Abstract Rationale In our earlier publications, we proposed an original mechanism by which IL-33 and its receptor ST2 regulate pulmonary fibrosis (PF). We postulated that each IL-33 and ST2 may bypass the conventionally considered IL-33/ST2 axis-driven regulation. This notion is supported in part by differential attenuation of PF in mice deficient of IL-33 vs mice deficient of ST2: IL-33 deficiency attenuates fibrosis more potently than ST2 deficiency. We sought to identify the mechanism that prevents ST2 deficiency from inducing a more complete attenuation of PF in mice. Methods Wild-type (WT), ST2 knockout (ST2KO), and dual ST2 and IL-33 knockout (DKO) mice were tested. The traditional intratracheal single-hit bleomycin model was used, as well as a double-hit fibrotic lung injury model. In the latter, the first hit was represented by adenovirus-mediated pulmonary overexpression of either mature IL-33 cytokine (MIL33) or full-length IL-33 precursor (FLIL33), followed by the second hit, intratracheal bleomycin injury. The levels of various cytokines were screened in lung homogenates utilizing Luminex multiplex and Olink technologies; the levels of selected cytokines were confirmed by ELISA. Total lung collagen was measured using QuickZyme approach. Neutralizing anti-IL-9 and anti-TSLP antibodies were used to assess the effects of these cytokines on pulmonary collagen accumulation. IL-9-producing cells were identified flowcytometrically and immunocytochemically. Results Among dozens of biomarkers screened by Luminex and Olink, a profibrotic cytokine IL-9 consistently stood out as unabated by ST2 deficiency. Instead, ELISA results showed that both ST2KO and DKO mice expressed more IL-9 than did WT mice. Overexpression of neither FLIL33 nor MIL33 affected the IL-9 expression pattern. ELISA results revealed that the levels of a known inducer of IL-9 expression, TSLP, were also elevated. Blockade of IL-9 attenuated fibrosis in WT but not ST2KO or DKO mice, whereas blockade of TSLP inhibited elevations in IL-9 and in pulmonary collagen across the mouse strains. The combined inhibitory effect of ST2 deficiency and TSLP blockade was particularly potent, bringing collagen to the basal levels seen in control mice. Overall, similar findings were made in ST2KO and DKO mice. Pulmonary CD4+ T cells were the main producers of IL-9. Conclusions These results point to ST2-mediated regulation of fibrosis being partially independent of IL-33. The incomplete attenuation of fibrosis by ST2 deficiency is driven by the simultaneous activation of the profibrotic TSLP - IL-9 axis. Therapeutic targeting of solely ST2 should be considered less promising than dual targeting of both ST2 and TSLP. This abstract is funded by: NIH R01AR077562; VA I01BX006173
Luzina et al. (Fri,) studied this question.