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Keywords:

  • IL-1 family;
  • IL-9 regulation;
  • TGF-β

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

TGF-β and IL-4 were recently shown to selectively upregulate IL-9 production by naïve CD4+ T cells. We report here that TGF-β interactions with IL-1α, IL-1β, IL-18, and IL-33 have equivalent IL-9-stimulating activities that function even in IL-4-deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T-cell activation. Based on intracellular IL-9 staining, all IL-9-producing cells were CD4+ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL-9, IL-13 and IL-17 were strongly stimulated by IL-1 and either inhibited (IL-13) or were unaffected (IL-17) by addition of TGF-β. IL-9 and IL-17 production also differed in their dependence on IL-2 and regulation by IL-1/IL-23. As IL-9 levels were much lower in Th2 and Th17 cultures, our results identify TGF-β/IL-1 and TGF-β/IL-4 as the main control points of IL-9 synthesis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

Since its discovery as a T-cell 1, 2 and mast-cell 3 growth factor that plays an essential role in helminth elimination 4 and in pulmonary mast cell and goblet cell accumulation 5, IL-9 has been considered a Th2 cytokine 6, 7, although, in asthmatic humans, eosinophils have also been identified as an IL-9 source 8. A recent publication, however, showed that, in the presence of IL-4 and TGF-β, naïve T cells could be programmed to selective IL-9 production 9 and failed to express T-bet, GATA3, Foxp3, or RORγt, suggesting that these cells could represent a novel Th subset, an idea confirmed in another report 10. These results changed our perception of the IL-9 stimulating activity of TGF-β/IL-4 reported by Schmitt 11. More recently, still, IL-9 production by CD4+ T cells stimulated in the presence of TGF-β/IL-6, the canonical Th17 combination 12–14, was also reported 15, 16.

All these experiments involved in vitro stimulation of pure CD4+ T-cell populations with anti-CD3/CD28 Ab. To evaluate the influence of TGF-β on IL-9 production in a more physiological configuration, we used both recall and primary responses to the conventional protein antigen, chicken OVA.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

TGF-β/IL–1 upregulates IL-9 production

Similar to IL-4, IL-1α has been shown to promote Th2 responses 17 and, although not essential for Th2 polarization, it is required for IL-4 secretion after Trichuris muris infection 18. However, in Leishmania major-infected BALB/c mice, IL-1α enhances Th1 immunity 19. These observations suggest that IL-1 can act as a nonpolarized enhancer of Th activity. We wondered what would be its effect on the production of IL-9 after antigenic stimulation. This question was addressed in antigen recall responses of lymph node cells of BALB/c and DBA/2 mice vaccinated with OVA in CFA. In both strains, IL-9 levels were minimally or not enhanced by IL-1α, contrary to what has been observed with long term Th cell lines 20. TGF-β also failed to modify IL-9 production but when both factors were combined, IL-9 levels raised more than ten-fold (Fig. 1A). This was observed in two experiments in each strain and also in C57BL/6 mice (data not shown). The IL-9 stimulating effect of TGF-β/IL-1α (or TGF-β/IL-1β) was confirmed in an experiment using spleen cells from DBA/2 mice immunized with OVA in alum (Fig. 1B), indicating that the inflammatory conditions elicited by CFA during the in vivo priming are not required for IL-9 upregulation by TGF-β/IL-1. Similar results were obtained in unprimed BALB/c spleen cells stimulated with anti-CD3/CD28 or Staphylococcal enterotoxin B (SEB) (Fig. 1C).

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Figure 1. Effects of TGF-β/IL-1, TGF-β/IL-18, and TGF-β/IL-33 on IL-9 production. (A) Lymph node cells from BALB/c and DBA/2 mice (pooled from five mice per group) primed s.c. with OVA in CFA or (B) pooled spleen cells from three DBA/2 mice primed i.p. with OVA and alum were collected 7 days after priming and cultured in triplicate with OVA together with the indicated cytokines for 4 days. (C) Pooled spleen cells from three BALB/c mice were stimulated in triplicate wells with either SEB or anti-CD3 plus anti-CD28 Ab and the indicated cytokines for 4 days. This experiment was repeated three times for SEB and two times for anti-CD3/CD28 (A–C). IL-9 production was measured and the data are mean±SEM of triplicates. *p<0.05, **p<0.01, and ***p<0.001 with respect to OVA, SEB, or anti-CD3/anti-CD28 stimulations only as calculated by ANOVA. (D) Unprimed D011.10 OVA-TCR BALB/c spleen cells pooled from two mice were incubated with OVA in the presence of the indicated factors in triplicate. IL-9 levels and thymidine incorporation were measured after 4 days (mean±SEM of triplicates). **p<0.01 and ***p<0.001 comparing cultures with and without TGF-β (t-test). (E) Lymph node cells from OVA-primed BALB/c mice were stained with CFSE before in vitro stimulation with OVA with or without cytokines as indicated and 7 days later were restimulated with PMA/ionomycin for intracellular staining as described in Material and methods. The plots are gated on CD4+ cells and analyzed for CFSE and IL-9 staining. This experiment was repeated three times with identical results.

