Proper regulation of sterol biosynthesis is critical for eukaryotic cellular homeostasis. Cholesterol and isoprenoids serve key roles in eukaryotic cells by regulating membrane fluidity and correct localization of proteins. It is becoming increasingly appreciated that dysregulated sterol metabolism engages pathways that lead to inflammation. Of particular importance are inflammasomes, which are multiplatform protein complexes that activate caspase-1 in order to process the pro-inflammatory and pyrogenic cytokines IL-1β and IL-18. In this review, we highlight recent research that links altered sterol biosynthetic pathway activity to inflammasome activation. We discuss how clues from human genetics have led to new insights into how alterations in isoprenoid biosynthesis connect to inflammation. We also discuss new mechanisms that show how macrophage cholesterol buildup can lead to inflammasome activation.

Type I interferon restrains interleukin-1β (IL-1β)-driven inflammation in macrophages by upregulating cholesterol-25-hydroxylase (Ch25h) and repressing SREBP transcription factors. However, the molecular links between lipid metabolism and IL-1β production remain obscure. Here, we demonstrate that production of 25-hydroxycholesterol (25-HC) by macrophages is required to prevent inflammasome activation by the DNA sensor protein absent in melanoma 2 (AIM2). We find that in response to bacterial infection or lipopolysaccharide (LPS) stimulation, macrophages upregulate Ch25h to maintain repression of SREBP2 activation and cholesterol synthesis. Increasing macrophage cholesterol content is sufficient to trigger IL-1β release in a crystal-independent but AIM2-dependent manner. Ch25h deficiency results in cholesterol-dependent reduced mitochondrial respiratory capacity and release of mitochondrial DNA into the cytosol. AIM2 deficiency rescues the increased inflammasome activity observed in Ch25h^-/-. Therefore, activated macrophages utilize 25-HC in an anti-inflammatory circuit that maintains mitochondrial integrity and prevents spurious AIM2 inflammasome activation.

Correct positioning of dendritic cells (DCs) is critical for efficient pathogen encounter and antigen presentation. Epstein-Barr virus-induced gene 2 (EBI2) has been identified as a chemoattractant receptor required for naïve CD4+DCIR2+ DC positioning in response to 7α,25-hydroxycholesterol (7α,25-HC). We now provide evidence that a second EBI2 ligand, 7α,27-HC, is involved in splenic DCIR2+ DC positioning and homeostasis. Cyp27a1, the enzyme uniquely required for 7α,27-HC synthesis, is expressed by stromal cells in the region of naïve DC localization. After activation, DCIR2+ DCs move into the T cell zone. We find that EBI2 is rapidly up-regulated in DCIR2+ DCs under certain activation conditions, and positioning at the B-T zone interface depends on EBI2. Under conditions of type I interferon induction, EBI2 ligand levels are elevated, causing activated DCIR2+ DCs to disperse throughout the T zone. Last, we provide evidence that oxysterol metabolism by Batf3-dependent DCs is important for EBI2-dependent positioning of activated DCIR2+ DCs. This work indicates that 7α,27-HC functions as a guidance cue in vivo and reveals a multitiered role for EBI2 in DC positioning. Deficiency in this organizing system results in defective CD4+ T cell responses.

Invasive fungal pathogens are major causes of human mortality and morbidity^1,2. Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans, the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages^3-8. Here, we identify CPL1 as an effector protein secreted by C. neoformans that drives alternative activation (also known as M2 polarization) of macrophages to enable pulmonary infection in mice. We observed that CPL1-enhanced macrophage polarization requires Toll-like receptor 4, which is best known as a receptor for bacterial endotoxin but is also a poorly understood mediator of allergen-induced type 2 responses^9-12. We show that this effect is caused by CPL1 itself and not by contaminating lipopolysaccharide. CPL1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signalling for its effect on infectivity. Notably, C. neoformans associates selectively with polarized interstitial macrophages during infection, suggesting a mechanism by which C. neoformans generates its own intracellular replication niche within the host. This work identifies a circuit whereby a secreted effector protein produced by a human fungal pathogen reprograms innate immunity, revealing an unexpected role for Toll-like receptor 4 in promoting the pathogenesis of infectious disease.

Candida albicans is the most common cause of fungal infection in humans. IL-17 is critical for defense against superficial fungal infections, but the role of this response in invasive disease is less understood. We show that C. albicans secretes a lipase, Lip2, that facilitates invasive disease via lipid-based suppression of the IL-17 response. Lip2 was identified as an essential virulence factor in a forward genetic screen in a mouse model of bloodstream infection. Murine infection with C. albicans strains lacking Lip2 display exaggerated IL-17 responses that lead to fungal clearance from solid organs and host survival. Both IL-17 signaling and lipase activity are required for Lip2-mediated suppression. Lip2 inhibits IL-17 production indirectly by suppressing IL-23 production by tissue-resident dendritic cells. The lipase hydrolysis product, palmitic acid, similarly suppresses dendritic cell activation in vitro. Thus, C. albicans suppresses antifungal IL-17 defense in solid organs by altering the tissue lipid milieu.

Cryptococcus neoformans is the leading cause of fungal meningitis and is characterized by pathogenic eosinophil accumulation in the context of type-2 inflammation. The chemoattractant receptor GPR35 is expressed by granulocytes and promotes their migration to the inflammatory mediator 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite. Given the inflammatory nature of cryptococcal infection, we examined the role of GPR35 in the circuitry underlying cell recruitment to the lung. GPR35 deficiency dampened eosinophil recruitment and fungal growth, whereas overexpression promoted eosinophil homing to airways and fungal replication. Activated platelets and mast cells were the sources of GPR35 ligand activity and pharmacological inhibition of serotonin conversion to 5-HIAA, or genetic deficiency in 5-HIAA production by platelets and mast cells resulted in more efficient clearance of Cryptococcus. Thus, the 5-HIAA-GPR35 axis is an eosinophil chemoattractant receptor system that modulates the clearance of a lethal fungal pathogen, with implications for the use of serotonin metabolism inhibitors in the treatment of fungal infections.

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14⁺ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies.