC) [50].

Immunofluorescence assay (IFA)

Eight to nine weeks old Il17rc^-/- and WT mice were infected with 100 PFU of WNV via f.p. inoculation. Mice were anesthetized using 30% isoflurane on D7 p.i. and transcardially perfused with 30 ml of ice-cold PBS. The brains of the infected animals were then collected, fixed with 4% PFA, and cryoprotected with 20% followed by 30% sucrose for 24 h at 4°C. The brains were then mounted with OCT compound by snap freezing with liquid nitrogen and incubated overnight at -80°C. Brain sections were cut sagittally using a cryostat (Leica Biosystems) at a thickness of 10 µm, transferred to charged glass slides (Fisher Scientific), and air-dried overnight. The brain sections were blocked with 5% goat serum and 0.5% Triton X-100 in PBS for 1 h at 4°C, probed with rabbit anti-WNV-E polyclonal antibody (1:100, PA1–41073, Invitrogen) overnight at 4°C. The sections were washed 3 times with PBST for 10 min each. Then, the sections were stained with Alexa Fluor 568-conjugated goat anti-rabbit IgG (1:500, Invitrogen, A-11011) for 1 h at RT. The sections were washed as above and stained with DAPI (4´, 6-diamidino-2-phenylindole, Life Technologies) [111,112]. The images were acquired at 40x magnification using a Nikon AIR confocal microscope (Leica Biosystems).

TUNEL assay

The TUNEL assay was conducted using a TACS 2 TdT-Fluor In Situ Apoptosis Detection Kit (R&D Systems). The brain samples were processed and sectioned into 10 µm thickness following the procedure mentioned in IFA. The cryosections are then rehydrated gradually with 100, 95, and 70% ethanol. After a PBS wash, the samples were treated with 50 µl Cytonin for 30 min at RT. The samples were then washed with labeling solution, followed by incubating with 50 µl of labeling mixture containing dNTP, TdT, manganese ion, and labeling buffer for 2 h at 37°C. The reactions were stopped with TdT stop buffer, washed twice, and stained with Strep-Fluorescein solution for 30 min in the dark at RT. Cell nuclei were stained with DAPI, and the images were acquired at 40x magnification using a Nikon AIR confocal microscope (Leica Biosystems).

Flow cytometry

Detection of immune cells from mouse brain.

Eight to nine weeks old Il17rc^-/- and WT mice were infected with 100 PFU of WNV via f.p. inoculation. The brains were collected after intracardial ice-cold PBS infusion on D7 p.i. and processed into a single-cell suspension using the 40 µm cell strainer. A discontinuous Percoll gradient (GE Healthcare) was used to isolate brain leukocytes after blocking the Fc receptors using 100 µg/ml of rat anti-mouse CD16/CD32 Ig (BD Biosciences) for 30 min. The leukocytes were probed with antibodies against mouse CD45 (PE-Cy7-conjugated, eBioscience, 25-0451-82), CD3 (FITC-conjugated, eBioscience, 11-0031-85 or APC-Cy7-conjugated), CD4 (PerCp Cy 5.5-conjugated, eBioscience, 45-0042-82), CD8 (PE-conjugated, eBioscience, 12-0081-82 or APC-conjugated, eBioscience, 17-0081-82), CD11b (APC-conjugated, BD Biosciences, 561690), and Ly6G surface markers (APC Cy7-conjugated, Invitrogen, A15424). The cells were then fixed with 4% paraformaldehyde (PFA) for 15 min and permeabilized with 1X permeabilization buffer for 30 min. The leukocytes were then probed with antibodies against mouse perforin (PE-conjugated, eBioscience, 12-9392-82) and granzyme B (FITC-conjugated, eBioscience, 11-8898-82). Data were acquired using BD LSRFortessa Cell Analyzer (BD Biosciences) and analyzed with FlowJo (v10.9.0.) software [50].

Assessment of immune cell development.

The spleens were collected from eight-week-old Il17rc^-/- and WT mice. The spleen samples were treated with red cell lysis (RBC) buffer and processed into a single-cell suspension using the 70 µm cell strainer. Then, the Fc receptors were blocked with 100 µg/ml of rat anti-mouse CD16/CD32 Ig (BD Biosciences) for 15 min. The splenocytes were stained with antibodies against mouse CD45 (PE-Cy7-conjugated, eBioscience, 25-0451-82), CD3 (FITC-conjugated, eBioscience, 11-0031-85), CD4 (PerCp Cy 5.5-conjugated, eBioscience, 45-0042-82), CD8 (PE-conjugated, eBioscience, 12-0081-82), CD11b (APC-conjugated, BD Biosciences, 561690), and Ly6G surface markers (APC Cy7-conjugated, Invitrogen, A15424). Data were acquired and analyzed as above.

