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Interleukin-22 Drives Endogenous Thymic Regeneration in Mice

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Science  06 Apr 2012:
Vol. 336, Issue 6077, pp. 91-95
DOI: 10.1126/science.1218004

Abstract

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence after stress, infection, or immunodepletion. However, the mechanisms governing this regeneration remain poorly understood. We detail such a mechanism, centered on interleukin-22 (IL-22) and triggered by the depletion of CD4+CD8+ double-positive thymocytes. Intrathymic levels of IL-22 were increased after thymic insult, and thymic recovery was impaired in IL-22–deficient mice. IL-22, which signaled through thymic epithelial cells and promoted their proliferation and survival, was up-regulated by radio-resistant RORγ(t)+CCR6+NKp46 lymphoid tissue inducer cells after thymic injury in an IL-23–dependent manner. Administration of IL-22 enhanced thymic recovery after total body irradiation. These studies reveal mechanisms of endogenous thymic repair and offer innovative regenerative strategies for improving immune competence.

Despite being exquisitely sensitive to insult, the thymus is remarkably resilient in young healthy animals. However, thymic renewal after immune depletion is a prolonged process, particularly in elderly patients, which substantially impairs the recovery of adaptive immunity (1, 2). This period of prolonged immune deficiency leads to an increase in opportunistic infections and higher treatment-associated morbidity and mortality (2, 3).

Thymopoiesis is a complex process involving cross-talk between developing thymocytes and the nonhematopoietic supporting stromal microenvironment, which comprises specialized thymic epithelial cells (TECs), endothelium, fibroblasts, and dendritic cells (DCs) (4, 5). TECs can be separated into two populations, cortical TECs (cTECs) and medullary TECs (mTECs), which differ in their spatial location and function within the thymus (46). Interleukin-22 (IL-22) is primarily associated with the maintenance of barrier function and induction of innate antimicrobial molecules at mucosal surfaces (7, 8). The principal sources of IL-22 are T helper 17 cells and innate lymphoid cell (ILC) subsets (912). Given the role of IL-22 in both promoting and reducing autoimmune pathology within epithelial compartments (8), we hypothesized that IL-22 would mediate epithelial regeneration after thymic injury.

At baseline, untreated wild-type mice and mice genetically deficient in IL-22 (Il22−/−) (13) demonstrated no difference in total thymic cellularity or in numbers of the various thymic cell populations (fig. S1, A to D). To explore the effects of IL-22 deficiency on thymic regeneration after insult (14), we exposed wild-type or Il22−/− mice to sublethal total body irradiation (SL-TBI). Il22−/− mice displayed significantly impaired thymic regeneration for up to 28 days after SL-TBI (Fig. 1A) with significantly reduced numbers of all developing thymocyte subsets, TECs and non-TECs (including endothelial cells and fibroblasts) (Fig. 1, B to D). We also performed syngeneic hematopoietic stem cell transplantation (HSCT) or allogeneic HSCT, and in both cases we observed significantly reduced thymic cellularity and reduced numbers of all thymic cell subsets in Il22−/− hosts (fig. S2). Upon long-term follow-up, we found that impaired thymic regeneration in Il22−/− mice persisted for up to 98 days after TBI (Fig. 1E). Il22−/− mice given a targeted dose of radiation to the thymus also exhibited significantly reduced thymic regeneration relative to wild-type controls at day 7 (Fig. 1F), which suggests that the systemic damage of TBI is not required for the impact of IL-22 deficiency on thymic regeneration.

