Effect of Pre-Treatment with a Recombinant Chicken Interleukin-17A on Vaccine Induced Immunity against a Very Virulent Marek’s Disease Virus

The host response to pathogenic microbes can lead to expression of interleukin (IL)-17, which has antimicrobial and anti-viral activity. However, relatively little is known about the basic biological role of chicken IL-17A against avian viruses, particularly against Marek’s disease virus (MDV). We demonstrate that, following MDV infection, upregulation of IL-17A mRNA and an increase in the frequency of IL-17A+ T cells in the spleen occur compared to control chickens. To elaborate on the role of chIL-17A in MD, the full-length chIL-17A coding sequence was cloned into a pCDNA3.1-V5/HIS TOPO plasmid. The effect of treatment with pcDNA:chIL-17A plasmid in combination with a vaccine (HVT) and very virulent(vv)MDV challenge or vvMDV infection was assessed. In combination with HVT vaccination, chickens that were inoculated with the pcDNA:chIL-17A plasmid had reduced tumor incidence compared to chickens that received the empty vector control or that were vaccinated only (66.6% in the HVT + empty vector group and 73.33% in HVT group versus 53.3% in the HVT + pcDNA:chIL-17A). Further analysis demonstrated that the chickens that received the HVT vaccine and/or plasmid expressing IL-17A had lower MDV-Meq transcripts in the spleen. In conclusion, chIL-17A can influence the immunity conferred by HVT vaccination against MDV infection in chickens.


Introduction
Since the discovery of murine and human T helper (Th) 17 cells, much progress has been made in defining their developmental origin, lineage, relative biological function, and respective expression of the IL-17 cytokine family members (IL-17A to IL-17F) [1,2]. It is clear that, in mice and humans, IL-17 has a role in immune responses associated with allergenic disease, autoimmune disease, malignancy, transplantation rejection, and modulation of host defense against both viral and bacterial microbes. While the expression of the avian IL-17 family of cytokines has been identified using intestinal intraepithelial lymphocytes (IELs), the functional role of these cytokines beyond differential gene expression has not been described [3].
IL-17 signaling is mediated through the IL-17A/F heterodimeric receptor complex (IL-17RA). The latter was demonstrated to have heterogeneous tissue distribution, which may serve to allow tissue-specific signal transduction [4]. As in mammalian hosts, in chickens, the IL-17RA molecule constitutes a distinct family of transmembrane proteins that are expressed by various immune system cells and fibroblasts [5,6]. IL-17A/IL-17RA interaction results in the induction of various cytokines ((IL-6, tumor necrosis factor (TNF)-α, and granulocyte monocyte colony stimulator factor (GM-CSF)), chemokines, antimicrobial peptides, and tissue-remodeling molecules. While Th17 cells are the best characterized source of IL-17, various innate immune system cells and T cell subsets can also express
(ii) Transfection/stably expressing cell lines: One million HEK 293-T cells were seeded per well in a 6 well plate. Overnight cells (>80% confluent) were transfected, according to Viruses 2023, 15, 1633 3 of 20 the manufacturer's recommendation, using Lipofectamine stem reagent (Life Technologies, Canada) with up to 3 µg of linearized pCDNA3.1/V5-HIS-TOPO empty vector or pCDNA3.1/rchIL-17A-V5-HIS TOPO. After 48 h (hrs), cells were passaged (0.5% trypsin; Life Technologies, Canada) into T25 flasks and treated with 800 µg/mL G418 in MEM complete medium (10% FBS and 0.1% penicillin-streptomycin). The medium was changed every 48 hrs until proliferating islands of cells were observed. After 2-3 weeks of antibiotic selection, stably expressing cells were cultured without G418 for the purpose of collecting both cells and supernatant to confirm mRNA expression, as well as extracellular secretion of recombinant chicken IL-17A (rchIL-17A), and the cells were stored at −80 • C until required.

Virus Preparation
The very virulent MDV-RB1B strain (vvMDV), was provided courtesy of Dr. K.A. Schat (Cornell University, Ithaca, NY, USA) [17]. vvMDV-RB1B virus titers were calculated on primary chicken kidney cells obtained from 2-to 3-week-old specific pathogen free (SPF) chickens to establish infectious doses of inoculums, as well as stocks (liquid nitrogen storage) [23]. The HVT vaccine strain (MD-Vac-CFL; Fort Dodge Animal Health, Fort Dodge, IA, USA) was diluted in the recommended diluent and stored on ice prior to use.