Detection of cytotoxic mediators in WNV-specific CD8⁺ T cells.

Eight to nine weeks old Il17a^-/-, Il17rc^-/-, and WT mice were infected with 20 PFU of WNV via f.p. inoculation. Mice were euthanized on D8 p.i. to collect splenocytes. The splenocytes (5 x 10⁶) were plated in 6-well plates and restimulated with 0.01 MOI of WNV for 24 h at 37°C. During the final 6 h of the stimulation, Brefeldin A solution (BD Biosciences) was added in 1:1000 to block cytokine secretion. To detect the WNV-specific CD8⁺ T cells, splenocytes were incubated with WNV tetramer (NS4B 2488–2496, SSVWNATTA) conjugated with APC (the NIH Tetramer Core Facility at Emory University) and antibody against CD8 (PE-conjugated, eBioscience, 12-0081-82) for 30 min at 4°C in dark. After washing twice with staining buffer, the splenocytes were fixed with 4% PFA for 10 min and permeabilized with 1x permeabilization buffer for 30 min at 4°C. The splenocytes were then stained intracellularly with antibodies against mouse perforin (FITC-conjugated, BioLegend, 154306) and granzyme A (PE-Cy7-conjugated, eBioscience, 25-5831-80) and incubated for 30 min at 4°C in the dark. After washing twice, splenocytes were resuspended in 300µl staining buffer. Data were acquired and analyzed as above.

Detection of PI3K-mTOR mediators in CD8⁺ T cells.

Eight to nine weeks old WT mice were infected with 20 PFU of WNV via f.p. inoculation. Mice spleens were collected on D8 p.i. Splenocytes (3 x 10⁶) were plated in 96-well round-bottom plates and probed with methanol-safe surface antibody against mouse CD3 surface marker (FITC-conjugated, eBioscience, 11-0031-85) for 30 min at 4°C in the dark. After washing with RPMI medium once, the splenocytes were treated with 100 ng/ml of mouse rIL-17A. Some rIL-17A-treated wells were also treated with either 12.5 µM of PI3K inhibitor LY294002 (Selleckchem, S1105) or 20 nM of mTOR inhibitor Rapamycin (Selleckchem, S1039) or RPMI medium as a control. The splenocytes were incubated for 1 and 6 h at 37°C in the dark. After incubation, the splenocytes were washed twice with RPMI, fixed with 4% PFA for 15 min, and permeabilized with 90 µl of ice-cold methanol for 30 min at 4°C. Then, the splenocytes were stained with antibodies against mouse CD8 (APC-conjugated, BioLegend, 100712), phospho-S6 (PE-Cy7-conjugated, Invitrogen, 25-9007-42), and phospho-4EBP1 (PE-conjugated, Invitrogen, 12-0107-42) for 30 min at 4°C in the dark. After washing twice, the splenocytes were resuspended in 300 µl staining buffer. Data were acquired and analyzed as above.

The limitation of our flow cytometric analysis is that a Live/Dead stain was not used to differentiate live cells for data analysis due to the limited color choices of the flow cytometer. We acknowledge that some dead cells may be present in our analysis; however, since we applied the same gating strategy for all the samples in the same experiment, this should not affect the overall data trend and the conclusions of the findings.

Generation of conditional knock-out mice

The Il17ra^fl/fl (JAX stock #031000) male mouse of C57BL/6J background [113], and Cd8a-Cre transgenic (JAX stock #008766) female mouse of C57BL/6N background [114] were purchased from the Jackson Laboratory (Bar Harbor, ME). The Il17ra^fl/fl male and Cd8a-Cre female mice were mated to get the first generation of pups, where 50% of them were Il17ra^fl/w containing the genotype of fl/w-cre/w. Then the Il17ra^fl/w mice were backcrossed to Il17ra^fl/fl to get the second generation of pups, where 25% of them were Il17ra^fl/fl containing the genotype of fl/fl-cre/w considered as the cre-KO, and the remaining littermates as cre-WT. The total DNA was extracted from blood samples using DNeasy Blood & Tissue Kits (Qiagen) for genotyping by PCR. For the confirmation of cre-KO and cre-WT, targeted DNA sequences were amplified using the primer sequences provided by the Jackson Laboratory.

