ABSTRACT
γδ T cells are prevalent at mucosal and epithelial surfaces and are a critical first line of defense against bacterial and fungal pathogens. γδ17 cells are a subset of γδ T cells which, in the presence of IL-23 and IL-1β, produce large quantities of interleukin-17A (IL-17A), a cytokine crucial to these cells' antibacterial and antifungal function. STAT6, an important transcription factor in Th2 differentiation and inhibition of Th1 differentiation, is expressed at high levels in the T cells of people with parasitic infections and asthma. Our group and others have shown that STAT6 attenuates IL-17A protein expression by CD4+ T cells. By extension, we hypothesized that STAT6 activation also inhibits innate γδ17 cell cytokine secretion. We show here that γδ17 cells expressed the type I IL-4 receptor (IL-4R), and IL-4 increased STAT6 phosphorylation in γδ T cells. IL-4 inhibited γδ17 cell production of IL-17A. IL-4 also decreased γδ17 cell expression of IL-23R as well as Sgk1. To determine whether STAT6 signaling regulates γδ17 cell numbers in vivo, we used a model of Klebsiella pneumoniae in mice deficient in STAT6. We chose K. pneumoniae for our in vivo model, since K. pneumoniae increases IL-17A expression and γδ17 numbers. K. pneumoniae infection of STAT6 knockout mice resulted in a statistically significant increase in the number of γδ17 cells compared to that of wild-type mice. These studies are the first to demonstrate that γδ17 cells express the type I IL-4R and that STAT6 signaling negatively regulates γδ17 cells, a cell population that plays a front-line role in mucosal immunity.
INTRODUCTION
Approximately 50% of the intraepithelial lymphocyte population is composed of γδ T cells, which constitute a critical first line of defense against bacterial and fungal pathogens (1). In contrast to adaptive αβ T cells, γδ T cells are capable of immediate cytokine release, providing an initial innate layer of protection at mucosal surfaces while influencing the development of subsequent adaptive responses (2, 3). γδ17 cells are a subset of γδ T cells that produce large quantities of interleukin-17A (IL-17A), a cytokine crucial to antibacterial and antifungal defense (4). γδ17 cells also produce high levels of IL-17A in various models of inflammation and autoimmunity, including experimental autoimmune encephalitis, ischemic brain injury, and psoriasis (2, 3, 5–7). While these data highlight the importance of understanding how γδ17 cell function is regulated, this process remains poorly understood.
γδ17 cell function is controlled by multiple immune cell populations and soluble molecules, particularly cytokines. Within 4 to 8 h in the presence of the inflammatory cytokines IL-23 and IL-1β, γδ17 cells secrete IL-17A without the need for T cell receptor (TCR) engagement (2). γδ17 cells constitutively express IL-23 receptor (IL-23R) and IL-1R1, providing for an efficient mechanism to induce rapid effector cytokine production. A recent study showed that the serine/threonine kinase Sgk1 is a novel, critical regulator of IL-23R expression (8).
Studies from our group and others have established that STAT6 negatively regulates IL-17A expression in Th17 cells (9–13). By extension, we hypothesized that STAT6 also inhibits innate γδ17 cell cytokine secretion. STAT6 is a transcription factor important for Th2 differentiation, inhibiting Th1 differentiation and activating the B cell response (14). IL-4 signals through both the type I IL-4 receptor (IL-4R), which consists of IL-4Rα and the common γ-chain, and the type II IL-4R, which consists of IL-4Rα and IL-13Rα, while IL-13 signals through only the type II IL-4R (15, 16). IL-4 binds to the IL-4Rα subunit and IL-13 binds to the IL-13Rα subunit of the IL-4 heterodimer receptor with high affinity, leading to the phosphorylation of STAT6 (17, 18). STAT6 is expressed at high levels in the settings of parasitic infections (19) and asthma, during which STAT6 induces Th2 differentiation, IgE antibody class switching, goblet cell metaplasia, alternative macrophage activation, mucus expression, and airway remodeling (20). Thus, STAT6 attenuation of γδ17 cell function may impair host defenses against bacterial and fungal infections in people with asthma or parasitic infections.