Experimental Design and Sampling
(i) Experimental animals: Two hundred ninety-eight-day-old SPF chickens (layers) were purchased from the Animal Disease Research Institute, Canadian Food Inspection Agency (Ottawa, ON, Canada), and were accommodated in the isolation unit at the University of Guelph. For the duration of the experiments, all chickens were given ad libitum access to food and water. All experiments were approved by the Animal Care Committee of the University of Guelph and were conducted according to their guidelines. (iii) Experimental outline: In the first experiment, 2-week-old chicks (n = 36) were injected intramuscularly (I/M) in the pectoral muscle with 100 µL of either CpG ODN 2007 (10 µg; n = 12) or Poly I:C (400 µg; n = 12). The control group (n = 12) received 100 µL of PBS (negative control). At 8 and 16 h post-inoculation, 6 chicks from each group were euthanized for sample collection.
In the second experiment, 1-day-old chicks (n = 108) were administered the HVT vaccine at one-half recommended dosage via a subcutaneous route. A reduced vaccine dose was used to observe the full potential of recombinant chicken IL-17A when used on its own or as a vaccine adjuvant. On day 3 post-vaccination, two groups of chicks were either inoculated intramuscular with 10 µg/chick of pCDNA3.1/V5-HIS-TOPO empty vector (n = 72) or pCDNA3.1/rchIL-17A-V5-His TOPO (n = 72). On day 4 post-vaccination, 198 chicks were infected with 250 plaque-forming units of the vvMDV-RB1B strain or sham/diluent treatment via the intraabdominal route.
(iv) Sampling: At various time points, tissue samples were collected for various analytical processes. Chicks (n = 6/group) from the first experiment were euthanized at 8 and 18 h post-TLR treatment, after which whole spleens were collected and stored (−20 • C) in RNA (Invitrogen, Toronto, ON, Canada). In the second experiment, whole spleens (n = 12/group) were collected in PBS containing penicillin (10 U/mL), and streptomycin (10 µg/mL) at 4, 10, and 21 days post infection (DPI), and parts of the whole spleen were either stored in RNA later for RNA extraction or were frozen in OCT embedding medium for immunostaining. Whole spleen samples were also collected from 5 control (non-infected chickens) and vvMDV-RB1B infected chickens at 4, 10, and 21 DPI and stored on ice for mononuclear cell isolation.

Spleen Mononuclear Cell Preparation and Stimulation
(i) Mononuclear cells: Whole spleens collected aseptically at various time points were applied onto 40-µm BD cell strainers (BD Biosciences, Mississauga, ON, Canada), crushed through using the rubber end of a 10-mL syringe plunger, and washed in RPMI 1640 with penicillin (10 U/mL) and streptomycin (10 µg/mL) [24]. Gradient suspensions were prepared by layering cells (2:1) onto histopaque 1077 (Millipore-Sigma, Oakville, ON, Canada) and centrifugation at 2100 rpm (600× g with no brakes) for 20 min to allow for the separation of mononuclear cells. Aspirated buffy coats were washed (2×) at 1500 rpm (400× g) for 5 min in RPMI 1640 with penicillin (10 U/mL), and streptomycin (10 µg/mL). Mononuclear cells were suspended in complete RPMI cell culture medium; RPMI 1640 medium contains 10% fetal bovine serum (Millipore-Sigma, Canada), penicillin (10 U/mL), and streptomycin (10 µg/mL). Cell numbers and viability were calculated using a hemocytometer and trypan the blue exclusion method. Mononuclear cells were suspended in complete RPMI cell culture medium at a density of 5 × 10 6 cells/mL and kept on ice.
(ii) In vitro stimulation assays: Spleen mononuclear cells were seeded in 96-well Ubottom plates at a density of 1.0 × 10 6 cells/200 µL and stimulated first with vehicle and then either CpG ODN 2007 (5 ug/mL) or lipopolysaccharides (LPS; 10 µg/mL). Cells were subsequently incubated (41 • C and 5% CO 2 ) overnight (18 h), and supernatants were collected for anti-IL-17A enzyme-linked immunosorbent assay (ELISA). All stimulation assays were performed at least three times in triplicate on different days. Second, cells were co-incubated with supernatant from pCDNA3.1/V5-HIS-TOPO empty vector or pCDNA3.1/rchIL-17A-V5-HIS TOPO stably expressing cell lines on ice (15 min in the dark) for FACS analysis.