Purification of CD8⁺ T cells

Eight to nine-week-old Il17a^-/-, Il17rc^-/-, and WT mice were infected with 20 PFU of WNV via f.p. inoculation, followed by collection of spleens on D8 p.i. Splenic CD8⁺ T cells were purified from the total splenocytes using a Mouse CD8a⁺ T cell Isolation Kit (Miltenyi Biotec) following the user’s manual.

RNA sequencing

Library preparation was performed according to the Smart-seq2 protocol [115]. Briefly, cDNA was generated from 50ng of total RNA using SuperScript IV reverse transcriptase (Thermo Fisher Scientific, Cat. #18091050) with oligo(dT) priming and template-switching oligo. Double-stranded cDNA was amplified via 12 cycles of PCR using KAPA HiFi HotStart ReadyMix (Roche) and ISPCR oligo. Amplified cDNA was fragmented using the Nextera XT DNA Library Prep Kit (Illumina), with adapter-ligated products size-selected (200–500 bp) via SPRI beads (Beckman Coulter). Then, libraries were quantified by qPCR (KAPA Biosystems) and pooled at equimolar ratios. Paired-end sequencing (2 × 37 bp) was performed on an Illumina NextSeq 500 platform using a High Output Kit v2 (75 cycles). Raw reads were quality-filtered (FastQC v0.11.9) and aligned to the mm10 with TopHat v2.1.1, Bowtie2 v2.2.8, and Samtools v1.3. Counts were normalized to Fragments per kilobase of exon per million mapped fragments (FPKMs), and differential expression analysis was conducted with Cufflinks v2.2.2. The RNA sequencing data were deposited in Dryad [62].

CD8⁺ T cell cytotoxicity assay

Eight-week-old Il17rc^-/- and WT mice were infected with 100 PFU of WNV via f.p. inoculation. The brains were collected after intracardial ice-cold PBS infusion on D7 p.i., processed into a single-cell suspension, and treated with RBC lysis buffer. A discontinuous Percoll gradient (GE Healthcare) was used to isolate brain leukocytes. Then, CD8⁺ T cells were purified from the infiltrated leukocytes using a Mouse CD8a⁺ T cell Isolation Kit (Miltenyi Biotec) via negative selection technique. These purified brain-infiltrated CD8⁺ T cells were co-cultured with target cells expressing the ectodomain of WNV-E (MC57GL_WNV-E) or control cells containing the expression vector (MC57GL_vector), incubated at 37°C for 8 h at the effector to target ratios of 10:1. After incubation, the cell supernatant was collected and determined the level of cytotoxicity by measuring the release of intracellular LDH using the CyQUANT LDH Cytotoxicity Assay (Invitrogen) following the user’s manual [50,79].

The Ex vivo rIL-17A treatment assay

The isolated splenic CD8⁺ T cells collected from WNV-infected (20 PFU, f.p.) mice were treated with either carrier-free mouse rIL-17A (50 ng/ml, eBioscience, diluted in RPMI medium) or RPMI medium as a control and incubated for 24 h at 37°C. The CD8⁺ T cells were centrifuged at 500 x g for 10 min. After discarding the supernatant, 250 ul of TRI Reagent was added to the cell pellets to conduct the RNA extraction from the CD8⁺ T cells. The gene expression of PIK3ca, mTOR, and S6K1 in the rIL-17A-treated or control CD8⁺ T cells was measured by SYBR-based RT-qPCR. The primer sequences used for this study are included in S1 Table.

Western blot

Eight to nine weeks old Il17rc^-/- and WT mice were infected with 20 PFU of WNV via the f.p. route followed by collecting spleens on D8 p.i. The CD8⁺ T cells were purified from splenocytes and treated with either rIL-17A (100 ng/ml) or RPMI (control) for 15 or 60 min at 37°C. After the treatment, the CD8⁺ T cells were lysed with a mixture containing RIPA lysis buffer, β-ME, and protease and phosphatase cocktail for 5 min. The protein separation was done by gel electrophoresis using 100 µg of total protein in 4–15% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred into a nitrocellulose membrane and probed with primary antibodies against mouse PI3K (p85, 1:500, Cell Signaling Technology, 4257), phosphorated (p)-PI3K (p85 [Tyr458]/p55 [Tyr199], 1:200, Cell Signaling Technology, 4228), AKT (1:500, Cell Signaling Technology, 9272), p-AKT (Thr308, 1:200, Cell Signaling Technology, 4056), mTOR (1:500, Cell Signaling Technology, 2972), p-mTOR (Ser2481, 1:200, Cell Signaling Technology, 2974), pS6K (1:500, Cell Signaling Technology, 9202), p-pS6K (Thr389, 1:200, Cell Signaling Technology, 9234), 4EBP1 (1:500, Cell Signaling Technology, 9644), p-4EBP1 (Thr37/46, 1:500, Cell Signaling Technology, 2855), and GAPDH as control (1:1000, Cell Signaling Technology, 5174) and incubated overnight at 4°C. The membranes were re-probed with horseradish peroxidase (HRP)-conjugated secondary antibody at RT for 2 h. The protein bands were developed by chemiluminescence substrate (Invitrogen), and images were acquired using the ChemiDoc MP system (Bio-Rad). The densitometric quantification of the protein bands was performed by Image Lab software.