We found that γδ17 cells expressed the type I IL-4R, and that IL-4 increased STAT6 phosphorylation in γδ17 cells. Furthermore, IL-4 signaling attenuated γδ17 cell production of IL-17A and IL-17F. IL-4 also decreased γδ17 cell expression of IL-23R as well as Sgk1. To determine whether STAT6 regulates γδ17 cell cytokine expression in vivo, we used a mouse model of Klebsiella pneumoniae lung infection in mice deficient in STAT6. We chose K. pneumoniae for our in vivo model, since K. pneumoniae increases IL-17A expression and the number of γδ17 cells (21–28). We found a significant increase in γδ17 cell numbers in STAT6-deficient mice following acute lung infection with K. pneumoniae in vivo compared to the level for wild-type (WT) mice. Together, these studies reveal that STAT6 negatively regulates γδ17 cells, a cell population that plays a front-line role in mucosal immunity.
MATERIALS AND METHODS
Mice.Eight- to 12-week-old female BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA). STAT6 knockout (KO), IL-4 KO, and IL-13 KO mice on a BALB/c background were purchased from The Jackson Laboratory, and breeding colonies were established (Bar Harbor, ME). In caring for the animals, investigators adhered to the revised 2011 Guide for the Care and Use of Laboratory Animals prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (29). Experiments were approved by the Vanderbilt Institutional Animal Care and Use Committee.
T cell isolation and culture.γδ T cells from the spleens of BALB/c WT mice were enriched using anti-murine γδ TCR magnetic microbeads (Miltenyi Biotec, Auburn, CA). γδ T cells were purified by flow cytometry by blocking with anti-FcR antibody (2.4G2; BD Biosciences, San Jose, CA) and using propidium iodide viability stain and surface markers against CD3 (145-2C11) and γδ TCR (GL3; BD Biosciences). The purified CD3+γδ TCR+ cells were resuspended at 3 × 105 cells/ml and were induced to produce IL-17A using IL-1β (10 ng/ml) and IL-23 (10 ng/ml) in 96-well plates for 3 days (2). CD3+ T cells were activated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml). IL-4 (0.01 to 1 ng/ml) or IL-13 (10 ng/ml) also was added in select cultures. Mouse recombinant IL-23 (rIL-23) was purchased from R&D Systems. Mouse rIL-1β, mouse rIL-4, and mouse rIL-13 were purchased from PeproTech (Rocky Hill, NJ).
Cytokine measurements.The secretion of cytokines (IL-17A, IL-17F, IL-22, and IL-13) in cell culture supernatants was analyzed by commercially available enzyme-linked immunoabsorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) by following the manufacturer's instructions. Any value below the lower limit of detection was assigned half the value of the lowest detectable standard for statistical comparisons.
Quantitative PCR.Total RNA was isolated using a Qiagen RNeasy micro kit (Valencia, CA) and cDNA was generated. A two-step real-time PCR assay using SYBR green mix (Bio-Rad, Hercules, CA) was used to detect IL-23R, Sgk1, Foxo1, MyD88, TRAF6 (proprietary information available from Qiagen), RORγT, and AhR as previously described (30), and results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The following primer sequences were used: IL-23R forward, 5′-GGTCCAAGCTGTCAATTCCCTAGG-3′; reverse, 5′-AGCCCTGGAAATGATGGACGCA-3′; RORγT forward, 5′-GCGGCTTTCAGGCTTCATGGAG-3′; reverse, 5′-GGGCGCTGAGGAAGTGGGAAAA-3′; AhR forward, 5′-CGGGGTACCAGTTCATCCACGCT-3′; reverse, 5′-GCAAACATGAAGGGCAGCGACGT-3′; GAPDH forward, 5′-GGCCCCTCTGGAAAGCTGTGG-3′; reverse, 5′-CCCGGCATCGAAGGTGGAAGA-3′.
Klebsiella infection.WT and STAT6 KO mice were anesthetized with ketamine-xylazine and then infected with 0.3 × 103 to 1 × 103 CFU of serotype 2 K. pneumoniae (ATCC 43816) in 50 μl phosphate-buffered saline (PBS) or 50 μl PBS alone by retropharyngeal instillation. Bacterial stocks of K. pneumoniae were inoculated into 100 ml of tryptic soy broth (TSB) and incubated for 18 h at 37°C and 225 rpm. One milliliter of this culture was transferred to 100 ml of TSB, and bacteria were grown to mid-log phase for 2 h at 37°C. Bacteria were pelleted, washed in sterile PBS, and diluted to the appropriate concentration as previously described (22, 31). The inoculum concentration was verified by serial dilution in PBS, plating on tryptic soy agar (TSA), and visual determination of colony counts.
