PI3K/mTOR pathway regulates IL-22 expression from γδ T cells in viral infection

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that belongs to the PI3K-related protein kinase family and regulates cell growth, proliferation, and differentiation^{10, 11, 22}. IL-23 is responsible for activating the PI3K/Akt pathway to promote IL-17 expression in CD4⁺ T cells²³. However, it remains unclear as to whether the signaling pathway regulates IL-22 production in viral infection. To address this question, we isolated intrahepatic lymphocytes (IHLs) from LCMV-infected mice at 3 dpi and treated these cells in vitro with IL-23, in the presence or absence of PI3K inhibitor (Ly294002) or mTOR complex 1 (mTORC1) inhibitor (rapamycin). No significant toxicity was observed by the treatments of these inhibitors in the indicated concentrations in vitro (Fig. 2A). IL-23 treatment significantly promoted both IL-22 and IL-17A expression in γδ T cells. Rapamycin and Ly294002 dramatically suppressed the stimulatory effects of IL-23 on IL-22 and IL-17A production (Fig. 2B). IL-22 as well as IL-17 levels in the supernatant were suppressed consistently by rapamycin and Ly294002 (Fig. 2C).

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Figure 2

PI3K/mTOR pathway regulates IL-22 and IL-17 expression in virus-exposed γδ T cells. (A to D) IHLs were isolated from Clone 13-infected B6 mice at 3 dpi and then cultured with indicated conditions overnight. IL-23 (20 ng/ml); Rapamycin (25 nM), mTOR inhibitor; Ly294002 (5 μM), PI3K inhibitor. (A) The living γδ T cells were stained with live dye and enumerated using flow cytometry. (B) IL-22 and IL-17A expression in γδ T cells was analyzed by flow cytometry. (C) IHLs were incubated with indicated conditions in vitro for 3 days, and then IL-22 and IL-17 levels in the supernatants were determined by ELISA. (D) IHLs of naïve or infected B6 mice were treated with IL-23 as indicated. Flow cytometry was used to detect pS6 expression in γδ T cells. (E and F) TSC-1-deficient (TSC-1^Δ) or control mice were challenged with Clone 13 (2×10⁶ pfu/mouse). IHLs were isolated at 3 dpi. (E) IHLs were cultured with or without IL-23 for 30 min. Expression of pS6 was examined by flow cytometry. (F) IHLs were cultured with or without IL-23 overnight. IL-22 and IL-17A expression in γδ T cells was detected by flow cytometry. Data are representative of three independent experiments. The results are expressed as the mean±SEM (n=3–5). *p<0.05; **p<0.01; ***p<0.001.

To further investigate the mechanistic action of the PI3K/mTOR signaling pathway in viral infection, we measured mTORC1 activation by monitoring the phosphorylation status of ribosomal protein S6 (pS6), a down-stream target of the mTORC1-mediated pathway²⁴. Hepatic γδ T cells of virus-infected mice had a higher level of pS6 (30% positive) compared to that in the naïve cells (18% positive) (Fig. 2D). IL-23 treatment in vitro greatly increased pS6 levels in virus-exposed hepatic γδ T cells, suggesting an IL-23-mediated activation of the mTORC1 signaling pathway in these cells. To further confirm that the activation of the IL-23/PI3K/mTOR axis affects IL-22 production in vivo, we depleted the expression of TSC1 (the up-stream negative regulator of mTORC1) by injecting TSC1 ^fl/fl mice with AAV5-Cre (TSC-1^Δ) or control vector (AAV5)^25–27, and then challenged these animals with LCMV. Hepatic γδ T cells from TSC-1^Δ mice had a higher level of pS6, which confirms the up-regulated mTORC1 activity (Fig. 2E). Importantly, TSC1-deficient γδ T cells exhibited increased IL-22 and IL-17A expression, and this promotion was augmented by IL-23 (Fig. 2F). Then taken together, these data from LCMV infection studies supported results showing that IL-22 production from γδ T cells was tightly regulated via the IL-23/PI3K/mTOR axis.

