ABSTRACT We understand very little of how invasive fungal pathogens overcome host blocks to infection, yet such infections are among the most challenging to diagnose and treat. Our focus is the most common cause of fungal meningitis, Cryptococcus neoformans. Macrophages and other myeloid cells are critical for killing pathogens and promoting immune responses. Within the initial site of Cryptococcal infection, the lung, multiple populations of myeloid cells exist, including alveolar macrophages (AMs), interstitial macrophages (IMs), monocytes, and neutrophils. Importantly, a type 2 immune response is triggered by C. neoformans infection and mediates increased susceptibility to Cryptococcal infection. We observed that incubation of C. neoformans with murine bone marrow- derived macrophages (BMDMs) results in induction of arginase-1 (Arg1) a marker for type 2 responses. Through forward genetics, we identified a C. neoformans secreted protein, CPL1, required for Arg1 induction and virulence in vivo. Remarkably, recombinant CPL1 protein is sufficient to induce Arg1 expression in BMDMs in vitro and to boost the bona fide M2 response to the type 2 cytokine IL-4. Correspondingly, CPL1 is critical for C. neoformans to activate Arg1 in interstitial macrophages in vivo. TLR4, known best as an LPS receptor but which is also a poorly-understood mediator of type 2 responses, is required for CPL1 to act on BMDMs. As TLR4 does not directly couple to the key type 2 transcription factor STAT6, we sought indirect mechanisms of action. Salmonella promotes M2 polarization via a secreted effector, steE, that activates STAT3 by mimicking cytokine signaling. Indeed, we found STAT3 to also be required for Arg1 induction by CPL1. These data lead to the following working model: 1) CPL1 promotes macrophage polarization indirectly by weakly activating TLR4 to produce cytokines that activate STAT3, and 2) cooperation between STAT3 and the key type 2 regulator STAT6 leads to enhanced macrophage polarization to drive virulence. To test this model, we have assembled a collaborative team with expertise in fungal pathogenesis and immunology. Through Aim 1, we will elucidate mechanisms by which CPL1 promotes macrophage polarization in vitro. We will test the model that CPL1 boosts the M2 response via STAT3-STAT6 cooperation, and we will investigate how CPL1 functions through TLR4. Through Aim 2, we will test the hypothesis that CPL1 polarizes lung interstitial macrophages in vivo, thereby producing a replicative niche. We will investigate the role of TLR4 in the macrophage/monocyte compartment, and we will use thick section imaging and other modalities to test predictions of the model that CPL1 acts locally to promote macrophage polarization to enable pathogen replication. These studies have the potential to transform our understanding how an invasive fungal pathogen reprogram the innate immune system to its advantage.