RNA Extraction and Reverse Transcription
RNA was extracted from in vitro mononuclear cell cultures, as well as spleens, using Trizol following the manufacturer's instructions and treated with DNA-free DNase (Ther-moFisher Scientific, Mississauga, ON, Canada) as previously described [25]. The quality, as well as the quantity, of RNA was estimated using NanoDrop ND-1000 spectrophotometry (NanoDrop Technologies, Wilmington, DE, USA). Subsequently, 1 µg of purified RNA was reverse transcribed to cDNA using Oligo(dT)12-18 primers (SuperScript II First-Strand Synthesis System; Invitrogen Life Technologies, Carlsbad, Ottawa, ON, Canada) according to the manufacturer's recommended protocol. The resulting cDNA was diluted 1:9 in diethyl pyrocarbonate-treated (DEPC) water.

Real-Time Polymerase Chain Reaction (RT-PCR)
Quantitative real-time PCR with SYBR green was performed on diluted cDNA using a LightCycler 480 II (Roche Diagnostics GmbH, Mannheim, GER) according to the manufacturer's recommendation. In brief, quantitative real-time PCR was performed with the following conditions: initial denaturation was performed at 95 • C for 5 min, followed by 40 cycles of denaturation at 95 • C for 10 s (s), primer annealing listed in Table 2 for 15 s, and extension at 72 • C for 20 s, with end-point melt-curve analysis. The relative fold change of target genes was calculated by 2−∆∆CT method. The Ct value for each sample was normalized against the GAPDH housekeeping gene for respective samples. Data represent the means from six biological replicates, using primers outlined in Table 2.

Flow Cytometry
Following a wash in FACS staining buffer (PBS with 0.5% Bovine Serum Albumin; BSA), 5.0 × 10 5 spleen mononuclear cells were counter-stained in two assays to demonstrate IL-17A functional interaction, as well as intracellular IL-17A cytokine staining. Antibodies used for the monocyte/macrophage panel (mouse anti-chicken Kul01-fluorescein isothiocyanate: FITC and mouse anti-chicken major histocompatibility complex (MHC) IIphycoerythrin: PE were all purchased from Southern Biotech, Birmingham, AL, USA) and T cell panel (mouse anti-chicken CD3ε-PB, mouse anti-chicken CD4-PE-CY7, mouse antichicken CD8α-FITC and mouse anti-chicken γδTCR-PE were all purchased from Southern Biotech, Birmingham, AL, USA).
(i) rchIL-17A surface binding: Mononuclear cells were incubated (4 • C for 15 min) in the presence of rchIL-17A. The cells were subsequently washed in FACS staining buffer (400× g for 5 min) and incubated (15 min at 4 • C) in the presence of unpurified mouse anti-chIL-17A antibody (IgG2a isotype), kindly provided by Dr. Thomas W. Göbel (Tierärztliche Fakultät, LMU München, Veterinärstrasse 13, 80539 München, Germany) [5]. The cells were washed again and incubated (15 min at 4 • C) in the presence of mouse anti-IgG2a-APC antibody prior to counter-staining per the APC or T cell panels listed. Following a final wash, mononuclear cells were counterstained with the live/dead marker 7-AAD (BDTM Pharmingen, Mississauga, ON, Canada) and acquired for analysis using a BD FACS Canto II. IFN-γ intracellular staining: One million splenocytes were cultured with Golgi Plug and Dnase I in a 96-well round-bottom plate for 4 h at 41 • C in 5% CO 2 . The cell staining protocol for flow cytometry was described elsewhere [32].
All samples were acquired on a BD FACS Canto II. Data were processed by FlowJo software, version V10.