AhR signaling is dispensable for IL-23-induced IL-22 production in γδ T cells

The transcriptional factor AhR has been considered as a positive inducer of IL-22 in CD4⁺ T cells and ILCs in the gut^{28, 29}. To determine whether AhR regulates IL-22 production from γδ T cells in viral infection, we measured the effects of AhR inhibitor on IL-22 expression in hepatic γδ T cells at 3 dpi. AhR inhibitor reduced IL-22 expression in γδ T cells in the absence of exogenous IL-23 (Fig. 3A). However, in the presence of IL-23, the AhR inhibitor did not impede IL-22 production from γδ T cells (Fig. 3A). Similar results were observed when we analyzed the supernatant IL-22 in the culture of IHLs (Fig. 3B). To confirm this finding, we isolated LCMV-exposed IHLs from wild-type (WT) and AhR ^−/− mice, and then cultured these cells with or without IL-23. AhR deficiency resulted in lower IL-22 production in the absence of IL-23 stimulation; however, IL-23 administration overcame AhR deficiency and restored the IL-22 secretion (Fig. 3C). These data indicated that in LCMV infection IL-23 regulates IL-22 production in an AhR-independent fashion.

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Figure 3

IL-22 expression in γδ T cells is independent of AhR signaling in the presence of IL-23. (A and B) Clone 13-induced IHLs at 3 dpi were cultured at the indicated conditions overnight. IL-23 (20 ng/ml); AhR i (3 μM), AhR inhibitor. (A) The percentage of IL-22-expressing γδ T cells was tested by flow cytometry. (B) IHLs were incubated under described conditions in vitro for 3 days, and then IL-22 levels in the supernatant were examined with ELISA. (C) IHLs from WT and AhR ^−/− mice were incubated with Clone 13 (1×10⁶ pfu/ml) and stimulated with or without IL-23 (20 ng/ml) in vitro for 3 days. IL-22 levels in the supernatants were determined by ELISA. Data are representative of two independent experiments. The results are expressed as the mean±SEM (n=3). *p<0.05; **p<0.01; ***p<0.001. ns, no significant difference.

IL-22 deficiency enhances expansion, recruitment and activity of T cells in acute viral infection

To explore whether IL-22 plays a role in acute viral infection, we infected WT and IL-22 ^−/− mice with the LCMV Armstrong (Arm) for 7 days. Although a lack of IL-22 had no effect on the size and weight of the liver, it resulted in significant splenomegaly and thymic proliferation (Fig. 4A). In this case, the total number of splenocytes and thymocytes was significantly increased in IL-22 ^−/− mice (Fig. 4B). No such difference was observed for the lymphoid organs in non-infected WT and IL-22 ^−/− mice (Fig. 4A and B). To identify the role of IL-22 in lymphoid organs, we analyzed IL-22Rα receptor expression in thymic and splenic stromal cells in infected mice. IL-22Rα was expressed in splenic stromal cells (CD45⁻), thymic non-epithelial stromal cells (CD45⁻), and thymic epithelial cells (CD45⁻MHCII⁺), with a frequency of 26%, 9% and 40%, respectively (Supplementary Fig. ^S2A). These data indicate that in viral infections the IL-22/IL-22R axis is crucial for host responses in lymphoid organs.

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Figure 4

IL-22 deficiency enhances effector T cell expansion in acute viral infection. WT and IL-22 ^−/− mice were infected with Arm (2×10⁵ pfu/mouse) for 7 days. (A) The size and weight of the liver, spleen and thymus were examined. (B) The total number of leukocytes in the liver, spleen and thymus were counted. (C) Plots for the percentage and number of hepatic CD4⁺ and CD8⁺ T cells. (D) Plots for the number of CD4⁺ and CD8⁺ T cells in the spleen and thymus, as well as thymic double-positive (DP) T cells by gating on CD3⁺ cells. (E) The expression of CD44, CXCR3, and Ki67 in splenic CD4⁺ T cells was analyzed by flow cytometry. Data are representative of three independent experiments. The results are expressed as the mean±SEM (n=9). *p<0.05; **p<0.01; ***p<0.001. ns, no significant difference.