Fig. 1

IL-22 is critical for endogenous thymic regeneration and is up-regulated upon thymic damage. (A to D) Wild-type mice (WT; gray bars, n = 11) and Il22−/− (black bars, n = 11) C57Bl/6 mice were given SL-TBI (550 cGy) with no hematopoietic rescue, and enzyme-digested thymus was analyzed: (A) total thymic cellularity at days 7 and 28 after TBI; (B and C) developing thymocyte subsets 28 days after SL-TBI; (D) developing stromal cell subsets 28 days after SL-TBI. (E) Total thymus cellularity in WT (n = 5) or Il22−/− (n = 6) mice 98 days after SL-TBI. (F) Total thymus cellularity 7 days after targeted thymic irradiation (850 cGy) of WT (n = 10) or Il22−/− (n = 7) mice. (G) Absolute amounts of intrathymic IL-22 were measured by enzyme-linked immunosorbent assay (ELISA) in untreated C57BL/6 (B6) (n = 22), untreated BALB/c (n = 5) or 7 days after SL-TBI without HSCT (550 cGy, n = 15) or L-TBI and syngeneic HSCT (C57Bl/6 HSCs into congenic Ly5.1+ C57Bl/6 hosts, 2 × 550 cGy, n = 10) or T cell–depleted allogeneic HSCT (B10.BR HSCs into MHC-mismatched C57Bl/6 hosts, 2 × 550 cGy, n = 10; or C57Bl/6 HSCs into MHC-mismatched BALB/c hosts, 2 × 425 cGy, n = 5). (H) Absolute amounts of IL-22 (solid black line) plotted with total thymic cellularity (dashed black line) over time after SL-TBI (n = 5 to 10 per time point). Dashed and solid red lines represent mean cellularity and IL-22 amount, respectively, at baseline. (I) Spearman correlation between absolute amounts of intrathymic IL-22 and total thymic cellularity in various models of thymic insult at 7 or 56 days. In (G) and (H), *P < 0.05, **P < 0.01 compared to untreated controls. Bar graphs represent means ± SEM of at least two or three independent experiments.

Thymic IL-22 production was measured in mice 7 days after SL-TBI without HSCT, lethal TBI and syngeneic HSCT, or T cell–depleted allogeneic HSCT. In each of these models, we found a factor of 2 to 3 increase in absolute amounts of IL-22 relative to control mice that were not irradiated (Fig. 1G). This result was striking, given the significant decrease in thymic cellularity seen in irradiated mice (fig. S3A) leading to a profound increase in the amount of IL-22 on a per-cell basis (fig. S3B). Absolute amounts of IL-22 peaked on day 5, corresponding closely with the lowest point of thymic cellularity, and returned to normal amounts by day 10 as thymic cellularity returned to baseline (Fig. 1H). These findings revealed an inverse correlation (r = –0.8345) between thymic size and absolute amount of intrathymic IL-22 (Fig. 1I).

We next titrated the radiation dose to further explore the coupling between thymic cellularity and IL-22. Although increasing doses of radiation led to more severe thymic insult (fig. S3C), peak absolute amounts of IL-22 were achieved at the lowest TBI dose (fig. S3D), which suggests that only a partial loss of thymic cellularity is necessary for increased expression of IL-22.

Mice given a range of radiation doses targeted directly to the thymus also significantly increased their intrathymic amounts of IL-22 (fig. S3E). In these same mice, there was no change in the amounts of IL-22 in the spleen after thymic irradiation, which suggests that up-regulation of intrathymic IL-22 is an intrinsic local response to thymic injury.

ILCs that express the transcription factor RORγ(t) have been identified as potent producers of IL-22 (11, 15). Moreover, CD4+CD3 thymic lymphoid tissue inducer (LTi) cells contribute toward TEC development and maturation (16). Three days after L-TBI (with no hematopoietic rescue), we identified a population of CD45+IL-7Rα+CD3CD8RORγ(t)+ thymic ILCs (tILCs) that up-regulated their production of IL-22 (Fig. 2A). No IL-22 expression was found by CD3+ or CD45 populations (fig. S4, A to C). Closer examination revealed that IL-22–producing tILCs in both untreated and TBI-treated mice uniformly expressed CD4 and CCR6 but not NKp46 (Fig. 2B)—a phenotype consistent with that of LTi cells (15).