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IL-18 and IL-33 are other members of the IL-1 family. We tested their effects on IL-9 production elicited by OVA in spleen cell cultures of D011.10 mice which carry an OVA-specific TCR and compared them with IL-1α and IL-1β and also with IL-4. This system was chosen also to verify the influence of these cytokine combinations on unprimed responses to a conventional protein antigen. None of these factors significantly modified IL-9 production unless combined with TGF-β (Fig. 1D). These results were confirmed in another experiment with D011.10 mice and in OVA-immunized BALB/c mice (data not shown). It is worth noting that these strong IL-9 productions were not the result of enhanced cell proliferation as indicated by thymidine incorporations measured after OVA stimulation which was inhibited by TGF-β (Fig. 1D).

To test whether the IL-9 detected in these cultures was produced by CD4+ T cells that, in spite of the growth inhibition by TGF-β, had proliferated, IL-9 intracellular staining was performed after in vitro antigen restimulation of CFSE-labeled lymph node cells from OVA-immunized BALB/c mice. IL-9 was detected in the CD4+ population and was inversely correlated with CFSE staining indicating that the majority of the IL-9-producing cells had proliferated. In agreement with IL-9 levels present in the supernatants (data not shown), the highest IL-9 staining was observed in TGF-β/IL-1 cultures (±2%) followed by TGF-β/IL-4 (±0.5%). After plain OVA stimulation, ±0.1% of the CD4+ cells were positive (Fig. 1E), a result similar to IL-1α, TGF-β, or IL-4 cultures (data not shown). These observations were reproduced in two other experiments.

TGF-β/IL-1 stimulates IL-9 production by CD4+ T cells independently of IL-4

The implication of IL-4 in IL-9 stimulation by TGF-β/IL-1 was tested in IL-4 KO (Fig. 2) and compared with TGF-β/IL-4. In these mice, the two combinations were equally potent, proving that TGF-β/IL-1 functioned in the absence of IL-4. However, in wild-type BALB/c mice, TGF-β/IL-1 was more potent than TGF-β/IL-4, suggesting that the two stimuli activate two nonredundant IL-9 programs. These results also prove that IL-4 is not required for IL-9-producing T-cell precursor development. These results were confirmed in another experiment and after stimulation with SEB (data not shown).

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Figure 2. TGF-β interaction with IL-1 stimulates IL-9 production independently of IL-4. Pooled lymph nodes of OVA-immunized BALB/c and IL-4 KO mice (three mice each) were stimulated with OVA±cytokines as indicated and IL-9 production measured (mean±SEM of triplicates). *p<0.05, **p<0.01, and ***p<0.001 as compared with the respective TGF-β group only (ANOVA). The experiment was repeated once with similar results.

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Comparison of TGF-β/IL-1 and other cytokines in the regulation of IL-9, IL-13, and IL-17

The influence of IL-1α or TGF-β/IL-1α on cytokine production was compared with that of Th1 (IL-12, anti-TGF-β and anti-IL-4), Th2 (IL-4, anti-TGF-β and anti-IFN-γ), “Th9” (TGF-β/IL-4/anti-IFN-γ and Th17 (TGF-β IL-6, anti-IFN-γ and anti-IL-4 culture conditions in recall responses of OVA-vaccinated BALB/c mice. TGF-β/IL-1α provided the strongest stimulus for IL-9 production followed by TGF-β/IL-4. Th2 and Th17 conditions did not significantly modify IL-9 levels as compared with control medium, whereas Th1 conditions made IL-9 completely undetectable (Fig. 3A). In this experiment, IL-1 also enhanced IL-9 levels but the difference with controls was not statistically significant and was not a constant observation (Fig. 1). On the contrary, for IL-13, IL-1 provided the strongest stimulus (stronger than Th2 conditions) but IL-13 levels dropped after addition of TGF-β. Similar results were obtained for IL-5 (data not shown). As to IL-17, it was maximal as expected in Th17 conditions but was also stimulated by IL-1. However, contrary to IL-9, it was not further increased by addition of TGF-β to IL-1. These results were confirmed in another experiment and in DBA/2 and C57BL/6 mice.

