Background: IL-33 is tissue-derived cytokine whose primary proposed function is to modulate immune responses when released during injury. Delivery of recombinant IL-33 protects cardiac allografts due to expansion of regulatory T cells (Treg), but how heart transplant (HTx) or recipient IL-33 shapes alloimmune responses or HTx outcomes is not known.

Methods: Modulation of IL-33 during HTx rejection was defined in pediatric recipient (n=39) endomyocardial biopsies using quantum dot-based immunofluorescence and serum samples by ELISA. A role for endogenous IL-33 in acute rejection was defined in heterotopic HTx studies using BALB/c, wild type (WT) C57BL/6 (B6) or IL-33^-/- B6 mice as recipients and donors. The impact of IL-33 on chronic HTx rejection was assessed in studies where: 1) B6 bm12 hearts were transplanted into WT or IL-33^-/- B6 mice or 2) IL-33^-/- male HTx in IL-33^-/- female B6 recipients were compared to equivalent WT B6 combinations. On day 7 (acute model) or day 120 (chronic models), HTx were stained with H+E or Trichrome. Frozen tissue was subjected to qRT-PCR, Western blot, and IF staining to define differences in cytokine expression. Splenocytes were characterized through flow cytometry.

Results: During rejection episodes in pediatric HTx recipients there was a 20x increase in HTx IL-33 (p=0.0049) and elevated serum IL-33 (9.7±1.8 ng/ml; p=0.0005) compared to patients without acute rejection (2.2 ±0.6 ng/ml). Increases in fibroblast IL-33 were obvious during acute and chronic mouse HTx rejection. IL-33 deficiency had minimal impact on acute rejection rates and associated alloimmune responses. However, bm12 HTx in IL-33^-/- recipients displayed increased HTx fibrosis that was associated with a decreased frequency of splenic Treg, especially an IL-33R⁺ CD44⁺ subset. IL-33^-/- female recipients mounted a more intensive rejection against male heart grafts, with higher percentages of activated (ICOS⁺), memory (CD44^hiCD62L^lo) and Th17 (ROR-γt⁺ and IL-17⁺) CD4 T cells. Augmented fibrosis in male heart grafts from IL-33^-/- female recipients was again observed.

Conclusions: Our data support a critical role for augmented endogenous IL-33 in regulation of T cell alloresponses and prevention of chronic rejection associated HTx fibrosis. Manipulating IL-33-related immune functions may lead to new therapies able to prevent or resolve chronic HTx rejection.

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