Fig. 2

IL-22 is produced by intrathymic ILCs under the control of IL-23. (A and B) Enzyme-digested thymus from untreated mice (n = 11) or mice 3 days after L-TBI (n = 15) was incubated with Brefeldin A (3 μg/ml) for 4 hours, but otherwise remained unstimulated. (A) Intracellular expression of IL-22 and RORγ(t) by CD45+IL-7Rα+CD3CD8 tILCs in untreated or L-TBI animals. (B) Expression of CCR6, NKp46, and CD4 on IL-22–producing tILCs. (C) IL-22 levels measured by ELISA in thymus of untreated mice or 7 days after SL-TBI in WT or Rorc−/− mice. (D and E) Absolute number (D) and frequency (E) of CD45+IL-7Rα+CD3CD8CD4+RORγ(t) LTi cells in untreated mice (n = 25) or 3 days after L-TBI (n = 10). (F) Expression of RANK ligand (RANKL), IL-23R, and RORγ(t) in LTi cells from untreated mice or mice 3 days after L-TBI. (G) C57Bl/6 mice were given SL-TBI (550 cGy) and absolute levels of IL-23 (solid black line) were measured by ELISA at days 1, 3, 5, 7, 10, 14, and 21 (n = 5 per time point). Compared with IL-22 kinetics (dashed black line) taken from Fig. 1H. Red lines represent mean at day 0 for IL-22 (dashed) and IL-23 (solid). (H and I) Absolute IL-22 levels measured by ELISA (H) and total thymic cellularity (I) in untreated mice (n = 11) or 7 days after SL-TBI in WT (n = 10) or Il12b−/− (n = 8) animals. (J) Untreated WT thymus was enzyme-digested and incubated with or without IL-23 (60 ng/ml) for 4 hours. After 1 hour of IL-23 incubation, Brefeldin A was added to all wells. IL-22 expression was examined in CD45+CD3CD8CD4+IL7Rα+RORγ(t)+ LTi cells. (K) Untreated (n = 10) or 3 days after L-TBI (n = 10) thymus cells were incubated for 4 hours in monensin to block Golgi export (2 μM) but otherwise remained unstimulated. Intracellular IL-23 expression in thymic DCs (CD45+CD11c+MHCII+) was measured. (L) Expression of CD103 on IL-23 and IL-23+ thymic DCs in untreated and L-TBI mice. *P < 0.05, **P < 0.01, ***P < 0.001. Bar graphs represent means ± SEM of two or three independent experiments. Fluorescence-activated cell sorter (FACS) plots were generated by concatenation of five individual observations from one of at least two independent experiments.

Apart from its role in ILC function, RORγ(t) is critical for thymocyte development and is widely expressed in the thymus (17). Mice deficient for Rorc, the gene encoding RORγ(t), contained normal amounts of intrathymic IL-22 (Fig. 2C) at baseline, indicating that steady-state amounts of intrathymic IL-22 do not require RORγ(t) or LTi cells. However, in contrast to wild-type mice, Rorc−/− mice did not significantly increase their intrathymic amounts of IL-22 in response to TBI (Fig. 2C), which suggests that RORγ(t)+ LTi cells are critical for intrathymic up-regulation in the production of IL-22 after thymic damage.

Thymic LTi cells were present immediately after radiation (Fig. 2D), indicating that they are radio-resistant for the period when the up-regulation of IL-22 is crucial for thymic regeneration, and could persist for up to 3 months after L-TBI and HSCT (fig. S4D). Furthermore, given the severe depletion of thymus cellularity early after TBI, the frequency of LTi cells increased significantly after L-TBI (Fig. 2E). After TBI, LTi cells also increased their expression of RANKL (Fig. 2F and fig. S4E), which has been reported to aid TEC maintenance and regeneration (16).