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Figure 3. Comparison of the control of IL-9, IL-13, and IL-17 production. (A) Lymph node cells pooled from three BALB/c mice immunized with OVA in CFA were cultured with OVA±cytokines as indicated and production of IL-9, IL-13, and IL-17 determined (mean±SEM of triplicates). *p<0.05, **p<0.01, and ***p<0.001 versus OVA only are shown (ANOVA). Similar results were obtained in two other experiments. (B) OVA-stimulated lymph node cells from OVA-vaccinated mice (pooled data from two BALB/C and two DBA/2 experiments, four mice per strain per experiment) were cultured under Th2, Th17, TGF-β/IL-1, and TGF-β/IL-4 conditions and IL-9 production measured (mean±SEM of triplicates). *p<0.05 versus OVA only stimulation (ANOVA). (C) Pooled spleen cells from two D011.10 BALB/c mice were incubated with OVA and the indicated cytokines with or without anti-IL-2Rα Ab (mean±SEM of triplicates). **p<0.01 and ***p<0.001 for the effect of anti-IL-2Rα Ab for each condition (t-test). These results were confirmed in three experiments. (D) Pooled spleen cells from two BALB/c mice were incubated with varying concentrations of IL-1α and IL-23 (Δ, 2 ng/mL; ◊, 1 ng/mL; □, 0.5 ng/mL; and ∘, 0 ng/mL) in the absence of other stimuli. IL-17 and IL-9 concentrations were measured after 4 days and data represent mean±SEM of triplicate cultures. This experiment was repeated once with identical results.

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Several publications have reported IL-9 production by purified Th17 CD4+ T cells activated with anti-CD3/CD28 15, 16. We carefully tested this condition in antigen recall responses of mice immunized with OVA. In pooled data of four experiments, the mean IL-9 concentrations were significantly enhanced only by TGF-β/IL-1α (30-fold) and by TGF-β/IL-4 (ten-fold) as compared with control OVA cultures. The minor increases triggered by IL-1α, Th17, or Th2 cultures were not significant (Fig. 3B).

IL-2 is required for IL-9 production by naïve CD4+ T cells 11 but was reported to constrain Th17 development 21. We tested the influence of the blocking anti-IL-2Rα Ab PC61 on IL-9 and IL-17 production. This Ab inhibited IL-9 production in TGF-β/IL-1 cultures by±75% but rather stimulated, although not significantly, that of IL-17 (Fig. 3C).

IL-1 and IL-23 were reported to initiate a strong IL-17 production by T cells even in the absence of other stimuli 22. We confirmed this synergistic interaction in a combined dose–response curve of the two factors in BALB/c spleen cells which produced more than 10 ng/mL of IL-17 when incubated with IL-1α and IL-23 and much lower concentrations when IL-1α and IL-23 were used separately (Fig. 3D). IL-9 remained undetectable under these conditions. No IL-17 was produced by SCID spleen cells treated with IL-1α and IL-23, attesting the T-cell origin of this IL-17 (data not shown). These results indicate that although T-cell IL-9 and IL-17 productions are both enhanced by TGF-β/IL-1, there are many differences in their regulation.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

The present results extend the implications of TGF-β in the control of IL-9 production and illustrate the modulation of its activities by the cytokine milieu. It is remarkable that the prototypic inflammatory cytokine IL-1, which amplifies both Th2 17 and Th17 responses 22, stimulates only IL-9 poorly but becomes a potent IL-9 inducer in the presence of TGF-β. The relevance of this finding for in vivo regulation of IL-9 is supported by the observations that IL-1α- and IL-1β-deficient mice have diminished IL-9 production and show increased susceptibility to T. muris17, an infection where IL-9 stimulates worm expulsion 4.

Our results also prove for the first time that the very selective IL-9 upregulation by TGF-β/IL-4 described for naïve CD4+ T cells activated in vitro9 operates also during antigen restimulation of in vivo primed CD4+ T cells, attesting the ability of this cytokine combination to transform immune T cells into selective IL-9 producers.