We further examined the effects of IL-22 on the output of lymphocytes in acute LCMV infection. CD8⁺ T cells were the dominant lymphocyte population (around 60%) in the livers of WT as well as IL-22 ^−/− mice, and the frequency and total number of CD8⁺ T cells in the liver of IL-22 ^−/− mice were comparable to those in WT mice (Fig. 4C). In contrast to CD8⁺ T cells, a lack of IL-22 resulted in a doubled frequency and number of CD4⁺ T cells in the liver compared to those found in WT mice (Fig. 4C). In addition to the non-lymphoid organs, IL-22 deficiency also strikingly doubled or even tripled the counts of splenic CD4⁺ or CD8⁺ T cells (Fig. 4D). In the thymus, IL-22 deficiency resulted in increased CD4⁺, CD8⁺ and double-positive (DP) cells when gated on either CD3⁺ cells or total thymocytes (Fig. 4D, Supplementary Fig. ^{S2B and C}). We then examined whether IL-22 deficiency impacts the activation, trafficking activities, and proliferation of T cells during acute infections. The absence of IL-22 resulted in an increased expression of the activation marker CD44, chemokine receptor CXCR3, and proliferation marker Ki67 in the splenic CD4⁺ T cells (Fig. 4E). In addition, the elevated T cell output was not a consequence of the cells being refractory to cell death, as IL-22 deficiency did not significantly reduce T cell apoptosis in the thymus and spleen (Supplementary Fig. ^S2D).

Next, we assessed whether a lack of IL-22 affects the function of effector T cells. Lymphocytes in the liver and spleen from LCMV Arm-infected mice at 7 dpi were stimulated with LCMV peptide GP 61 or GP 33 for viral-specific CD4⁺ or CD8⁺ T cells, respectively. IL-22 deficiency resulted in higher frequency of epitope-specific IFN-γ⁺TNF-α⁺ and IFN-γ single-positive CD4⁺ and CD8⁺ T cells in the liver and spleen (Fig. 5A and B). The percentage of TNF-α⁺ population was also increased in IFN-γ⁺CD4⁺ T cells of IL-22 ^−/− mice (Supplementary Fig. ^S2E). Thus, IL-22 affects both polyfunctional and IFN-γ single-positive T cell subtypes. Since few cytokine-producing cells were detected without peptide stimulation (Fig. 5A and B), these data suggest to us that the elevated T cell response is driven by an antigen-specific mechanism rather than by a generalized inflammatory environment. In addition, the absence of IL-22 slightly decreased ALT and AST levels in the serum (Fig. 5C) and slightly attenuated liver histological injuries (Supplementary Fig. ^S2F). Importantly, intensive viral-specific CD4⁺ and CD8⁺ T cell functions were associated with an obviously decreased viral load in liver, lung and spleen tissues at 7 dpi (Fig. 5D). These data suggest that, during acute LCMV infection, the lack of IL-22 boosts anti-viral T cell responses without significant immunological injuries in inflamed tissues.

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Figure 5

IL-22 deficiency augments T cell anti-viral activities during acute viral infection. WT and IL-22 ^−/− mice were infected with LCMV Arm (2×10⁵ pfu/mouse) for 7 days. (A and B) Leukocytes were isolated and incubated with or without LCMV peptide GP 61 or GP 33 for 5h. IFN-γ and TNF-α production in viral-specific CD4⁺ and CD8⁺ T cells in the liver (A) and spleen (B) was determined by flow cytometry. (C) Serum ALT and AST levels were tested. (D) Expression of viral mRNA in the liver, lung and spleen tissues at 7 dpi was determined by qPCR. Data are representative of three independent experiments. The results are expressed as the mean±SEM (n=9–12). *p<0.05; **p<0.01; ***p<0.001.