Harvest and bacterial quantitation.Lungs were harvested 36 h after K. pneumoniae infection. Mice were euthanized by intraperitoneal injection of pentobarbital sodium (Vortech Pharmaceuticals, Dearborn, MI). The right lung was harvested into 1 ml of RPMI medium and analyzed by flow cytometry. The left lung was harvested into 1 ml of sterile PBS and organs were homogenized. Serial dilutions in PBS were plated onto TSA and grown overnight at 37°C, and colonies were counted.
Flow cytometry.CD3+ T, γδ T, and γδ17 cells were harvested, and cells were blocked with anti-FcR antibody (BD Biosciences) and, in select experiments, stained with surface markers against CD3, γδ TCR, IL-4Rα (mIL4R-M1), IgG2a, κ isotype control (BR2a), and common γ-chain (TUGm) or IgG2b, κ isotype control (27-35; BD Biosciences). For experiments examining STAT6 phosphorylation, select wells were stimulated with IL-4 (10 ng/ml) for 1 h. Cells were blocked with normal mouse serum (eBioscience, San Diego, CA) and stained with surface markers against CD3 and γδ TCR. Cells then were permeabilized and fixed with 4% paraformaldehyde and methanol, respectively, washed thoroughly, and stained for phospho-STAT6 (J71-773.58.11) or IgG1κ isotype control (MOPC-31C; BD Biosciences).
Lungs were harvested, minced, and digested in RPMI medium containing 5% fetal bovine serum (FBS), 1 mg/ml collagenase type IV, and 0.02 mg/ml DNase I for 40 min at 37°C. The digestion was stopped with 100 μl of 0.5 M EDTA, and a single-cell suspension was generated by straining these digestions through a 70-μm strainer. Cells were restimulated in RPMI medium containing 10% FBS, 50 ng/ml phorbol myristate acetate (PMA; Sigma-Aldrich, St. Louis, MO), 1 μM ionomycin (Sigma-Aldrich), and 0.07% GolgiStop (BD Biosciences) for 4 h at 37°C and 5% CO2. Following restimulation, 6 × 106 cells were stained with Live/Dead blue (Life Technologies, Carlsbad, CA) and blocked with anti-FcR antibody and surface markers against CD3, CD4 (H129.19), and γδ TCR. Cells were fixed/permeabilized for 12 h in Cytofix/Cytoperm (BD Biosciences) and stained for IL-17A (TC11-18H10; BD Bioscience). Anti-FcR antibody (BD Biosciences) was used to prevent nonspecific staining. A total of one million cells were analyzed using an LSR II flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Statistical analyses.Data are presented as means ± standard errors of the means (SEM). For Fig. 1 to 4, data shown represent a combined analysis of cell culture wells from three independent experiments. For Fig. 5, data shown represent a combined analysis of individual mice from three independent experiments. Groups were compared using one-way analysis of variance (ANOVA) with Bonferroni's posttest or Student's t test using Prism (version 5; GraphPad Software, San Diego, CA) with values being considered significant at a P value of <0.05.
RESULTS
The type I IL-4R is expressed on γδ17 cells.STAT6 negatively regulates IL-17A expression in CD4+ Th17 cells (9–11). By analogy, we hypothesized that γδ17 cells express the IL-4R, that IL-4 phosphorylates STAT6 in γδ T cells, and that IL-4 inhibits γδ17 cell cytokine secretion. To test this hypothesis, we first enriched γδ T cells from mouse spleens using a commercially available magnetic sorting kit (Miltenyi Biotec, Auburn, CA), and cells were sorted for viable CD3+ γδ TCR+ cells (see Fig. S1 in the supplemental material). IL-4Rα was clearly detected on γδ T cells and also on CD3+ T cells, which are known to express the IL-4R. Common γ-chain expression also was clearly detected on both γδ T cells and CD3+ T cells (Fig. 1A). As optimal IL-17A production from γδ17 cells is induced following culture with both IL-1β and IL-23 (2), γδ T cells were cultured with IL-1β (10 ng/ml) and IL-23 (10 ng/ml) for 3 days. γδ17 cells or CD3+ T cells then were collected and IL-4R expression was examined by flow cytometry. Both IL-4R and common γ-chain were detected on induced γδ17 cells and activated CD3+ T cells (Fig. 1B). Combined, these data demonstrate that γδ17 cells express the subunits of the type I IL-4R, suggesting a potential role for IL-4 in regulating these cells.