Regulation of IL-22 production has been closely associated with DC-produced IL-23, and ex vivo incubation of ILCs with IL-23 stimulates production of IL-22 (13, 1820). Three days after L-TBI, we found increased expression of IL-23 receptor (IL-23R) and RORγ(t) by LTi cells (Fig. 2F and fig. S4E), consistent with the importance of IL-23 in regulating IL-22 (21, 22). We then assessed intrathymic amounts of IL-23 in vivo and observed increased IL-23 production after SL-TBI, mirroring the kinetics of IL-22 (Fig. 2G). Mice genetically deficient in Il12b, the gene that encodes the p40 subunit of IL-12 and IL-23, showed no change in IL-22 production (Fig. 2H) and exhibited a defect in thymic regeneration after SL-TBI (Fig. 2I), demonstrating that intrathymic TBI-induced production of IL-22 requires p40. Consistent with this finding, IL-22 expression was increased by thymic LTi cells after IL-23 stimulation in vitro (Fig. 2J).

We next sought to identify the source of elevated intrathymic IL-23 after TBI. Although some thymic DCs expressed IL-23 at baseline, a greater frequency expressed IL-23 after L-TBI (Fig. 2K). IL-23 expression was found in both CD103+ and CD103 thymic DCs in untreated mice; however, there was significant enrichment of IL-23+ thymic DCs expressing CD103 (Fig. 2L) in irradiated animals. This is consistent with the finding that mucosal CD103+ DCs are potent IL-23 producers (23).

To further explore the relationship between IL-22 and thymocyte cellularity (Fig. 1G), we examined mutant animals with well-defined blocks in intrathymic T cell development (24) for production of IL-22 and IL-23 (Fig. 3A). Mice blocked within the CD4CD8 double negative (DN) stage of thymocyte differentiation, prior to developing CD4+CD8+ double positive (DP) thymocytes, expressed significantly more intrathymic IL-22 and IL-23 than wild-type controls (Fig. 3B). In contrast, mice deficient for TCRα or CCR7, which lack mature CD4 or CD8 single positive (SP) thymocytes but have no loss of DP thymocytes (5, 24), exhibited no up-regulation of IL-22 and IL-23 (Fig. 3B). Stable intrathymic IL-22 and IL-23 were also observed in mice deficient for CD40 ligand (Cd40l−/−) (Fig. 3B), which have a defect in mTECs but have normal numbers of DP and SP thymocytes (25). Consequently, there was a strong inverse correlation between the number of DP thymocytes and amounts of intrathymic IL-22 and IL-23 (Fig. 3C), further suggesting that depletion or absence of DP leads to up-regulation of IL-22 and IL-23. This was confirmed by treatment with dexamethasone (Dex), which specifically depletes DP thymocytes (26) and led to up-regulation of IL-22 and IL-23 in wild-type mice (Fig. 3D). Strikingly, increased IL-22 expression was detected in freshly isolated LTi cells from Dex-treated mice without incubation, in stark contrast to the low or undetectable levels in untreated mice (Fig. 3E). Furthermore, consistent with our findings in the TBI model, we observed significantly increased expression of RORγ(t) in LTi cells isolated from Dex-treated mice relative to untreated controls (Fig. 3F). Although IL-7 signaling has been implicated in LTi cell maintenance (27), similar numbers of LTi cells were found in Il7−/− and Rag1−/− mice, and there was an increase in Il7ra−/− mice (fig. S4F). Furthermore, both the frequency of LTi cells (fig. S4G) and their baseline production of IL-22 (fig. S4H) were increased relative to wild-type controls. In all our models of thymic damage and mutant mouse strains, there was a strong correlation (r = 0.9554) between amounts of thymic IL-22 and IL-23 (fig. S5).