Over-expression of IL-22 shrinks lymphoid organs and dampens T cell response in acute viral infection

To establish the role of IL-22 in the thymic and splenic pools during acute viral infection, we investigated the effect of IL-22 over-expression on T cell expansion and maintenance in lymphoid pools by the hydrodynamic injection of IL-22 plasmid. IL-22 levels were identified in the thymus, spleen, liver, and lung tissues as well as the serum (Supplementary Fig. ^S3A). Over-expression of IL-22 resulted in apparent atrophy in the spleen and thymus during acute LCMV infection (Fig. 6A), accompanied by decreased numbers of splenocytes and thymocytes (Fig. 6B). Moreover, IL-22 over-expression decreased the frequency of CD8⁺ T cells in the spleen and liver and also reduced the counts of single-positive T cells, as well as DP cells in the thymus (Fig. 6C, Supplementary Fig. ^S3B). Excessive IL-22 significantly reduced the number of viral-specific IFN-γ⁺TNF-α⁺ CD4⁺ and CD8⁺ T cells in the spleen and liver (Fig. 6D) and also resulted in a lower proportion of cytokine-producing T cells in the spleen (Supplementary Fig. ^S3C), indicating that IL-22 dampened production of multi-functional T cells in acute viral infection. Consequently, over-expression of IL-22 resulted in a delayed viral clearance in tissues (Fig. 6E).

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Figure 6

Over-expression of IL-22 dampens effector T cell response in acute viral infection. (A to E) B6 mice were infected with LCMV Arm (2×10⁵ pfu/mouse) at day 0, and then hydrodynamically injected with pRK5-mIL-22 (pIL-22) or pRK5 (control) at 3 and 5 dpi. Mice were sacrificed at 7 dpi. (A) The spleen and thymus were weighed. (B) The number of leukocytes in the spleen and thymus was counted. (C) The percentage of CD4⁺ and CD8⁺ T cells in the spleen and liver were analyzed by flow cytometry. (D) The number of IFN-γ⁺TNF-α⁺ CD4 and CD8 T cells in the spleen and liver was determined with flow cytometry. (E) Expression of viral mRNA was determined by qPCR. (F to H) B6 mice were infected with LCMV Arm and then i.p. injected with recombinant IL-22 protein (5 µg/mouse) or PBS at 1, 3 and 5 dpi. Mice were sacrificed at 7 dpi. (F) Plots for the number of splenic or hepatic CD4⁺ and CD8⁺ T cells. (G) The number of IFN-γ⁺TNF-α⁺ CD4 and CD8 T cells in the spleen and liver was analyzed by flow cytometry. (H) Expression of viral mRNA in the liver was determined by qPCR. Data are representative of two independent experiments. The results are expressed as the mean±SEM (n=4–5). *p<0.05; **p<0.01; ***p<0.001.

To confirm our findings with IL-22-expressing plasmid, we next injected infected mice with recombinant IL-22 protein (rIL-22). Similarly, we found that CD4⁺ and CD8⁺ T cell numbers in the spleen and liver were reduced by rIL-22 (Fig. 6F). Administration of rIL-22 resulted in decreased numbers of thymocytes and also slightly reduced the counts of single- and double-positive T cells in the thymus (Supplementary Fig. ^{S3D and E}). Importantly, rIL-22 inhibited the proportion and number of viral-specific IFN-γ⁺TNF-α⁺ CD4⁺ and CD8⁺ T cells in the spleen and liver (Fig. 6G, Supplementary Fig. ^S3F). The rIL-22 treatment also resulted in decreased percentages of TNF-α⁺ cells when gated on IFN-γ⁺ T cells (Supplementary Fig. ^S3F). Accordingly, a higher viral burden in the liver tissues was observed in rIL-22-treated mice (Fig. 6H). Therefore, these data demonstrated that IL-22 possesses impressive features to temper antiviral T cell responses in both lymphoid and non-lymphoid tissues during acute viral infection.