Confocal Microscopy
(i) Sample preparation: OCT-embedded frozen spleen tissue samples were cut using a cryostat at approximately 10 µm in thickness. Samples were mounted onto slides and stored (−80 • C) for immunostaining. The slides were subsequently fixed (1 h in the dark at RT) in 4% paraformaldehyde (PFA). They were washed (PBS), permeabilized with 0.1% triton-X buffer solution (10 min in the dark at RT), and blocked (1 h in the dark at RT) with 1% bovine serum albumin (BSA-PBS). The slides were then washed 3× (PBS) and incubated (1 h in the dark at RT) with the mouse anti-chIL-17A antibody (IgG2a; 500 µL in 0.5% BSA-PBS). Following another wash (3× with PBS), the slides were again incubated overnight (4 • C in the dark at RT) with a goat anti-mouse IgG2a-488 (from ThermoFisher Scientific, ON, Canada) antibody (0.5 µg/500 µL in 0.5% BSA-PBS). The next day, the slides were washed (3× with PBS), and nuclei were labeled with Vectashield containing 4 ,6-diamidino-2-phenylindole (DAPI from Vector laboratories/Cedarlane, ON, Canada). Tissue samples were sealed with round coverslips for fluorescence imaging.
(ii) Visualization: Tissue samples were viewed using a Leica SP5 laser scanning confocal microscope, and optical sections were recorded using either 405 or 488 nM with a numerical aperture of 1.4 or 1.25, respectively. All data were collected sequentially to minimize cross talk between fluorescence signals. The data are presented as maximum projections of each channel and overlaid for analysis with LAS AF software.

Statistical Analysis
Graph-Pad Prism software, version 8 for Windows, was utilized to generate graphs and perform statistical analysis. All data are presented as means + standard deviations (SDs) and analyzed by Tukey's post hoc test, the Kruskal-Wallis non-parametric test, Wilcoxon's test (Mann-Whitney,) or Fisher's exact test with the results shown as the mean ± standard deviation by Graph-Pad Prism software, version 8. The results were considered statistically significant at p < 0.05 (*).

Expression of IL-17A, COX-2, TGF-β, and IFN-γ in vvMDV-RB1B Infected Chickens
The expression of IL-17A in the spleens of vvMDV-RB1B-and non-infected chickens was probed using confocal microscopy and real-time PCR (Figure 1). Our results demonstrated the detection of IL-17A in the spleens of vvMDV-RB1B-infected and not in non-infected chickens at 4 and 10 dpi ( Figure 1A,B). IL-17A could be detected in the spleens of non-infected chickens at 21 dpi ( Figure 1C). Based on punctate fluorescence, expression of IL-17A (488 nm) at the protein level was the highest at 21 days post infection (dpi) ( Figure 1C) in vvMDV-RB1B-infected chicken when compared to 4 ( Figure 1A) and 10 dpi ( Figure 1B). The expression of IL-17A was also confirmed at the mRNA level based on real-time PCR ( Figure 1D). The results demonstrated that IL-17A expression was significantly higher (p < 0.005) at 4 dpi compared to non-infected chickens. No difference was observed at 10 dpi. At 21 dpi, the expression of IL-17A was significantly lower (p < 0.005) compared to non-infected chickens.

