Project Summary The interleukin 1 (IL-1) cytokine superfamily member IL-33 drives host protection against gastrointestinal (GI) pathogens through mechanisms that remain unclear. Paradoxically, IL-33 responsiveness in different lymphocyte subsets dictates its pro- vs. anti-inflammatory roles evinced by its ability to promote expansion of group 2 innate lymphoid cells (ILC2) or Foxp3+ regulatory T cells (Tregs), respectively. Whether source of IL-33 influences its biological role has been largely ignored due the current paradigm that IL-33 is only released from damaged structural cells (e.g. epithelial cell lineages). However, our recently published data show that intestinal epithelial cell (IEC)-derived IL-33 drives ILC2 responses and GI helminth immunity, whereas dendritic cell (DC)- derived IL-33 drives ST2+Foxp3+ Treg responses and helminth susceptibility. This proposal seeks to understand the mechanisms responsible for IL-33 expression and production in the myeloid lineage and further investigates whether IL-33 cellular context controls the balance between inflammation vs. immunosuppression during protozoan infections as it does in helminth infections. Our project tests the central hypothesis that cellular source of IL-33 determines whether IL-33 serves a protective or pathogenic role during GI parasite infection through controlling intestinal Treg subset expansion. We will test this hypothesis in two specific aims (SA). Experiments proposed in SA1 will: a) transcriptionally profile IL-33+ vs. IL-33- DC to determine whether IL-33 expressing APC constitute a distinct subset, b) employ microscopy and imaging mass spectrometry to understand the tissue niches where these cells reside, and c) determine whether DC-derived IL-33 is critical for the maintenance and/or induction of distinct subsets of Foxp3+ Treg through microbial-dependent mechanisms. SA2 will generate mice that allow IL-33 deletion in distinct DC-subsets and tissue macrophage populations to determine their impact on intestinal tissue homeostasis and host protection against GI pathogens. Lastly, we will determine whether epithelial vs. myeloid derived-IL-33 controls microbial dysbiosis and the pathological outcome following oral Toxoplasma gondii infection. Taken together, this project seeks to expand the basic understanding of how the cellular context of cytokine production impacts immunity against important human GI pathogens.