Animals and viruses

C57BL/6 (B6) mice (6-8-weeks-old) were purchased from the Jackson Laboratory (Bar Harbor, ME). IL-22 ^−/− mice on the B6 background were kindly provided by Dr. Wenjun Ouyang of Amegen (Thousand Oaks, CA). AhR ^−/− mice (C57BL/6-Ahr ^tm1.2Arte) were obtained from Taconic (Hudson, NY). TSC1 ^fl/fl mice (TSC1 ^tm1Djk/J) were from The Jackson Laboratory. LCMV strains Clone 13 and Arm were gifts from Dr. Maria Salvato (University of Maryland, MD). To induce acute hepatitis, we i.v. injected mice with Clone 13 (2×10⁶ pfu/mouse) or Arm (2×10⁵ pfu/mouse) for 7 days. To induce a persistent viral infection, we i.v. injected mice with Clone 13 (2×10⁶ pfu/mouse) for 30–60 days. All animal experiments were performed according to the National Institutes of Health Guide for Care and Use of Experimental Animals. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch, Galveston.

Propagation and quantitation of viruses

The LCMV stocks were prepared and titrated according to procedures in a previous report⁵¹. Briefly, viruses were incubated with baby hamster kidney cells (BHK, a gift from Dr. Lihong Zhang, University of Texas Medical Branch at Galveston, TX) for 72 h. Then the culture medium was centrifuged for 10 min at 350×g, 4°C, and the supernatant was stored at −70°C. For quantitation of the virus, Vero cells (a gift from Dr. Lihong Zhang, University of Texas Medical Branch at Galveston) were cultured with a series of 10-fold virus dilutions for 60 min. An overlay containing 0.8% methylcellulose (Sigma-Aldrich) was added into the wells. After 4 days’ culture, immunocytochemistry was performed by using mouse anti-LCMV polyclonal antibody (gift from Dr. Robert Tesh, University of Texas Medical Branch, Galveston, TX)⁵² and the number of positive clusters was counted, followed by the calculation of viral titers.

Hydrodynamic IL-22 delivery

B6 mice were infected with LCMV Arm (2×10⁵ pfu/mouse) at day 0, and then hydrodynamically injected with IL-22-expressing plasmid (pRK5-mIL-22, pIL-22, 10 µg/mouse) or control vector (pRK5, 10 µg/mouse) at 3 and 5 dpi, according to procedures in a previous report³². The pRK5-mIL-22 and pRK5 plasmids were gifts from Dr. Yangxin Fu (UT Southwestern Medical Center, Dallas, TX).

TSC-1 knockout mice model

According to previous methods^25–27, TSC-1 ^fl/fl mice were i.v. injected with Cre-expressing vector (AAV5/Cre, 1×10⁷ pfu/mouse) or control vector (AAV5, 1×10⁷ pfu/mouse). Three weeks later these mice were challenged with Clone 13 (2×10⁶ pfu/mouse). AAV5/Cre and AAV5 vectors were purchased from the Gene Therapy Center Vector Core, the University of North Carolina at Chapel Hill (Chapel Hill, NC).

Isolation of splenocytes, thymocytes and intrahepatic lymphocytes

The spleen and thymus were mechanically dissociated. The supernatant was collected and incubated with red blood cell lysis buffer (BD Biosciences) to remove red blood cells. IHLs were isolated according to our previous method⁵². Briefly, liver tissue was pressed and digested with collagenase IV (0.05%, Roche Applied Science, Indianapolis, IN) at 37°C for 30 min. After digestion, cell suspensions were passed through 70-μm nylon cell strainers to yield single-cell suspensions. Cells were purified by centrifugation (400×g) at room temperature for 30 min over a 30%/70% discontinuous Percoll gradient (Sigma-Aldrich). Then cells were collected from the interphase, thoroughly washed, and re-suspended in complete RPMI-1640 medium containing 10% FBS. The relative percentages of lymphocyte subpopulations were measured by flow cytometry, and their absolute numbers were calculated according to their percentages and the total numbers.

