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. 2016 Aug;100(2):381-9.
doi: 10.1189/jlb.3A1015-474R. Epub 2016 Apr 5.

Stromal fibroblasts support dendritic cells to maintain IL-23/Th17 responses after exposure to ionizing radiation

Anna Malecka  1 , Qunwei Wang  1 , Sabaria Shah  1 , Ruhcha V Sutavani  2 , Ian Spendlove  3 , Judith M Ramage  3 , Julie Greensmith  4 , Hester A Franks  1 , Michael J Gough  5 , Anja Saalbach  6 , Poulam M Patel  1 , Andrew M Jackson  7
Affiliations

Stromal fibroblasts support dendritic cells to maintain IL-23/Th17 responses after exposure to ionizing radiation

Anna Malecka et al. J Leukoc Biol. 2016 Aug.

Abstract

Dendritic cell function is modulated by stromal cells, including fibroblasts. Although poorly understood, the signals delivered through this crosstalk substantially alter dendritic cell biology. This is well illustrated with release of TNF-α/IL-1β from activated dendritic cells, promoting PGE2 secretion from stromal fibroblasts. This instructs dendritic cells to up-regulate IL-23, a key Th17-polarizing cytokine. We previously showed that ionizing radiation inhibited IL-23 production by human dendritic cells in vitro. In the present study, we investigated the hypothesis that dendritic cell-fibroblast crosstalk overcomes the suppressive effect of ionizing radiation to support appropriately polarized Th17 responses. Radiation (1-6 Gy) markedly suppressed IL-23 secretion by activated dendritic cells (P < 0.0001) without adversely impacting their viability and consequently, inhibited the generation of Th17 responses. Cytokine suppression by ionizing radiation was selective, as there was no effect on IL-1β, -6, -10, and -27 or TNF-α and only a modest (11%) decrease in IL-12p70 secretion. Coculture with fibroblasts augmented IL-23 secretion by irradiated dendritic cells and increased Th17 responses. Importantly, in contrast to dendritic cells, irradiated fibroblasts maintained their capacity to respond to TNF-α/IL-1β and produce PGE2, thus providing the key intermediary signals for successful dendritic cell-fibroblasts crosstalk. In summary, stromal fibroblasts support Th17-polarizing cytokine production by dendritic cells that would otherwise be suppressed in an irradiated microenvironment. This has potential ramifications for understanding the immune response to local radiotherapy. These findings underscore the need to account for the impact of microenvironmental factors, including stromal cells, in understanding the control of immunity.

Keywords: cancer; cytokine; immunity.

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Figures

Figure 1.
Figure 1.. FBs promote the IL-23–Th17 axis during irradiation.
IR suppresses IL-23. DCs were irradiated before addition of LPS/IFN-γ and IL-23 release measured by ELISA. (A) IL-23 suppression was dependent on the dose of IR (a representative donor from 3). (B) A representative experiment at 6 Gy. (C) A summary of 18 donors. (D) FBs rescue IL-23 secretion by irradiated DCs. Coculture of irradiated FBs with irradiated DCs up-regulated IL-23 secretion (summary of 8 donors). (E) Fold change of IL-23 secretion by irradiated DC/FB cocultures at 0 and 6 Gy. (F) Primary dermal FBs demonstrate similar IL-23-enhancing activity to the BJ6 cell line (data from a representative donor or 5). (G) FBs permit irradiated DCs to promote Th17 responses. IL-17A secretion by naïve CD4+ T cells activated with anti-CD3/anti-CD28 in the presence of supernatants of the indicated DC/FB cultures. T cells were stimulated for 5 d and IL-17A secretion determined after restimulation (collective data from 3 donors). Error bars indicate sd of triplicate experiments. **0.01 > P ≥ 0.001, ***0.001 > P ≥ 0.0001, ****P < 0.0001; ns, not significant. Results are presented as means ± sd.
Figure 2.
Figure 2.. Effect of IR on DCs.
Changes in IL-23 secretion were associated with decreased transcription of the (A) IL-23A gene and (B) IL-12B gene, as shown using quantitative RT-PCR in 3 donors. Statistical significance was measured by the Student's t test. (C) Despite exposure to up to 6 Gy IR, there was no decrease in viability of DCs as assayed by annexin V/PI staining with flow cytometry up to 72 h after irradiation. Representative result for 1 of 3 donors tested. ****P < 0.0001. Results are presented as means ± sd.
Figure 3.
Figure 3.. FBs are functionally resistant to IR.
Irradiation of FBs did not impair their ability to up-regulate IL-23 secretion from nonirradiated DC. (A) A representative donor. (B) Summary of 3 donors. (C) PGE2 secretion (determined by ELISA) by FBs stimulated with rTNF-α and rIL-1β (1 ng/ml) is unaffected by IR; BJ6 cell line. (D) Representative results for 1 of 2 primary FB donors. (E) IR (6 Gy) does not affect FB viability up to 24 h after irradiation, as assessed by annexin V/PI staining; representative data from 3 experiments. ***0.001 > P ≥ 0.0001. Results are presented as means ± sd.
Figure 4.
Figure 4.. Mechanisms of IL-23 recovery from irradiated DCs by FBs.
FB-dependent IL-23 secretion from irradiated DCs is mediated by soluble factors and was independent of cell-cell contact. (A) Fold increase in IL-23 secretion by DCs separated from FBs with Transwell (0.3 μm) treated with IR (summary of 3 donors). (B) Addition of PGE2 to irradiated DCs up-regulates IL-23 secretion (summary of 5 donors). (C) Up-regulation of IL-23 secretion from irradiated DCs by FBs is COX2 dependent. FBs were treated with indomethacin for 24 h before (and throughout) coculture with irradiated DCs (data from 3 experiments). (D) FBs did not affect pATM in irradiated DCs. Irradiated mDCs cultured with FBs (blue lines) or without FB (orange lines) show similar levels of pATM. In both of these settings, IR increased ATM activation when compared with nonirradiated mDCs (red lines), as demonstrated by intracellular staining for flow cytometry at 2 and 12 h after IR (6 Gy, representative donor of 3). (E) Stimulation of TLR4-activated DCs with the cAMP agonist forskolin immediately following exposure to IR increased IL-23 secretion (data from 3 representative donors.) *0.05 > P ≥ 0.01, **0.01 > P ≥ 0.001. Results are presented as means ± sd.
Figure 5.
Figure 5.. Model of DC-FB crosstalk governing IL-23-dependent Th17 responses after irradiation.
(A) TLR activation of DCs in monoculture elicits secretion of IL-1β, -6, and -23, which prime Th17 responses. (B) The addition of FB to DC provides an important feedback loop that serves to enhance IL-23 secretion and thus, augments Th17 responses. (C) Irradiation of DC monocultures selectively inhibits IL-23 secretion. (D) However, the presence of FBs ensures that irradiated DCs continue to secrete sufficient IL-23 to generate Th17 responses. Importantly, the irradiation of FBs does not hinder their reinforcement of IL-23 responses.
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