Finally, we show that in the presence of antigen and APC, Th2, and Th17 culture conditions induce much lower IL-9 production than TGF-β/IL-4 or TGF-β/IL-1, indicating that IL-9 is neither a Th2 nor a Th17 cytokine but obeys distinct regulation rules.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

Mice

BALB/c, DBA/2, C57BL/6, and D011.10 BALB/c mice which express an OVA-specific TCR 23 were maintained under SPF conditions at our animal facility. BALB/c IL-4-KO 24 were bred in the University of Cape Town (South Africa). The experimental protocol and animal handling was approved by the ethical committee of our Faculty.

In vitro stimulation and cytokine analysis

Mice were immunized with 200 μg OVA (Sigma-Aldrich) emulsified in CFA s.c. at the base of the tail or i.p. in Imject Alum (Pierce). Inguinal and para-aortic lymph node or spleen cells were collected after 7 days and cultured in Iscove-Dulbecco's medium supplemented as described previously 1. Cells were incubated with OVA (100 μg/mL), SEB at 20 ng/mL or anti-CD3 Ab (1 μg/mL) and anti-CD28 Ab (10 μg/mL) for 4 days in flat bottomed 96-well plates in the presence of cytokines (5–10 ng/mL) and Ab at 10 μg/mL (anti-IL-4: 11B11, anti-TGF-β: 1D11, anti-IFN-γ: FXIVF3 (a kind gift of H. Heremans, KUL, Belgium) 25, anti-IL-2Rα PC61).

Cytokines were from R&D Systems, eBioscience or home made. IL-5 and IL-13 ELISA were from R&D Systems, IL-9 and IL-17 ELISA used Ab made in our laboratory.

IL-9 intracellular staining

BALB/c lymph nodes were collected 7 days after immunization with OVA in CFA. Cells were labeled with CFSE before in vitro stimulation with OVA for 5–7 days. They were then restimulated with PMA/ionomycine in the presence of monensin for 6 h and labeled with anti-CD4-APC and anti-IL-9 RM9A4-PE Ab, both from Biolegend, using the BD Cytofix/Cytoperm kit. Data were acquired on a FACScalibur (BD) and analyzed with the FlowJo software.

Statistical analysis

All data represent mean±SEM of triplicate cultures and significance was tested by ANOVA or t-tests (*p<0.05; **p<0.01; ***p<0.001).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References