Metabolomic study

For in vivo study, eight to nine weeks old Il17rc^-/- and WT mice were infected with 20 PFU of WNV via f.p. followed by collection of spleens on D8 p.i. CD8⁺ T cells were isolated from splenocytes, snap-frozen by liquid nitrogen, and kept at -80°C until ready for analysis. For ex vivo study, the isolated CD8⁺ T cells from WNV-infected WT mice were treated with either carrier-free mouse rIL-17A (100 ng/ml, R&D Systems, diluted in RPMI medium) or RPMI medium as control and incubated for 24 h at 37°C. After washing twice with ice-cold PBS, the CD8⁺ T cells were snap-frozen with liquid nitrogen and kept at -80°C until ready for analysis. The metabolomic assessment of CD8⁺ T cells was performed using liquid chromatography-mass spectrometry (LC-MS) by Gigantest.

Statistical analysis

Data were compared using the Mann-Whitney U test, log-rank test, or one- or two-tailed Student’s t-tests with the GraphPad Prism software (version 10.2.3), whichever was applicable.

Supporting information

S1 Fig. Viral load in different tissues in Il17rc^-/- mice in WNV infection.

Eight to nine weeks old Il17rc^-/- (n = 4–8) and WT (n = 4–9) mice were infected with 100 PFU of WNV via the f.p. route. Blood, liver, and spleen tissues were collected, followed by measuring the viral titer by RT-qPCR and expressed as WNV-E to mouse β-actin. (A) Viral load in blood on D2, 4 p.i. (B) Viral load in liver on D2, 4, 6 p.i. (C) Viral load in spleen on D2, 4, 6 p.i. Data were analyzed using two-tailed Student’s t-tests and presented as means.

https://doi.org/10.1371/journal.ppat.1013218.s001

(TIF)

S2 Fig. The antiviral and inflammatory responses in the plasma of Il17rc^-/- mice.

Eight to nine weeks old Il17rc^-/- (n = 10) and WT mice (n = 8) were infected with 100 PFU of WNV via f.p. inoculation, plasma was collected on D2 p.i. and the expression of different anti-viral and inflammatory genes was measured by RT-qPCR and normalized to mouse β-actin. The gene expressions of IFN-α (A), IFN-β (B), IFN-γ (C), IL-1β (D), CXCL-2 (E), CXCL-10 (F), and IL-6 (G). Data were analyzed by two-tailed Student’s t-tests and presented as mean with ** denotes p < 0.01.

https://doi.org/10.1371/journal.ppat.1013218.s002

(TIF)

S3 Fig. Detection of immune cells in spleen and blood in Il17rc^-/- mice.

Spleen and blood samples were collected from eight to nine weeks old Il17rc^-/- (n = 3–4) and WT mice (n = 7–10) and characterized by flow cytometry after probing with antibodies against CD45, CD11b, Ly6G, CD3, CD4, and CD8, and measured the cell population. (A-E) Spleen: (A) Neutrophil (CD45⁺CD11b⁺Ly6G⁺); (B) monocyte (CD45⁺CD11b⁺); (C) B cells (CD45⁺CD3^-CD19⁺); (D) CD4⁺ T cells (CD45⁺CD3⁺CD4⁺); and (E) CD8⁺ T cells (CD45⁺CD3⁺CD8⁺) in Il17rc^-/- and WT mice. (F-J) Blood: (F) Neutrophil (CD45⁺CD11b⁺Ly6G⁺); (G) monocyte (CD45⁺CD11b⁺); (H) B cells (CD45⁺CD3^-CD19⁺); (I) CD4⁺ T cells (CD45⁺CD3⁺CD4⁺); and (J) CD8⁺ T cells (CD45⁺CD3⁺CD8⁺) in Il17rc^-/- and WT mice Data were analyzed by Mann Whitney U tests t-tests and presented as mean ± s.e.m, p > 0.05.

https://doi.org/10.1371/journal.ppat.1013218.s003

(TIF)

S4 Fig. The expression of granzyme B in infiltrated CD8⁺ T cells in the brain of Il17rc^-/- mice.