Fig. 3

Absence of CD4+CD8+ double positive thymocytes triggers the up-regulation of IL-23 and IL-22. (A to C) Mutant mouse strains with blocks at different stages of the T cell development were assessed for their production of IL-22 and IL-23. (A) Schematic of T cell developmental stage blocked in various mutant strains and methods used. (B) Absolute IL-22 and IL-23 at baseline in thymus of untreated WT (n = 15), Il7Ra−/− (n = 9), Il7−/− (n = 11), Rag1−/− (n = 22), Tcrb−/− (n = 10), Tcra−/− (n = 18), Ccr7−/− (n = 6), and Cd40l−/− (n = 10) mice. Statistical comparisons were made with the Kruskal-Wallis test with posttest comparison to WT controls (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Spearman correlation between number of DP thymocytes and amounts of IL-22 or IL-23 in various mutant mouse strains. (D to F) C57Bl/6 mice were treated with phosphate-buffered saline (PBS) (n = 10) or Dex (20 mg/kg, n = 11). (D) Thymocyte profiles and absolute amounts of thymus IL-22 and IL-23 were assessed 5 days after treatment. (E) Freshly isolated LTi cells from untreated WT (n = 12) or Dex-treated (n = 13) mice were analyzed for intracellular IL-22 with no incubation period. (F) Mean fluorescence intensity (MFI) of RORγ(t) in LTi cells isolated from untreated or Dex-treated mice. Bar graphs represent means ± SEM; all data were generated from two or three independent experiments. FACS plots were generated by concatenation of at least five individual observations from one of at least two independent experiments.

IL-22R is a heterodimer of IL-10Rβ and IL-22Rα (8). Its expression has been reported to be restricted to nonhematopoietic cells (8). No IL-22Rα was detectable on developing thymocytes or nonepithelial stromal cells (fig. S6A). In contrast, IL-22Rα was expressed on cTECs as well as on major histocompatibility complex (MHC) class II high- and low-expressing mTECs (mTEChi and mTEClo, respectively), a marker of mTEC maturation (6, 25) (Fig. 4A). To test whether IL-22 could functionally signal through IL-22R expressed by TECs, we stimulated the TE-71 TEC cell line with IL-22. Consistent with findings in the mucosal epithelium (28), IL-22 stimulation of TE-71 cells led to activation-induced phosphorylation of the transcription factors STAT-3 and STAT-5 (fig. S6B) (29).

Fig. 4

Exogenous administration of recombinant murine IL-22 enhances thymopoiesis by promoting the proliferation and viability of TECs. (A) Wild-type thymus was enzyme-digested and enriched for CD45 cells. Expression of IL-22Rα on cTECs (UEA-1lo), mTEClo (UEA-1hiMHCIIlo), and mTEChi (UEA-1hiMHCIIhi) is shown. All populations were gated on CD45EpCAM+. (B to D) CD45 or MHCII+ enriched thymus cells were incubated for 24 hours with or without IL-22 (100 ng/ml). (B) Expression of EpCAM in uncultured CD45 cells (n = 5) and in CD45 cells incubated for 24 hours with IL-22 (n = 10) or media alone (n = 10). (C) Proportion of specific TEC subsets in CD45 cells incubated for 24 hours with or without IL-22. (D) Expression of 4′,6-diamidino-2-phenylindole (DAPI) and the marker of proliferation Ki-67, (Ki-67) on TEC subsets on MHC class II–enriched thymus cells after 24 hours of incubation with IL-22 (n = 10) or media alone (n = 10). For in vitro experiments with enriched cells, each individual observation represents three or four pooled thymuses. (E to H) C57Bl/6 mice were given SL-TBI (550 cGy), then treated with PBS (gray bars, n = 10) or IL-22 (black bars, 200 μg/kg per day, n = 10 to 15) at days –1, 0, and +1 and assessed at days 7 and 28. (E) Total thymus cellularity. (F) Absolute number of thymocyte subsets. (G) Absolute number of TEC subsets. (H) Proportion of Ki-67–expressing cTECs, mTEClo, and mTEChi. Bar graphs represent means ± SEM of at least two independent experiments. FACS plots were generated by concatenation of at least five individual observations from one of at least two independent experiments.

To assess the impact of IL-22 on primary TECs, we enriched and incubated CD45 cells with IL-22 or media alone for 24 hours. Although there was significant attrition of epithelial cell adhesion molecule (EpCAM) expression in untreated cells, those incubated with IL-22 maintained greater EpCAM expression, and viability of TEC subsets, in culture (Fig. 4, B and C). Consistent with these findings, the presence of IL-22 improved TEC survival and increased proliferation of cTECs and mTEClo (Fig. 4D). There was no change in the expression of apoptosis-related Annexin V or Bcl-2 proteins (fig. S6C). These findings demonstrate that IL-22 signals through IL-22R on the surface of TECs, and in particular in cTECs and mTEClo. It is within this latter population that immature mTEC populations are currently thought to reside (6, 25). Although it is possible that IL-22 acts as a maturation signal for mTECs, it is more likely that IL-22 primarily functions to induce proliferation and viability, given the uniform expression of IL-22R on immature mTEClo and mature mTEChi, and given the preferential promotion of proliferation among mTEClo.

