Image-Based Analysis of Phagocytosis: Measuring Engulfment and Internalization.

The process of phagocytosis is crucial for fighting infection in innate immunity, for maintaining homeostasis through clearing cell debris, and for tissue remodeling in development. Here we describe two semi-automated image-based assays for the quantitative characterization of the early stages of phagocytosis and pathogen entry. A feature of these assays is the ability to detect and assess molecules or agents that subtly affect the stages both before and after cup closure or internalization.

Sequential recruitment of Rab GTPases during early stages of phagocytosis.

The phagocytosis and destruction of pathogens and dead cells by macrophages is important for innate immunity and tissue maintenance. Multiple Rab family GTPases engage effector molecules to coordinate the early stages of phagocytosis, which include rapid changes in actin polymerization, membrane phospholipids, trafficking and the activation of receptors. Defining the spatiotemporal, sequential recruitment of these Rabs is critical for insights into how phagocytosis is initiated and coordinated. Here, we screened GFP-tagged Rabs expressed in fixed and live cells to identify and stratify those recruited to early phagocytic membranes at stages defined by phospholipid transitions. We propose a sequence of Rabs 35, 13, 8a, 8b, 27a, 10, and 31 that precedes and accompanies phagocytic cup closure, followed after closure by recruitment of endosomal Rabs 5a, 5b, 5c, 14, and 11. Reducing the expression of individual Rabs by siRNA knockdown, notably Rabs 35 and 13, disrupts phagocytosis prior to phagocytic cup closure, confirming a known role for Rab35 and revealing anew the involvement of Rab13. The results enhance our understanding of innate immune responses in macrophages by revealing the sequence of Rabs that initiates phagocytosis.

Distinct Roles for APPL1 and APPL2 in Regulating Toll-like Receptor 4 Signaling in Macrophages.

Macrophages are activated by contact with pathogens to mount innate immune defenses against infection. Toll-like receptor 4 (TLR4) at the macrophage surface recognizes and binds bacterial lipopolysaccharide (LPS), setting off signaling and transcriptional events that lead to the secretion of pro- and anti-inflammatory cytokines; these in turn control inflammatory and antimicrobial responses. Although the complex regulatory pathways downstream of TLR4 have been extensively studied, further molecules critical for modulating the resulting cytokine outputs remain to be characterized. Here we establish potential roles for APPL1 and 2 signaling adaptors as regulators of LPS/TLR4-induced signaling, transcription, and cytokine secretion. APPL1 and 2 are differentially localized to distinct signaling-competent membrane domains on the surface and in endocytic compartments of LPS-activated macrophages. By depleting cells of each adaptor respectively we show separate and opposing functions for APPL1 and 2 in Akt and MAPK signaling. Specifically, APPL2 has a dominant role in nuclear translocation of NF-KB p65 and it serves to constrain the secretion of pro- and anti-inflammatory cytokines. The APPLs, and in particular APPL2, are thus revealed as adaptors with important capacity to modulate inflammatory responses mounted by LPS/TLR4 during infection.

RORα and 25-Hydroxycholesterol Crosstalk Regulates Lipid Droplet Homeostasis in Macrophages.

Nuclear hormone receptors have important roles in the regulation of metabolic and inflammatory pathways. The retinoid-related orphan receptor alpha (Rorα)-deficient staggerer (sg/sg) mice display several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia, and increased susceptibility to atherosclerosis. In this study we demonstrate that macrophages from sg/sg mice have increased ability to accumulate lipids and accordingly exhibit larger lipid droplets (LD). We have previously shown that BMMs from sg/sg mice have significantly decreased expression of cholesterol 25-hydroxylase (Ch25h) mRNA, the enzyme that produces the oxysterol, 25-hydroxycholesterol (25HC), and now confirm this at the protein level. 25HC functions as an inverse agonist for RORα. siRNA knockdown of Ch25h in macrophages up-regulates Vldlr mRNA expression and causes increased accumulation of LDs. Treatment with physiological concentrations of 25HC in sg/sg macrophages restored lipid accumulation back to normal levels. Thus, 25HC and RORα signify a new pathway involved in the regulation of lipid homeostasis in macrophages, potentially via increased uptake of lipid which is suggested by mRNA expression changes in Vldlr and other related genes.

Dynamic imaging of the recycling endosomal network in macrophages.

Recycling endosomes (REs) form an extensive and complex network of subcompartmentalized vesicular and tubular elements that connect with the cell surface and other endosomes in macrophages. As surveillance and defense cells of the innate immune system, macrophages are highly dependent on REs for their active and voluminous cell surface turnover and endocytic, exocytic, and recycling of membrane and cargo. Here we set out three approaches for imaging and analyzing REs in macrophages, based on the expression of fluorescently labeled RE-associated proteins and the uptake of fluorescent cargo. Subcompartments of the REs are identified by co-expression and co-localization analysis of RE associated Rab GTPases. Transferrin is a well-known cargo marker as it recycles through REs and it is compared here to other cargo, revealing how different endocytic routes intersect with REs. We show how the movement of transferrin through REs can be modeled and quantified in live cells. Finally, since phagosomes are a signature organelle for macrophages, and REs fuse with the maturing phagosome, we show imaging of REs with phagosomes using a genetically encoded pH-sensitive SNARE-based probe. Together these approaches provide multiple ways to comprehensively analyze REs and the important roles they play in these immune cells and more broadly in other cell types.

