Epimetabolic regulation of macrophage activation in the intra-alveolar compartment during acute lung injury - Acute lung injury (ALI) and its clinical manifestation, acute respiratory distress syndrome (ARDS), carry significant morbidity and mortality. However, there are intrinsic innate protective mechanisms. Harnessing those poorly understood mechanisms could enable us to exploit them therapeutically. Alveolar type II (ATII) cells are key players in acute lung injury. Alveolar macrophages (AM) are critical to the pathogenesis of ALI, where local microenvironmental cues shape their inflammatory or anti-inflammatory properties, which can be fluid and amenable to manipulation. How metabolic intermediates (such as lactate, the end product of glycolysis), released into the microenvironment, can affect macrophage phenotypes is unknown, especially in the setting of ALI. A key innate protective mechanism in ALI involves enhanced glycolysis in ATII cells. Our published work shows that macrophages co-cultured with ATII cells display a blunted response to LPS stimulation, which is dependent on lactate produced by the alveolar epithelial cells. Furthermore, locally intratracheally (i.t.) applied lactate can attenuate ALI in mouse models. AMs take up lactate via the monocarboxylase transporter (MCT) 1. Lastly, our preliminary data suggest lactate can induce an increase in histone lactylation. Histone lactylation is a recently described histone modification that renders promoters of genes accessible to transcription. The overall goal of this proposal is to delineate how lactate produced by alveolar epithelial type II cells is protective in ALI by shifting the AM towards an anti-inflammatory phenotype. We hypothesize that in the intra-alveolar compartment, lactate released by ATII cells is necessary and sufficient to induce the generation of anti-inflammatory cytokines by AM in ALI via epimetabolic regulation of transcription: we will test if uptake of ATII-cell-derived lactate by AMs via MCT1 induces an anti-inflammatory macrophage phenotype (Aim 1). We will test if ATII derived lactate specifically targets AMs and if, in AMs, lactate funnels into the Krebs cycle (Aim 2). Lastly, we will study whether lactate induces the expression of anti-inflammatory cytokines by increasing the accessibility of transcription factors through histone lactylation (Aim 3). Acid aspiration, i.t. LPS and Staph. aureus pneumonia will be used as murine models of ALI that directly target the intra-alveolar compartment. We will use a comprehensive genetic approach utilizing tissue specific knock out mice targeting macrophages and AT II cells. We will utilize primary human AMs for translational studies. Flow cytometry, ELISA assays, RNA single-cell sequencing, and qPCR will be used to characterize AMs. Functional studies with metabolomic tracer experiments with 13C-labeled lactate will be performed to characterize the metabolic flux. We will study H3 histone lactylation modifications and unbiased transcription factor analysis experiments. This proposal is novel by expanding the scope of understanding of the role of epimetabolic crosstalk between ATII cells and AMs in ALI. It involves state-of-the-art in vitro and in vivo experimental approaches and incorporates a therapeutic approach with potential for translation.