IL-4Rα and common γ-chain are expressed in freshly isolated (A) and induced (B) γδ17 cells. CD3+ T cells were stimulated with plate-bound anti-CD3 and anti-CD28, and γδ T cells were induced to produce IL-17A with IL-1β and IL-23 for 3 days. Representative histograms are shown of IL-4Rα and common γ-chain (γc) expression in CD3+ or γδ TCR+ cells compared to the isotype control. Histograms are representative of 3 independent experiments; n = 5.
IL-4 increases STAT6 phosphorylation in γδ17 cells.Since the type I IL-4R was expressed on γδ17 cells, we hypothesized that the downstream transcription factor STAT6 is phosphorylated in the presence of IL-4. To test this hypothesis, we stimulated cultured γδ T cells or CD3+ T cells for 1 h with IL-4 (0 or 10 ng/ml). CD3+ T cells stimulated with IL-4 had increased STAT6 phosphorylation compared to that seen in CD3+ T cells with no IL-4 added. Similarly, γδ17 cells stimulated with IL-4 had increased STAT6 phosphorylation compared to that seen in γδ17 cells with no IL-4 added (Fig. 2). These data demonstrate that a functional IL-4R is expressed by γδ17 cells.
IL-4 increases STAT6 phosphorylation in γδ T cells. CD3+ T cells or γδ T cells were stimulated with IL-4 (10 ng/ml) for 1 h and examined for phospho-STAT6 expression by flow cytometry. Representative histograms of phospho-STAT6 expression in CD3+ (A) or γδ TCR+ (B) cells compared to untreated cells or an isotype control. Histograms are representative of 3 independent experiments; n = 5.
IL-4, but not IL-13, directly attenuates IL-17A production from γδ17 cells.Based on the expression of a functional type I IL-4R on γδ17 cells and the known inhibitory effect of IL-4 on CD4+ Th17 cells, we hypothesized that IL-4 directly decreases γδ17 cell cytokine secretion. Treatment of γδ17 cells with IL-4 in vitro (0.01 to 1 ng/ml) induced a significant dose-dependent decrease in γδ17 cell IL-17A protein expression (Fig. 3A). IL-4 also attenuated γδ17 cell production of IL-17F (Fig. 3B) in a dose-dependent manner. In three independent experiments, IL-4 also decreased γδ17 cell IL-22 production in a dose-dependent manner, although the decrease in IL-22 production was not statistically different when the three experiments were combined (data not shown).
IL-4 but not IL-13 attenuates IL-17A and IL-17F production from γδ17 cells. γδ T cells were induced to produce IL-17, and IL-4 (0.01 ng/ml to 1 ng/ml) and IL-13 (10 ng/ml) were added at the time of γδ T cell induction. IL-17A (A), IL-17F (B), or IL-13 (C) protein expression in cultured supernatants was quantified by ELISA 3 days after induction. Data are combined from 3 independent experiments; n = 5. *, P < 0.05 versus untreated γδ T cells (no IL-4), determined by ANOVA (A and B) or Student's t test (C).
Since the Th2 cytokine IL-13 also negatively regulates IL-17A expression in CD4+ Th17 cells (10, 32), we hypothesized that IL-13 also directly inhibits γδ17 cell cytokine secretion. However, we found that IL-17A expression was not statistically different between γδ17 cells treated with IL-13 and vehicle (Fig. 3C). These data demonstrate that IL-4, but not IL-13, negatively regulated IL-17A and IL-17F protein expression from γδ17 cells at the time of induction.