Rab31 and APPL2 enhance FcγR-mediated phagocytosis through PI3K/Akt signaling in macrophages.

Membrane remodeling in the early stages of phagocytosis enables the engulfment of particles or pathogens and receptor signaling to activate innate immune responses. Members of the Rab GTPase family and their disparate effectors are recruited sequentially to regulate steps throughout phagocytosis. Rab31 (Rab22b) is known for regulating post-Golgi trafficking, and here we show in macrophages that Rab31-GTP is additionally and specifically recruited to early-stage phagosomes. At phagocytic cups, Rab31 is first recruited during the phosphoinositide transition from PI(4,5)P2 to PI(3,4,5)P3, and it persists on PI(3)P-enriched phagosomes. During early phagocytosis, we find that Rab31 recruits the signaling adaptor APPL2. siRNA depletion of either Rab31 or APPL2 reduces FcγR-mediated phagocytosis. Mechanistically, this corresponds with a delay in the transition to PI(3,4,5)P3 and phagocytic cup closure. APPL2 depletion also reduced PI3K/Akt signaling and enhanced p38 signaling from FcγR. We thus conclude that Rab31/APPL2 is required for key roles in phagocytosis and prosurvival responses of macrophages. Of interest, in terms of localization and function, this Rab31/APPL2 complex is distinct from the Rab5/APPL1 complex, which is also involved in phagocytosis and signaling.

Rab8a interacts directly with PI3Kγ to modulate TLR4-driven PI3K and mTOR signalling.

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses. The TLR4-induced release of pro- and anti-inflammatory cytokines generates robust inflammatory responses, which must then be restrained to avoid disease. New mechanisms for the critical regulation of TLR-induced cytokine responses are still emerging. Here we find TLR4 complexes localized in LPS-induced dorsal ruffles on the surface of macrophages. We discover that the small GTPase Rab8a is enriched in these ruffles and recruits phosphatidylinositol 3-kinase (PI3Kγ) as an effector by interacting directly through its Ras-binding domain. Rab8a and PI3Kγ function to regulate Akt signalling generated by surface TLR4. Rab8a and PI3Kγ do not affect TLR4 endocytosis, but instead regulate mammalian target of rapamycin signalling as a mechanism for biasing the cytokine profile to constrain inflammation in innate immunity.

Single-step protease cleavage elution for identification of protein-protein interactions from GST pull-down and mass spectrometry.

The study of protein-protein interactions is a major theme in biological disciplines. Pull-down or affinity-precipitation assays using GST fusion proteins have become one of the most common and valuable approaches to identify novel binding partners for proteins of interest (bait). Non-specific binding of prey proteins to the beads or to GST itself, however, inevitably complicates and impedes subsequent analysis of pull-down results. A variety of measures, each with inherent advantages and limitations, can minimise the extent of the background. This technical brief details and tests a modification of established GST pull-down protocols. By specifically eluting only the bait (minus the GST tag) and the associated non-specific binding proteins with a simple, single-step protease cleavage, a cleaner platform for downstream protein identification with MS is established. We present a proof of concept for this method, as evidenced by a GST pull-down/MS case study of the small guanosine triphosphatase (GTPase) Rab31 in which: (i) sensitivity was enhanced, (ii) a reduced level of background was observed, (iii) distinguishability of non-specific contaminant proteins from genuine binders was improved and (iv) a putative new protein-protein interaction was discovered. Our protease cleavage step is readily applicable to all further affinity tag pull-downs.

Disruption of Rorα1 and cholesterol 25-hydroxylase expression attenuates phagocytosis in male Rorαsg/sg mice.

We and others have previously demonstrated that congenital deficiency of the nuclear hormone receptor, Rorα1, in staggerer (sg/sg) mice results in resistance to diet-induced obesity and increased insulin sensitivity. Paradoxically, the sg/sg mice are susceptible to atherosclerosis and display impaired innate immunity, underscoring the regulatory links between metabolic disease, inflammation, and susceptibility to infection. Here, we present novel evidence that Rorα1 regulates innate immune function by demonstrating impaired phagocytosis in sg/sg mice. The early stages of Fc-γ receptor-mediated phagocytosis in lipopolysaccharide-activated sg/sg bone marrow-derived macrophages (BMMs) were significantly impaired compared with wild-type cells. Moreover, in sg/sg BMMs, the phagocytic cup membranes had reduced levels of cholesterol. Expression profiling revealed dysregulated expression of genes involved in inflammation and lipid metabolism in sg/sg BMMs. Notably, we identified decreased expression of the mRNA encoding cholesterol 25-hydroxylase (Ch25h), an enzyme that converts cholesterol to 25-hydroxycholesterol (25HC), an oxysterol with emerging roles in immunity. Treatment of sg/sg BMMs with 25HC rescued phagocytosis in a dose-dependent manner, whereas small interfering RNA knockdown of Ch25h mRNA expression in wild-type cells attenuated phagocytosis. Hence, we propose that 25HC is essential for optimizing membrane internalization during phagocytosis and that aberrant Ch25h expression in Rorα1-deficient sg/sg macrophages disrupts phagocytosis. Our studies reveal new roles for Rorα1, Ch25h, and 25HC in phagocytosis. Aberrant 25HC underpins the paradoxical association between insulin sensitivity and impaired innate immunity in Rorα1-deficient mice, heralding a wider and essential role for this oxysterol at the nexus of metabolism and immunity.