Cloning, Expression and Evaluation of the rchIL-17A Bioactivity Based on Receptor Binding
The full-length coding sequence of chIL-17A with signal peptide was subcloned into a pCDNA3.1/V5-HIS-TOPO plasmid by digestion/ligation reactions from a pDRIVE-IL-17A vector (TA cloning) ( Figure 3A,B). HEK 293 T cells were transfected with the linearized pCDNA3.1/V5-HIS-TOPO and pCDNA3.1/rchIL-17A-V5-HIS TOPO to generate supernatant containing the recombinant chIL-17a from stably expressing cell lines. Expression of rchIL-17A in the culture medium was confirmed by an anti-V5 ELISA ( Figure 3C). To demonstrate that a biologically active rchIL-17A protein ( Figure 3B) is expressed by a eukaryotic expression system, chicken spleen mononuclear cells were co-incubated with supernatants from pCDNA3.1/V5-HIS-TOPO (control) or pCDNA3.1/rchIL-17A-V5-HIS-TOPO (IL-17A+)-transfected HEK-293 T cells. Spleen mononuclear cells were further phenotypically differentiated as either APCs (KUL01+MHCII+) or T cells (CD3+ αβ or γδ) per FACS analysis to demonstrate specific interactions ( Figure 3D). Surface binding of rchIL-17A to its biological receptor, IL-17RA, was probed, and the gating strategy demonstrated detection of the rchIL-17A on the cell surfaces of both APCs and various T cell subsets following incubation with both control and IL-17A+ supernatant ( Figure 3D). The results demonstrated that rchIL-17A can bind to a cell surface receptor found on both APCs and CD3+ T cells (αβ and γδ subsets). Co-incubation with the IL-17A+ supernatant delineated the frequency of APCs ( Figure 3E), γδ ( Figure 3F), and αβ ( Figure 3G,H) CD3+ T cell subsets that express the cell surface IL-17RA molecule. Significantly higher (p < 0.05) frequencies of both CD4+ ( Figure 3G) and CD8α+ ( Figure 3H) αβ T cells were observed as expressing the IL-17RA complex.
TOPO (IL-17A+)-transfected HEK-293 T cells. Spleen mononuclear cells were further phenotypically differentiated as either APCs (KUL01+MHCII+) or T cells (CD3+ αβ or γδ) per FACS analysis to demonstrate specific interactions ( Figure 3D). Surface binding of rchIL-17A to its biological receptor, IL-17RA, was probed, and the gating strategy demonstrated detection of the rchIL-17A on the cell surfaces of both APCs and various T cell subsets following incubation with both control and IL-17A+ supernatant ( Figure 3D). The results demonstrated that rchIL-17A can bind to a cell surface receptor found on both APCs and CD3+ T cells (αβ and γδ subsets). Co-incubation with the IL-17A+ supernatant delineated the frequency of APCs ( Figure 3E), γδ ( Figure 3F), and αβ ( Figure 3G, H) CD3+ T cell subsets that express the cell surface IL-17RA molecule. Significantly higher (p < 0.05) frequencies of both CD4+ ( Figure 3G) and CD8α+ ( Figure 3H) αβ T cells were observed as expressing the IL-17RA complex.   The results are based on at least 5-6 biological replicates in each group. Wilcoxon's non-parametric test (Mann-Whitney) was used to test significance with the results shown as means ± standard deviations. * (p ≤ 0.05) and *** (p ≤ 0.001) indicates a significant difference.

Neither CpG ODN 2007 Nor Poly:IC Treatment Induce IL-17A Expression
The class B CpG ODN 2007 (5 µg/mL) and LPS (10 µg/mL) were used for in vitro stimulation of spleen mononuclear cells (Figure 4). After overnight culture, supernatants were collected for indirect ELISA and detection of IL-17A ( Figure 4A). The results demonstrated that LPS, but not CpG ODN 2007, is an inducer of IL-17A expression. We further demonstrated that TLR3 or TLR21 signaling following intramuscular inoculation of 2-week-old chicks with either poly:(IC) or CpG ODN, respectively, did not lead to IL-17A expression at 8 and 18 hrs post-inoculation compared to PBS-treated chickens ( Figure 4B).
vvMDV-RB1B-infected chickens did not result in changes in expression of either IL-12p40 ( Figure 7E) or IFN-γ ( Figure 7F) transcripts compared to vvMDV-RB1B-infected chickens only. The combination with HVT resulted in an increase in both IL-12p40 ( Figure 7E) and IFN-γ ( Figure 7F) transcripts. Specifically, chickens that were inoculated with HVT and/or that were rchIL-17A or empty vector treated and subsequently infected (vvMDV-RB1B) had significantly higher expression of both IL-12p40 ( Figure 7E; p < 0.01) and IFN-γ (Figure 7F; p < 0.01) genes compared to the non-infected chickens.  The results are based on at least 6 biological replicates in each group. Wilcoxon's non-parametric test (Mann-Whitney) was used to test significance with the results shown as the means ± standard deviations. * (p ≤ 0.05) ** (p ≤ 0.01) and *** (p ≤ 0.001) indicates a significant difference.