IL-4 decreases IL-23R and Sgk1 expression in γδ17 cells.IL-4 reduced γδ17 cell IL-17A protein expression. Since IL-23 signals through the IL-23R to induce IL-17A production in γδ T cells, we hypothesized that IL-4 administered during γδ17 cell induction decreases the expression of IL-23R and Sgk1, a critical regulator of IL-23R expression (8). We found that IL-4 decreased IL-23R mRNA expression in γδ17 cells 3 days following γδ17 cell induction (Fig. 4A). We also found that γδ17 cells exposed to IL-4 had decreased Sgk1 mRNA expression 1 day following γδ17 cell induction (Fig. 4B). The decreases in IL-23R and Sgk1 mRNA expression paralleled the IL-4-mediated decrease in γδ17 cell IL-17A production. Sgk1 positively regulates IL-23R expression by deactivating Foxo1, a direct repressor of IL-23R (8); however, we did not find statistically significant differences in Foxo1 mRNA expression between IL-4 and vehicle-treated γδ17 cells in vitro (data not shown). We also determined mRNA expression for downstream components of the IL-1R signaling pathway, MyD88 and TRAF6, as well as for the canonical Th17-related transcription factors RORγT and AhR, which control γδ17 cell development (4, 33). We did not find statistically significant differences in MyD88, TRAF6, RORγT, or AhR mRNA expression between IL-4 and vehicle-treated γδ17 cells in vitro (data not shown). Combined, these data suggest that IL-4 attenuates γδ17 cell IL-17A production by decreasing IL-23R expression.
IL-4 signaling through IL-4R decreases IL-23R and Sgk1 mRNA expression in γδ17. Total RNA was isolated from γδ17 cells 3 (A) or 1 (B) day after induction. IL-23R (A) and Sgk1 (B) mRNA expression in γδ T cells induced in the presence of IL-4 is shown. All mRNA levels were assayed by quantitative real-time RT-PCR, with each sample normalized to GAPDH and relative expression compared to that of γδ T cells induced without IL-4 present. Data are combined from 3 independent experiments; n = 5. *, P < 0.05, versus untreated γδ T cells (no IL-4) by ANOVA.
STAT6 deficiency increases the number of mouse lung γδ17 cells in response to K. pneumoniae infection.Based on our in vitro data that IL-4 inhibits IL-17A protein expression by γδ17 cells, we hypothesized that STAT6 reduces the number of lung γδ17 cells in vivo. We chose mice deficient in STAT6, which is essential for signaling downstream of the IL-4R. We infected mice with K. pneumoniae administered by retropharyngeal instillation and sacrificed the mice 36 h later. We chose K. pneumoniae for our in vivo model because K. pneumoniae increases IL-17A expression and γδ17 cell numbers (21–25). We chose this time course with the consideration that there was unlikely to be a robust adaptive immune response after only 36 h of K. pneumoniae infection, and that instead this protocol would allow us to assess the innate response during which γδ T cells are important producers of IL-17A. We determined the number of lung γδ17 cells following 36 h of K. pneumoniae infection. Following digestion of the lung, the number of γδ17 cells was determined following cell surface and intracellular cytokine staining in K. pneumoniae-infected WT and STAT6 KO mice (Fig. 5A). There was a statistically significant 2-fold increase in the percentage of lung γδ T cells that expressed IL-17A in the K. pneumoniae-infected STAT6 KO mice compared to the WT mice (Fig. 5C). In addition, there was a statistically significant 3-fold increase in the total numbers of lung γδ T cells that expressed IL-17A in the K. pneumoniae-infected STAT6 KO mice compared to the WT mice (Fig. 5D). In our experiments with K. pneumoniae, we were unable to detect IL-4 or IL-13 protein. However, our data suggest that there was sufficient endogenous IL-4 and/or IL-13 expression to decrease γδ17 cell numbers. To determine whether IL-4 or IL-13 alone had effects on γδ17 cells during acute K. pneumoniae infection, we also detected γδ17 cells in K. pneumoniae-infected IL-4 KO and IL-13 KO mice. The deletion of either IL-4 or IL-13 alone did not have an effect on γδ17 cell numbers during acute K. pneumoniae infection (see Fig. S2 in the supplemental material). Therefore, STAT6 suppression of γδ17 cells during acute K. pneumoniae infection likely is due to the combined effect of both IL-4 and IL-13 signaling through STAT6. There were no significant differences in the percentage or number of γδ17 cells in STAT6 KO, IL-4 KO, or IL-13 KO mice compared to that of WT mice at a baseline level in mice not infected with K. pneumoniae (see Fig. S3 in the supplemental material).