To examine the clinical effectiveness of IL-22 as a regenerative strategy, we administered recombinant IL-22 to mice given SL-TBI. We found significantly increased thymic cellularity at days 7 and 28, relative to controls, in mice receiving SL-TBI (Fig. 4E). Increases were also observed in all developing thymocyte subsets (Fig. 4F) and TEC subsets (Fig. 4G). There was also a significant increase in the proliferation of cTECs and mTEClo at early time points after IL-22 treatment (Fig. 4H). IL-22–treated animals receiving L-TBI in combination with syngeneic HSCT also showed significantly enhanced thymic recovery at day 7 (fig. S7). In otherwise untreated animals given IL-22, we observed no change in total thymic cellularity, although there was a small increase in cTEC and mTEClo proliferation (fig. S8).

These studies suggest that after thymic injury, and specifically after the depletion of DP thymocytes, up-regulation of IL-23 by radio-resistant CD103+ thymic DCs induces IL-22 production by LTi cells. This cascade of events leads to regeneration of the supporting epithelial microenvironment, and ultimately to enhanced thymopoiesis (fig. S9). We previously demonstrated in knockout studies that keratinocyte growth factor is also required for thymic regeneration and is redundant for thymic ontogeny (30). In this instance, however, depletion of DP cellularity triggers a thymic molecular network to aid in its own regeneration. Interestingly, once thymus cellularity has been restored, IL-22 production stabilizes. This is consistent with the fact that administration of IL-22 was a highly effective regenerative strategy after radiation damage but had little effect in untreated mice or those with substantial recovery after syngeneic HSCT.

Prolonged thymic deficiency after cytoreductive conditioning is a major clinical challenge. These studies not only identify a mechanism governing endogenous thymic regeneration, but also offer an innovative therapeutic strategy for immune regeneration in patients whose thymus has been irrevocably damaged.

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1218004/DC1

Materials and Methods

Figs. S1 to S9

Reference (31)

References and Notes

  1. See supporting material on Science Online.
  2. Acknowledgments: We thank A. Farr for the gift of the TE-71 TEC cell line; D. Littman, W. Ouyang, A. Beaulieu, J. Sun, and S. Prockop for the gift of mice; R. Essner and D. Schwartz for technical assistance; E. Velardi and Y. Shono for helpful discussion; and L. Reilly, B. Morcerf, and B. Rojas for administrative assistance. M.R.M.v.d.B. also thanks his former mentor J. J. van Rood. Supported by NIH R01 awards HL069929, CA107096, AI080455, and HL095075, U.S. Department of Defense USAMRAA award W81XWH-09-1-0294, the Radiation Effects Research Foundation of the National Institute of Allergy and Infectious Diseases, the Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center (funded by W. H. Goodwin and A. Goodwin), the Lymphoma Foundation, Alex’s Lemonade Stand, and the Peter Solomon Fund. J.A.D. was supported by fellowships from the Australian National Health and Medical Research Council (CJ Martin Biomedical Research Fellowship, 1012401) and the Leukemia and Lymphoma Society (5534-11). A.M.Ha. was supported by a Research Training Award for Fellows from the American Society of Hematology and a New Investigator Award from the American Society for Blood and Marrow Transplant. A.G. was supported by supported by a Judah Folkman Fellowship from the American Association of Cancer Research. A provisional patent application has been filed on the use of IL-22 as a thymopoietic growth factor (U.S. 61/487,517) with J.A.D, A.M.Ha., and M.R.M.v.d.B. listed as inventors.
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