Discussion
The IL-17A cytokine is a T cell-derived proinflammatory cytokine that has an important role in inflammation and immunity [1,7]. IL-17A is believed to be mainly produced by Th17 cells, which constitute a unique helper T-cell subset well defined in mice and humans [33]. While avian Th17 cells have yet to be characterized, IL-17A gene expression has recently been observed in mitogen stimulated avian αβ and γδ T cells and further detected via intracellular cytokine staining in various T cell subsets [5,6]. In this study, we demonstrated that: (i) infection with MDV resulted in early induction of IL-17A and IFN-γ expression in chicken splenocytes; (ii) based on FACS analysis, IL-17A+ T cells could be detected in splenocytes from vvMDV-RB1B infected chickens with the highest proportion of IL-17A+ T cells peaking at 21 dpi; and (iii) in vivo inoculation with a recombinant chIL-17A post-HVT vaccination significantly reduced tumor incidence compared to infected-only chickens compared to HVT+IL-17A or HVT+vector control. This outcome was associated with a reduction in COX-2, TGF-β, and MDV-Meq gene expression and an increase in IL-12p40 and IFN-γ expression.
The function and expression of IL-17A in the avian lung mucosal tissue following infection with MDV have not been previously defined. While the avian host's early response (4 dpi) to MDV infection results in overexpression of both IL-17A and IFN-γ, their respective roles in chickens are not well understood. Due to the cell-associated nature of MDV, T cells play an essential role in immunity against this virus. We previously reported that the frequency of IFN-γ+CD8α+ γδ T cells in vvMDV-RB1B-infected chickens was significantly higher at 4 (p < 0.05) and 10 dpi (p < 0.01) than in the control group [32]. Pre-treatment with a recombinant chIFN-γ in an MDV infection model was associated with a reduction in tumor incidence [19]. However, subsequent IFN-γ knockdown based on an siRNA treatment had limited to no effects on MDV replication and tumor incidence [34]. More recently, Bertzbach et al. showed that chIFN-γ can have direct anti-viral effects against MDV and was induced by NK cells in vitro following MDV infection [35,36]. This finding indicated that other mechanisms exist that can limit MDV replication. In the present study, we demonstrated a relationship between the specific stages of MDV pathogenesis (4, 10, and 21 dpi) and percentage of IFN-γ+ and IL-17A+ T cells. Based on intracellular cytokine staining, the frequency of IFN-γ+ αβ T cells peaked at 10 dpi and was significantly lower at 4 and 21 dpi compared to 10 dpi. IL-17A+ T cells could be detected in splenocytes from vvMDV-RB1B infected chickens with the highest proportion of IL-17A+ αβ and γδ T cells peaking at 21 dpi. It is unclear whether the reduction in IFN-γ+ and increase in IL-17A+ T cells at 21 dpi relate to CD4+ T cell expansion and aggravation of lymphoma. In mice, IFN-γ is the major cytokine produced by CD27+ γδ T cells, and their CD27− counterparts preferentially secrete IL-17A [37,38]. In the present study, double positive IL-17A+IFN-γ+ T cells could not be defined due to limitations in antibody availability. Both IL-17A and IFN-γ can regulate the lineage differentiation of naïve T cells into Th1 or Th17 cells, respectively [33]. The presence of both IFN-γ+ and IL-17A+ T cells during MDV infection suggests a potential role in MDV pathogenesis.
The biological role of IL-17A has yet to be fully characterized in chickens but has been defined in mice and humans [39]. We observed a discrepancy between the abundance of IL-17A cognate protein and transcripts following MDV infection. Protein and RNA represent different steps of the multi-stepped cellular process, in which they are expressed, translated, and degraded. Specific regulatory processes can suppress mRNA transcription, thereby leading to no or limited protein expression. MDV is known to express and stimulate the expression of an array of micro RNAs that impact the host cellular machinery [40]. At mucosal sites, IL-17 may have protective roles by enhancing the Th1 response in host defenses against infectious diseases and promoting the induction of cytotoxic T lymphocyte (CTL) responses [33]. HVT-vaccinated chickens that were pre-treated with the rIL-17A had an increase in IL-12p40 and IFN-γ expression at 21-dpi compared to vvMDV-RB1B-infected chickens. This outcome was associated with a reduction in the expression of IL-10 and TGF-β at 21 dpi in IL-17A-pre-treated and HVT-vaccinated chickens. To gain some insight into the immune mechanisms triggered by treatment with IL-17A in chickens, we analyzed the cytokine expression profiles in the spleen. Both negative and positive feedback loops regulate the local and system effects of IL-17A.