Eight to nine weeks old Il17rc^-/- (n = 4) and WT mice (n = 4) were infected with 20 PFU of WNV via f.p. inoculation, and brain tissue was collected on D8 p.i. The brain leukocytes were characterized by flow cytometry after probing with antibodies against CD45, CD3, CD8, and granzyme B. (A) percentages of granzyme B expression in CD8⁺ T cells (CD45^hiCD3⁺CD8⁺granzyme B⁺); (B) MFI of granzyme B expression in CD8⁺ T cells (CD45^hiCD3⁺CD8⁺granzyme B ⁺). Data were presented as mean ± s.e.m.

https://doi.org/10.1371/journal.ppat.1013218.s004

(TIF)

S1 Table. Primer sequences used in the study.

https://doi.org/10.1371/journal.ppat.1013218.s005

(DOCX)

Acknowledgments

The authors thank Dr. Anne Le at Gigantest for analyzing the metabolomic results, Dr. Richard A. Flavell at Yale University School of Medicine for providing the breeding pairs of Il17a^-/- mice, Dr. Sarah L. Gaffen at the University of Pittsburg for providing the breeding pairs of Il17ra^-/- mice, Dr. John F. Anderson at the Connecticut Agricultural Experiment Station for providing the WNV isolate CT2741, and Dr. Michael S. Diamond at Washington University School of Medicine in St. Louis for providing the MC57GL_WNV-E and MC57GL_vector cells. We also thank the NIH-sponsored Mississippi INBRE (P20GM103476) for using their research facility.

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Figures

Abstract

Author summary

Introduction

Results

IL-17RC is essential to protect mice from WNV infection

IL-17A-IL-17RC signaling promotes infiltration and cytotoxicity of CD8+ T cells in the brain

IL-17A-IL-17RA signaling increases the cytotoxicity of WNV-specific CD8+ T cells

IL-17A-IL-17RC signaling increases the cytotoxicity of WNV-specific CD8+ T cells

IL-17A signaling activates PI3K-mTOR signaling in CD8+ T cells

IL-17A signaling stimulates metabolism in CD8+ T cells during WNV infection

IL-17A-mediated PI3K-mTOR signaling increases the cytotoxicity of CD8+ T cells

Discussion

Methods

Ethical statement and biosafety

Cells and viruses

Mice and animal studies

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Immunofluorescence assay (IFA)

TUNEL assay

Flow cytometry

Detection of immune cells from mouse brain.

Assessment of immune cell development.

Detection of cytotoxic mediators in WNV-specific CD8+ T cells.

Detection of PI3K-mTOR mediators in CD8+ T cells.

Generation of conditional knock-out mice

Purification of CD8+ T cells

RNA sequencing

CD8+ T cell cytotoxicity assay

The Ex vivo rIL-17A treatment assay

Western blot

Metabolomic study

Statistical analysis

Supporting information

S1 Fig. Viral load in different tissues in Il17rc-/- mice in WNV infection.

S2 Fig. The antiviral and inflammatory responses in the plasma of Il17rc-/- mice.

S3 Fig. Detection of immune cells in spleen and blood in Il17rc-/- mice.

S4 Fig. The expression of granzyme B in infiltrated CD8+ T cells in the brain of Il17rc-/- mice.

S1 Table. Primer sequences used in the study.

Acknowledgments

References

IL-17A-IL-17RC signaling promotes infiltration and cytotoxicity of CD8⁺ T cells in the brain

IL-17A-IL-17RA signaling increases the cytotoxicity of WNV-specific CD8⁺ T cells

IL-17A-IL-17RC signaling increases the cytotoxicity of WNV-specific CD8⁺ T cells

IL-17A signaling activates PI3K-mTOR signaling in CD8⁺ T cells

IL-17A signaling stimulates metabolism in CD8⁺ T cells during WNV infection

IL-17A-mediated PI3K-mTOR signaling increases the cytotoxicity of CD8⁺ T cells

Detection of cytotoxic mediators in WNV-specific CD8⁺ T cells.

Detection of PI3K-mTOR mediators in CD8⁺ T cells.

Purification of CD8⁺ T cells

CD8⁺ T cell cytotoxicity assay

S1 Fig. Viral load in different tissues in Il17rc^-/- mice in WNV infection.

S2 Fig. The antiviral and inflammatory responses in the plasma of Il17rc^-/- mice.

S3 Fig. Detection of immune cells in spleen and blood in Il17rc^-/- mice.

S4 Fig. The expression of granzyme B in infiltrated CD8⁺ T cells in the brain of Il17rc^-/- mice.