The authors are indebted to Brigitta Stockinger and Marc Veldhoen (Division of Molecular Immunology, Medical Research Council National Institute for Medical Research, London, UK) for critical reading and experimental suggestions. They also thank Dominique Donckers and Isabelle Bar for expert technical assistance, Julie Klein, and Suzanne Depelchin for editorial contribution, Dr. Laure Dumoutier for suggestions regarding intracellular staining, Professor Jean-Christophe Renauld for discussions, and Dr. Sherrie Zhang (Biolegend) for helpful interactions. This work was supported in part by a grant from the Fonds National de la Recherche Scientifique, Belgium.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  • 1
    Uyttenhove, C., Simpson, R. and Van Snick, J., Functional and structural characterization of P40, a mouse glycoprotein with T cell growth factor activity. Proc. Natl. Acad. Sci. USA 1988. 85: 69346938.
  • 2
    Schmitt, E., van Brandwijk, R., Van Snick, J., Siebold, B. and Rüde, E., TCGF III/P40 is produced by naive murine CD4+T cells but is not a general T cell growth factor. Eur. J. Immunol. 1989. 19: 21672170.
  • 3
    Hültner, L., Druez, C., Moeller, J., Uyttenhove, C., Schmitt, E., Rüde, E., Dörmer, P. and Van Snick, J., Mast cell growth enhancing activity (MEA) is structurally related and functionally identical to the novel mouse T cell growth factor P40/TCGFIII (interleukin-9). Eur. J. Immunol. 1990. 20: 14131416.
  • 4
    Richard, M., Grencis, R. K., Humphreys, N. E., Renauld, J.-C. and Van Snick, J., Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia in Trichuris muris-infected mice. Proc. Natl. Acad. Sci. USA 2000. 97: 767772.
  • 5
    Townsend, J. M., Fallon, G. P., Matthews, J. D., Smith, P., Jolin, E. H. and McKenzie, N. A., IL-9-deficient mice establish fundamental roles for IL-9 in pulmonary mastocytosis and goblet cell hyperplasia but not T cell development. Immunity 2000. 13: 573583.
  • 6
    Else, K. J., Hultner, L. and Grencis, R. K., Cellular immune responses to the murine nematode parasite Trichuris muris. II. Differential induction of TH-cell subsets in resistant versus susceptible mice. Immunology 1992. 75: 232237.
  • 7
    Gessner, A., Blum, H. and Rollinghoff, M., Differential regulation of IL-9-expression after infection with Leishmania major in susceptible and resistant mice. Immunobiology 1993. 189: 419435.
  • 8
    Gounni, A. S., Nutku, E., Koussih, L., Aris, F., Louahed, J., Levitt, R. C., Nicolaides, N. C. and Hamid, Q., IL-9 expression by human eosinophils: regulation by IL-1beta and TNF-alpha. J. Allergy Clin. Immunol. 2000. 106: 460466.
  • 9
    Veldhoen, M., Uyttenhove, C., van Snick, J., Helmby, H., Westendorf, A., Buer, J., Martin, B. et al., Transforming growth factor-beta ‘reprograms’ the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset. Nat. Immunol. 2008. 9: 13411346.
  • 10
    Dardalhon, V., Awasthi, A., Kwon, H., Galileos, G., Gao, W., Sobel, R. A., Mitsdoerffer, M. et al., IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+ IL-10+ Foxp3(−) effector T cells. Nat. Immunol. 2008. 9: 13471355.
  • 11
    Schmitt, E., Germann, T., Goedert, S., Hoehn, P., Huels, C., Koelsch, S., Kühn, R. et al., IL-9 production of naive CD4+ T cells depends on IL-2, is synergistically enhanced by a combination of TGF-β and IL-4, and is inhibited by IFNg. J. Immunol. 1994. 153: 39893996.
  • 12
    Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T. B., Oukka, M., Weiner, H. L. and Kuchroo, V. K., Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006. 441: 235238.
  • 13
    Mangan, P. R., Harrington, L. E., O'Quinn, D. B., Helms, W. S., Bullard, D. C., Elson, C. O., Hatton, R. D. et al., Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 2006. 441: 231234.
  • 14
    Veldhoen, M., Hocking, R. J., Atkins, C. J., Locksley, R. M. and Stockinger, B., TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006. 24: 179189.
  • 15
    Elyaman, W., Bradshaw, E. M., Uyttenhove, C., Dardalhon, V., Awasthi, A., Imitola, J., Bettelli, E. et al., IL-9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural regulatory T cells. Proc. Natl. Acad. Sci. USA 2009. 106: 1288512890.
  • 16
    Nowak, E. C., Weaver, C. T., Turner, H., Begum-Haque, S., Becher, B., Schreiner, B., Coyle, A. J. et al., IL-9 as a mediator of Th17-driven inflammatory disease. J. Exp. Med. 2009. 206: 16531660.
  • 17
    Helmby, H. and Grencis, R. K., Interleukin 1 plays a major role in the development of Th2-mediated immunity. Eur. J. Immunol. 2004. 34: 36743681.
  • 18
    Humphreys, N. E. and Grencis, R. K., IL-1-dependent, IL-1R1-independent resistance to gastrointestinal nematodes. Eur. J. Immunol. 2009. 39: 10361045.
  • 19
    Von Stebut, E., Ehrchen, J. M., Belkaid, Y., Kostka, S. L., Molle, K., Knop, J., Sunderkotter, C. and Udey, M. C., Interleukin 1alpha promotes Th1 differentiation and inhibits disease progression in Leishmania major-susceptible BALB/c mice. J. Exp. Med. 2003. 198: 191199.
  • 20
    Schmitt, E., Beuscher, U., Huels, C., Monteyne, P., Van Brandwijk, R., Van Snick, J. and Ruede, E., IL-1 serves as a secondary signal for IL-9 expression. J. Immunol. 1991. 147: 38483854.
  • 21
    Laurence, A., Tato, C. M., Davidson, T. S., Kanno, Y., Chen, Z., Yao, Z., Blank, R. B. et al., Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation. Immunity 2007. 26: 371381.
  • 22
    Sutton, C., Brereton, C., Keogh, B., Mills, K. H. and Lavelle, E. C., A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis. J. Exp. Med. 2006. 203: 16851691.
  • 23
    Murphy, K. M., Heimberger, A. B. and Loh, D. Y., Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 1990. 250: 17201723.
  • 24
    Noben-Trauth, N., Kohler, G., Burki, K. and Ledermann, B., Efficient targeting of the IL-4 gene in a BALB/c embryonic stem cell line. Transgenic Res. 1996. 5: 487491.
  • 25
    Billiau, A., Heremans, H., Vandekerckhove, F. and Dillen, C., Anti-interferon-gamma antibody protects mice against the generalized Shwartzman reaction. Eur. J. Immunol. 1987. 17: 18511854.