Flow cytometry

Intracellular staining was performed with flow cytometry according to our previously reported methods⁵³. Briefly, for detecting IFN-γ and TNF-α, cells were incubated 5 h with LCMV GP61 peptide (5 μg/ml, AnaSpec, Fremont, CA) or GP33 peptide (5 μg/ml, AnaSpec) for viral-specific CD4⁺ or CD8⁺ T cells, respectively. For IL-22 and IL-17A detection, cells were cultured with or without rIL-23 (20 ng/ml) overnight. Brefeldin A solution (eBioscience) was added at the last 4h of culture. After incubation, cells were collected, stained with fixable viability dye, blocked with Fc receptors blocker (CD16/32), and stained for specific surface molecules. After surface staining, cells were fixed, permeabilized, and stained for intracellular cytokines by using a fixation/permeabilization kit (eBioscience). For phospho-S6 detection, cells were cultured in 96-well plates (2×10⁶ cells in 50 μl RPMI-1640 medium with 2% FBS) for 2 h, and then stimulated with rIL-23 (20 ng/ml) for 0, 15, 30, and 60 min, respectively. These cells were collected and fixed with phosflow lyse/fix buffer (BD Biosciences) for 15 min at 37°C, followed by spinning down and adding phosflow perm buffer III (BD Biosciences) on ice for 30 min. After washing, cells were stained for surface markers and phospho-S6. All samples were processed on an LSRII FACS Fortessa (Becton Dickinson, San Jose, CA) and analyzed by using FlowJo software (TreeStar, Ashland, OR).

Real-time PCR

Frozen tissues were used to extract total RNA, for which we used an RNeasy Mini kit (Qiagen, Valencia, CA), followed by digestion with DNase I (Ambion, Thermo Scientific, Hudson, NH). The cDNA was synthesized by using an iScript Reverse Transcription Kit (Bio-Rad, Hercules, CA). Then cDNA was amplified in a 10-µl reaction mixture containing 5 µl of iTaq SYBR Green Supermix (Bio-Rad) and 0.9µM each of gene-specific forward and reverse primers. The PCR assays were denatured for 30s at 95°C, followed by 40 cycles of 15s at 95°C, and 60s at 60°C, by utilizing the CFX96 Touch real-time PCR detection system (Bio-Rad). Relative quantitation of mRNA expression was calculated, by using the 2^−ΔΔCt method, as the fold increase in expression. The sequences of the forward and reverse gene-specific primers used are listed in Supplementary Table ^S1.

ELISA assays

For detecting IL-22 and IL-17 levels in the supernatants of cell cultures, cells were incubated for 3 days, and then the supernatants were collected, followed by an ELISA assay with commercial kits (eBioscience). For IL-22 detection in tissues, briefly, proteins were extracted from tissues by homogenization on ice in the RIPA Buffer (Cell Signaling Technology) with a protease inhibitor cocktail (Sigma-Aldrich). After centrifugation at 20,000×g for 20min, the supernatants were collected, and protein concentrations measured with a protein assay kit (Bio-Rad). Equal amounts of proteins (100 μg) were loaded for ELISA assays by using commercial kits (eBioscience).

We gratefully thank Dr. Wenjun Ouyang of Amgen for the IL-22 ^−/− mice; Dr. Maria Salvato of University of Maryland, MD for providing us LCMV stocks; and Dr. Yangxin Fu of UT Southwestern Medical Center for providing pRK5-mIL-22 and pRK5 plasmids. We additionally thank Dr. Lihong Zhang of The University of Texas Medical Branch at Galveston (UTMB) for providing BHK and Vero cell lines; Dr. Robert Tesh from UTMB for providing mouse anti-LCMV polyclonal antibody; and Dr. Robert Tigelaar from the Yale School of Medicine for providing the Vγ6 antibody. We thank Ms. Mardelle Susman, Dr. Linsey Yeager, and Dr. Lynn Soong for assistance with manuscript preparation. We thank other members of the UTMB Joint Immunology Working Group (Drs Soong, Cong, Stephens, Rajsbaum and their trainees) for many helpful discussions. Panpan Yi was supported by the Department of Infectious Diseases, Xiangya Hospital, China and the Hunan Provincial Natural Science Foundation of China (#14JJ6003). Yan Chen was supported by the grant from the NIH (EY026999). This work was supported by grants from the NIH (AI109100 and AI126371).