Increased airway γδ17 cell numbers in STAT6 KO compared to WT mice following acute lung infection with K. pneumoniae. Lung cells were restimulated with PMA and ionomycin in the presence of GolgiStop. (A and B) Representative contour plots that are CD3 gated. (C) Percentages of IL-17A+ CD3+ γδ TCR+ or IL-17A+ CD3+ CD4+ cells. (D) Total numbers of cells that are IL-17A+ CD3+ γδ TCR+ or IL-17A+ CD3+ CD4+. Total cell numbers were calculated by multiplying the percentage of IL-17A+ CD3+ γδ TCR+ or IL-17A+ CD3+ CD4+ cells by the total cell number. Data are combined from 3 independent experiments; n = 15 mice for each group. *, P < 0.05 by ANOVA.
We also used flow cytometry to determine the number of CD4+ T cells producing IL-17A in our model (Fig. 5B). We found that there was a 2-fold increase in γδ17 cells compared to CD4+ IL-17A-expressing cells in WT mice and a 6-fold increase in γδ17 cells compared to CD4+ IL-17A-expressing cells in STAT6 mice, showing that γδ17 cells were more abundant than CD4+ IL-17A+ cells at this time point after infection in both WT and STAT6 KO mice (Fig. 5D). The percentage of CD4+ IL-17A+ cells was not statistically different between the K. pneumoniae-infected STAT6 KO and WT mice. Similarly, the number of CD4+ IL-17A+ cells was not statistically different between the K. pneumoniae-infected STAT6 KO and WT mice (Fig. 5D). Lung K. pneumoniae burden did not differ significantly between the K. pneumoniae-infected STAT6 KO and WT mice (data not shown). These results reveal that endogenous STAT6 signaling significantly inhibited the number of γδ17 cells in response to K. pneumoniae infection. The results also support that γδ17 cells, and not CD4+ cells, were the primary producers of IL-17A in response to this acute K. pneumoniae infection protocol.
DISCUSSION
Our data are the first to demonstrate that γδ17 cells expressed both IL-4Rα and common γ-chain, the heterodimeric components of the type I IL-4R. γδ17 cell expression of the type I IL-4R was functional, as IL-4 induced STAT6 phosphorylation in γδ T cells. IL-4 negatively regulated γδ17 cell production of IL-17A and IL-17F in vitro. This confirms a recent finding that also demonstrates that IL-4 activates STAT6 and attenuates IL-17A expression in γδ T cells (51). IL-4-mediated attenuation of IL-17A production also paralleled decreases in IL-23R and Sgk1 expression. Since IL-23 is required for γδ17 cell production of IL-17, IL-4 may inhibit γδ17 cell function by decreasing IL-23R expression. This mechanism of γδ17 cell inhibition via IL-23R downregulation could mirror how IL-4 inhibits Th1 differentiation by decreasing IL-12R expression (34). The serine/threonine kinase Sgk1 recently was found to stabilize the Th17 cell phenotype by stabilizing IL-23R expression (8). We found that Sgk1 expression was decreased 1 day following γδ17 cell induction in culture, while IL-23R expression was decreased 3 days following γδ17 cell induction. Therefore, the observed IL-4-mediated decreases in IL-17A production could be caused by decreased Sgk1 expression leading to destabilized IL-23R expression on γδ17 cells.
An important finding in this report is that IL-4 and IL-13 differentially regulate CD4+ Th17 and γδ17 cell cytokine production. While a concentration of IL-4 as low as 0.1 ng/ml significantly decreased γδ17 cell IL-17A production, 100 times more IL-4 is required to inhibit IL-17A production from Th17 cells (10). This indicates that the IL-4R on γδ17 cells is either more sensitive to the presence of IL-4 or more robustly induces downstream IL-4 signaling compared to the IL-4R on Th17 cells. While IL-4 directly inhibited IL-17A production from both Th17 and γδ17 cells, IL-13 directly inhibited IL-17A production from Th17 cells but not from γδ17 cells (10, 32). These results suggest that IL-4 and IL-13 use different mechanisms to inhibit γδ17 cells.
