Comprehensive lipidomics of tissue macrophages reveal LTE4–driven eosinophil survival

Reviewer #1 (Public review):

Summary:

Recent findings have established that macrophage function is tailored to individual tissues through upregulation of tissue-specific transcription factors in response to local microenvironmental signals. However, how these transcriptional pathways affect macrophage lipid metabolism and the importance of this for homeostasis of neighbouring immune cells remains relatively uncharted. One exemplary pathway is the specific expression of GATA-6 by macrophages within the serous cavities that is triggered by local retinoic acid production. Here, Czubala et al have used mice with macrophage-specific deletion of GATA6 (GATA-6KO-mye) to study the importance of tissue-specific macrophage programming in regulating the macrophage and tissue lipidome and the functional importance of this for the regulation of eosinophil numbers in the tissue.

Strengths and Weaknesses:

The authors show accumulation of lipid-rich vesicles in the absence of GATA6, which lipidomic analysis suggests are largely comprised of sphingolipids and glycophospholipids. Using published transcriptional data identifies candidate genes in GATA6-deficient cells that may underlie these changes. Manipulating two of these candidate genes, Gba2 and Smpd1, in a macrophage cell line leads to similar changes in sphingolipid composition to those in GATA6-deficient macrophages in vivo, supporting the hypothesis that tissue specialisation of peritoneal macrophages induces transcriptional changes via GATA6 that directly control sphingolipid metabolism. GATA6 deficiency is then shown to affect the oxylipin content of peritoneal macrophages and peritoneal fluid, including higher levels of LTE4 in fluid. Elevated expression of the Ltc4s in GATA6-deficient cells is predicted as the likely mechanism leading to elevated LTE4.

To determine the functional effects of altered lipid metabolism, and specifically LTE4, the authors focus on the elevated accumulation of peritoneal eosinophils previously reported to occur in GATA-6KO-mye mice. They show that eosinophils undergo less apoptosis in these mice and the absence of a measurable increase in known eosinophil chemokines leads them to conclude that eosinophil numbers arise through increased longevity. However, this point remains to be formally demonstrated, and directly measuring the longevity of eosinophils in the cavity would greatly strengthen their conclusions. The authors then examine known regulators of eosinophil survival, IL-5 and GM-CSF. They convincingly demonstrate a role for IL-5 in the regulation of peritoneal eosinophil numbers but conclude that survival factors other than IL-5 and GM-CSF likely control the differential numbers in control and GATA-6KO-mye mice, given IL-5 was observed to be a general survival signal in both genotypes and that no difference in the levels of these growth factors was observed in lavage fluid between genotypes. The authors then blocked production of prostaglandins using the inhibitor indomethacin. This treatment also led to a general reduction in survival and number of eosinophils in both control and GATA-6KO-mye mice, leading to the conclusion that altered prostaglandin production is not the underlying mechanism regulating elevated eosinophil numbers in the absence of GATA6.

One weakness in these conclusions is that if the GATA-6-KO-mye phenotype does lead to increased production of a homeostatic growth factor for eosinophils, then inhibition/blockade of such a factor would be expected to lead to loss of eosinophils in both WT and GATA-6KO-mye mice. Furthermore, cytokines, chemokines, and lipid mediators can be rapidly bound and removed or metabolised in vivo by their receptors, meaning detecting an increase in production in body fluids can be difficult.

Finally, they block production of LTE4 using an inhibitor of the upstream enzyme 5-LO. This treatment reduces eosinophil survival and number in GATA-6KO-mye mice, from which the key conclusion is drawn that elevated LT4E is responsible for the increased survival and accumulation of eosinophils in GATA-6KO-mye mice. The major weakness here is that the equivalent experiment in control mice to determine if inhibition of 5-LO leads to a general reduction in survival/number of eosinophils or if this effect is restricted to the GATA-6KO-mye appears not to have been performed.

Impact and context:

Overall, this study demonstrates key alterations in lipid metabolism and lipid mediator release resulting from loss of GATA6 expression in peritoneal macrophages, and links this to the elevated survival/accumulation of eosinophils that occurs concurrently in GATA6-KO-mye mice. The role of endogenous LTE4 in regulation of eosinophil survival and/or migration into tissues is exciting and opens up a new avenue of research for understanding the importance of this pathway in regulation of eosinophils across tissues and during disease. Furthermore, unlike in the mouse, GATA6-expressing macrophages represent only a minor proportion of macrophages in the human peritoneal cavity, while the dominant GATA6-negative population is more equivalent to the GATA6-KO-mye cells studied here (PMID: 38102487). Hence, the data presented in the current manuscript could have important implications for how eosinophil numbers and lipid metabolism may be regulated by these cells in people.

Reviewer #2 (Public review):

Summary:

This manuscript examines how GATA6-dependent programming of resident peritoneal macrophages regulates their lipidome and, in turn, eosinophil homeostasis, combining lipid imaging, mass spectrometry, transcriptional analysis and in vivo pharmacology. BODIPY/CARS microscopy with targeted lipidomics convincingly shows substantial lipid changes following myeloid GATA6 deficiency, particularly in sphingolipids, with Smpd1 and Gba2 manipulations providing mechanistic support.