Compared to negative control groups, vaccination with HVT does not result in a significant increase in IL-17A expression. Similarly, Hao et al. demonstrated that vaccination with the CVI988-RISPENS vaccine does not induce IL-17A expression [41]. This finding suggests that HVT vaccination elicits a cytokine-specific pattern of expression that works in synergy with pCDNA:IL-17A to modulate the IL-17A-mediated immunopathology observed post-MDV infection. In chickens, infection with a vvMDV-RB1B virus led to a significant increase in IL-17A expression at 4 dpi with no changes at 10 dpi and a significant reduction at 21 dpi compared to control chickens. It is possible that the increased production of proinflammatory cytokines in the absence of IL-17A during vaccination is a compensatory mechanism implemented to help protect the avian host against MDV infection. This theory suggests that IL-17A could be involved in modulating the lung mucosal environment. Viruses can utilize IL-17A to modulate the host immune system to support productive infection. Gammaherpesviruses, such as murine gammaherpesvirus 68 (MHV68), preferentially modulate the mouse immune system following primary infection, leading to IL-17A expression [42]. As such, during early infection, IL-17A can antagonize Th1 activity while promoting productive MHV68 viral infection. IL-17A directly suppresses the expression of IFN-γ, T-bet, and eomesodermin in T cells [43]. In MDV infection, we observed an increase in both IL-17A and IFN-γ during the early stage of infection. While antigen-specific T cell responses based on IFN-γ could be detected in MDV-infected chickens, it is not clear whether overexpression of IL-17A could limit antigen-specific T cell responses [18]. However, following MDV infection, IL-17A expression was induced during the early phase (4 dpi). More work is required to define whether IL-17A could be involved in the pathogenesis of MD.
In mammals, T cells of the γδ subset immediately produce IL-17A and induce inflammation upon pathogenic infections [44]. It is understood that immunity against herpes simplex virus type (HSV) 1 is partly imparted by the functional activity of IL-17A+ γδ T cells [45]. γδ T cells exert their immune-modulatory activity by secreting various innate factors, as well as Th1 and Th17 cytokines, which promote direct cytotoxic activity against infected cells [46]. During the early phase of MDV pathogenesis, high counts of both macrophages and T cells can be seen in the lungs of MDV-infected chickens [47]. Our FACS results indicated that γδ T cells are a major source of IL-17A during the later stages of MDV infection. It cannot be ruled out that the differentiation of IL-17A-producing γδ T cells occurs within the tumor microenvironment, but this possibility requires further experimental confirmation. IL-17A has been implicated in protective γδ T cell responses in other cancer models. Mechanistically, tumor-infiltrating IL-1β activated IL-17A+ γδ T cells, enhancing the priming and recruitment of IFN-γ-producing CD8+ T cells that exert antitumor effects [48]. We have previously reported that avian γδ T cells are activated during MDV infection and retain effector functions, based on expression of IFN-γ and cytotoxic (CD107a+) activity [32]. It is not known whether the increase in IL-17A at 4 dpi relates to an increase in T cell infection and modulation of antigen-presenting cell functions, such as macrophages [33]. However, IL-17 can promote the recruitment of innate cells to the site of inflammation [49]. We identified an increase in the percentage of IL-17A+ T cells at 21 dpi in the spleens of MDV-infected chickens. Chronic IL-17A production, at the protein level, has been shown to support tumor formation and progression [50]. This effect is derived from IL-17A-mediated signaling, which results in recruitment of innate immune cells to the site of injury [51]. IL-17-induced inflammatory mediators, such as IL-1β, IL-6, and TGF-β, promote a proangiogenic and immune suppressive tumor environment that enhances tumor growth. It is likely that the host response to MDV infection, which results in IL-17A progression, contributes to tumor formation. MDV is an oncogenic virus causing CD4+ T cell lymphoma in chickens. Transformation of CD4+ T cells has been linked to MDV-Meq function [52]. MDV-Meq has been shown to modulate the PI3K/AKT pathway [53]. In mice, the PI3K/AKT pathway regulates IL-17A expression and the induction of Th17 cells [54]. However, the mechanism for induction of IL-17A in MDV-infected chickens is unknown. Furthermore, it has yet to be shown whether IL-17A-mediated signaling during MDV results in tumor progression. Taken together, induction of IL-17A during MDV infection can have multifactorial effects.

Conclusions
In summary, our data revealed the effects of IL-17A during MDV infection and improved our understanding of adaptive immune responses against MDV. Preventive strategies that aim to induce strong Th1 cell immunity should be able to effectively control MDV infection. Along with Th1 responses, MDV infection also induces a Th17 immune response. More work is required to demonstrate this response in avian Th17 cells and the modulatory activity of IL-17A in avian innate and adaptive immune cell types.