We show that STAT6 signaling reduced lung γδ17 cell numbers in vivo. IL-4 binds to the IL-4Rα portion and IL-13 binds to the IL-13α subunit of the IL-4 receptor, leading to the phosphorylation of STAT6. STAT6 deficiency increased both the percentage of lung γδ T cells that expressed IL-17A as well as the total number of γδ T cells in the lung that expressed IL-17A protein in response to acute K. pneumoniae infection. We confirmed that γδ17 cells, and not CD4+ T cells, were the major contributors of IL-17A in our model of acute K. pneumoniae infection. To our knowledge, there are no studies in which CD4+ T cells or IL-13 has been depleted during acute K. pneumoniae infection to determine whether this cell type or cytokine has a role during the immune response. IL-13 did not have a direct effect on γδ17 cell cytokine production in vitro. However, the genetic deletion of either IL-4 or IL-13 alone did not have an effect on γδ17 cell numbers during acute K. pneumoniae infection. Therefore, STAT6 suppression of γδ17 cells during acute K. pneumoniae infection likely is due to the combined effect of both IL-4 and IL-13 signaling through STAT6. IL-13 may regulate γδ17 cells indirectly, as IL-13 suppresses dendritic cell expression of γδ17 cell-promoting factors such as IL-1β and IL-23 (35).
Future lines of research should address STAT6 downregulation of γδ17 cell function in other tissues and in additional models of immune-mediated inflammation and infection. Tissue localization dictates γδ TCR diversity (36). Similarly, γδ17 cells may differentially express the IL-4R, and STAT6 may distinctly regulate γδ17 cell cytokine production in other sites where γδ T cells are prevalent, such as in the gut or skin. Increasing STAT6 activity could represent an immunotherapeutic strategy to treat γδ17-mediated diseases, such as psoriasis and multiple sclerosis, by decreasing inflammatory IL-17A production (37, 38). In contrast, STAT6 is expressed at high levels in patients with asthma (20), and phase II randomized clinical trials have investigated the effects of blocking IL-4 and IL-13 signaling. The IL-4Rα antibody, AMG 317, reduced IgE levels and the number of exacerbations in patients with moderate-to-severe asthma (39). The recombinant human IL-4 variant, pitrakinra, which competitively inhibits IL-4 or IL-13 binding to IL-4Rα, resulted in a smaller decrease in forced expiratory volume 1 (FEV1) in patients with mild asthma (40). However, the impact of these anti-IL-4 and anti-IL-13 therapies on γδ17 cells is unknown. Based on the findings of our study, anti-IL-4 and anti-IL-13 therapies used to treat asthmatic patients could have the adverse effect of exacerbating coexisting immune-mediated inflammatory diseases, such as multiple sclerosis, in these patients. Blocking IL-4 and IL-13 activity also could decrease infection severity from pathogens for which strong, early γδ17 cell responses are critical for immunity. During the immune response to pathogens such as Mycobacterium tuberculosis, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Candida albicans (41–44), γδ17 cells stimulated neutrophil recruitment, antibacterial peptide production, and maintenance of epithelial barriers (45). Notably, people with asthma, whose T cells express high levels of STAT6 (46), are at increased risk for invasive bacterial infections and bacterial pneumonia (47–50). However, the mechanisms by which asthma impairs host defense against bacterial infection are unknown. Our results may provide one explanation for why asthmatic subjects have a significantly greater risk of invasive bacterial disease than nonasthmatic subjects.
In summary, we are the first to show that γδ17 cells express a functional IL-4R, and we demonstrate a novel role for the transcription factor STAT6 in negatively regulating γδ17 IL-17A production. These findings have significant and broad implications for how γδ17 cells, an important cell population that plays a front-line role in mucosal immunity, are regulated in the spleen and lung.
ACKNOWLEDGMENTS
We greatly appreciate the commentary of Weisong Zhou and Shinji Toki on the manuscript.
FOOTNOTES
- Received 15 May 2015.
- Returned for modification 16 June 2015.
- Accepted 23 February 2016.
- Accepted manuscript posted online 7 March 2016.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00646-15.
- Copyright © 2016, American Society for Microbiology. All Rights Reserved.