Strengths:

The authors also connect these changes to eosinophil biology, confirming increased peritoneal eosinophils in Gata6-deficient mice with reduced apoptosis (via two methods) rather than increased production. Testing of alternative explanations (chemokines, IL-5, prostaglandins, 12/15-LOX products) strengthens the argument by narrowing candidate mechanisms. The identification of increased LTE4 is notable as it correlates with eosinophil abundance, and zileuton reduces LTE4, eosinophil numbers, and increases apoptosis. This supports a role for 5-LOX/cysteinyl-leukotriene signalling.

Weaknesses:

The principal weakness is specificity: zileuton affects the broader leukotriene pathway, not LTE4 alone, so correlation with LTE4 doesn't establish causality. This matters more given the LTE4 receptor remains unidentified (to the best of my knowledge). Similarly, the proposed transcellular biosynthesis mechanism (ImmGen data suggesting complementary enzyme expression across cell types converting LTC4 to LTE4) is inferential; direct evidence of cellular source and transfer is lacking.

Design limitations include reliance on pooled animals in some lipidomic measurements, small replicate numbers, and a stronger eosinophil phenotype in females that shifts subsequent analysis toward females. Indeed, this sex dependence deserves more discussion given it limits generalisability.

Overall, this is a technically strong, conceptually interesting study. The core conclusions, that GATA6-dependent regulation of the macrophage lipidome and a role for cystl Lts in eosinophil survival, are well supported. The more specific claim that LTE4 is the causal factor via a defined transcellular pathway is plausible but not yet firmly established. Experimental strengthening or moderated claims would improve the study.

Reviewer #3 (Public review):

Summary:

The authors sought to define how GATA6-dependent programming of resident peritoneal macrophages regulates lipid metabolism and, in turn, eosinophil survival. By integrating a myeloid-restricted GATA6-deficiency model with cellular phenotyping, lipidomic analyses, and measurements of lipid mediators, the study attempts to connect macrophage transcriptional identity to sphingolipid and cysteinyl leukotriene pathways that may shape eosinophil persistence. The work also appears intended to provide a mechanistic bridge between prior observations from this group and others regarding GATA6-positive macrophages, lipid metabolism, and eosinophil homeostasis.

Strengths:

(1) The study addresses an important and understudied question: how tissue-resident macrophage identity controls the local lipid environment and thereby influences eosinophil survival.

(2) The use of a genetically defined GATA6-deficiency model provides a biologically relevant framework for testing the contribution of macrophage programming.

(3) The lipidomic data broaden the analysis beyond a single mediator and identify coordinated changes in sphingolipids and glycerophospholipids that may generate useful hypotheses for the field.

(4) The finding that GATA6 deficiency promotes eosinophil survival is clear, potentially important, and consistent with prior work cited by the authors.

(5) The study is performed by a knowledgeable team and brings together macrophage biology, eosinophil biology, and lipid metabolism in a way that is likely to interest several research communities.

Weaknesses:

(1) The central mechanistic chain-GATA6 deficiency leading to altered sphingolipid abundance, altered LTE4 production, and consequently increased eosinophil survival-is not fully demonstrated. The data support associations among these features, but the causal order remains uncertain.

(2) The cited literature linking sphingolipid and cysteinyl leukotriene biosynthesis does not substitute for direct testing in this model. Perturbation or rescue experiments targeting sphingolipid synthesis and cysteinyl leukotriene production would be needed to establish necessity and directionality.

(3) The broader lipidomic changes complicate the emphasis on sphingolipids. Because glycerophospholipids are also increased, the phenotype may reflect more extensive membrane-lipid remodeling, altered phospholipase activity, changes in the Lands cycle, or shifts in free fatty-acid availability.

(4) The manuscript would benefit from a clearer distinction between observations made directly in GATA6-deficient peritoneal macrophages and mechanistic inferences extrapolated from prior studies.

(5) The physiological and pathological relevance is not yet sufficiently established. It remains unclear whether enhanced eosinophil survival translates into altered eosinophil accumulation, activation, or tissue injury during inflammatory disease in the peritoneal cavity or lung.

Author response:

We would like to thank all the reviewers and the editors for their considerate evaluation of our study.

We are pleased that overall the reviewers were positive about the bulk of our study establishing a role of tissue macrophage programming/specialisation in regulating the macrophage lipidome, in the peritoneum, including the exemplar sphingolipid class. The reviewers raise understandable issues about the specificity of the available inhibitory compounds, such as zileuton meaning that conclusive statements about the role of LTE4 are not possible.

In a revised manuscript, we will address all points but predominantly focus on the second aspect of the study, ensuring that reviewers comments are addressed appropriately, detailing and weaknesses, or ambiguities, with our study. This will include, but will not be limited to:

- Further commentary on the regulation of eosinophil numbers within the tissue;

- Addressing the specificity of zileuton and the implications of this for interpretation of our results with respect to eosinophil biology;

- More careful framing of the transcellular biosynthesis potential;

- A detailed discussion of sex dependency with regard to eosinophil numbers in general and any potential effect on the reported Gata6-dependent phenomenon;

We are grateful for the constructive comments.