Regulation of adrenal homeostasis by macrophages – ADREMAC

How We Studied It

To remove macrophages, we used two approaches. One involved genetic engineering, deleting the survival signals that macrophages need. The other was drug-based, using a treatment that wipes out macrophages but can be reversed when the drug is stopped.

With these models, we could then ask:

• Do macrophages help clear away dying cells, like the fetal zone that disappears during puberty?

• Do they guide the organization of the adrenal’s layers or influence hormone production?

• Do they sustain the progenitor cells that keep the adrenal renewing itself?

Finally, by sequencing thousands of individual adrenal cells, we looked at how macrophages and adrenal cells exchange signals — a sort of molecular dialogue.

What We Found

Our findings were both surprising and humbling.

• Macrophages are diverse. We discovered several distinct types of macrophages in the adrenal, some of which differed between males and females. This suggests that they may carry out specialized roles depending on sex and context.

• Deleting macrophages isn’t catastrophic. Whether removed genetically or with drugs, their absence did not stop the adrenal from developing normally or maintaining its hormone balance. Early on, we saw some delays in the shaping of zones, but the gland eventually corrected itself.

• The fetal zone clears itself. Even though macrophages accumulate when the fetal cells disappear, removing them didn’t prevent this process. Other mechanisms must be responsible.

• Subtle effects on hormones. Without macrophages, hormone production was largely intact, but small differences emerged: in males, the stress hormone response increased, while in females the salt-regulating hormone response weakened slightly.

• Renewal continues without them. The constant turnover of adrenal cells did not depend on macrophages.

• Regeneration after stress shows sex differences. When the adrenal was stressed and then allowed to recover, males and females followed different patterns, with macrophages piling up in males. But even here, removing them didn’t dramatically change the outcome, though it slightly slowed recovery in males.

What It All Means

At first glance, these results might seem like a negative finding — if macrophages are so abundant in the adrenal gland, why doesn’t their removal have a bigger impact? But science often advances by uncovering what doesn’t happen, and here the story is more nuanced.

Our project set out to study macrophages — immune cells often described as the body’s “clean-up crew” — in the adrenal gland. We found that these cells live in sex-specific niches and are recruited during key phases of adrenal growth, renewal, and repair. Yet, surprisingly, removing macrophages did not prevent normal adrenal development or hormone balance. These findings suggested that macrophages may be dispensable in everyday adrenal health but are mobilized in unusual or stressful conditions.

This raised a new question: could macrophages play a role in adrenal cancer?

We explored this in mouse models of adrenocortical carcinoma (ACC), a rare but aggressive cancer starting in the adrenal cortex. Unlike most cancers, ACC is more common in women. About 20% of patients carry mutations in ZNRF3, a brake on cell growth. In mice, deleting this gene caused females to develop tumors, while males did not. In males, testosterone triggered tumor cells into senescence (a growth arrest), which in turn recruited specialized macrophages (MERTKhi/TREM2hi). These macrophages cleared the senescent cells and stopped tumors from progressing.

Analysis of patient data confirmed this pattern: such macrophages were more common in male ACC patients and linked to better outcomes. Published in Science Advances, this work showed that macrophages help explain why ACC behaves differently in men and women. We also found that testosterone acts indirectly, blocking the cancer-driving gene MYC in tumor cells, which weakens them and allows macrophages to finish the job. These results are now being expanded in a new study for Cell Reports Medicine.

We next studied a harsher model where both ZNRF3 and TP53 were deleted. Here, males and females alike developed aggressive, metastatic tumors. Macrophages no longer acted as protectors: instead, they became immunosuppressive and senescent, helping the cancer spread. Using single-cell and spatial technologies, we found these altered macrophages both in primary tumors and in lung metastases, especially near hormone-producing cells.

We confirmed their presence in human ACC samples. Crucially, treating mice with ABT-737, a drug that removes senescent cells, slowed tumor growth and strongly reduced metastasis. This points to a new therapeutic strategy: targeting senescent macrophages in aggressive ACC. These results are being prepared for Nature Cancer.

Encouraged by these findings, we are now testing ways to reprogram macrophages into tumor fighters. One approach uses CD40 antibodies to push them into an active, pro-inflammatory state. Another blocks CD47, a “don’t-eat-me” signal displayed by tumor cells, encouraging macrophages to engulf them. Combined with the standard drug mitotane, which makes tumor cells more vulnerable, this could further enhance macrophage-driven clearance.

The adrenal cortex is a major regulator of body homeostasis through secretion of glucocorticoids in zona fasciculata (zF) and aldosterone in zona glomerulosa (zG). Alterations of the endocrine activity of the cortex are associated with either adrenal insufficiency, which can be lethal if untreated or adrenal hyperactivity, which is associated with obesity, insulin resistance, immunosuppression and hypertension. Proper control of adrenal cortex endocrine activity relies on maturation of the cortex in the perinatal period. This is characterised by regression of the foetal cortex and morphogenesis of the mature zG, which involves formation of glomeruli. Once established, the structure of the adult cortex has to be maintained through cortical cell renewal, which involves proliferation, centripetal migration and sequential differentiation of progenitor cells located in the outer cortex. Although the last 15 years have witnessed an impressive increase in our understanding of the mechanisms of adrenal cortex development, differentiation and function, some key aspects of post-natal maturation and maintenance of cortical homeostasis remain elusive. This is particularly the case for mechanisms involved in clearance of foetal cells, matrix remodelling to allow functional maturation of zG glomeruli and coordination of progenitors recruitment and cell migration for renewal, which cannot easily be explained by cell-autonomous mechanisms. Macrophages (Mf) are the most abundant immune cell type in the adrenal cortex. They are concentrated in both the developing zG and at the cortico-medullary junction where apoptotic foetal cells are cleared out post-partum. In the adult cortex, Mf are concentrated in the capsular/subcapsular area where adrenal progenitors reside and within the zF. Beyond their role in innate immunity, Mf play key roles in tissue patterning and remodelling as well as stem cell homeostasis in a large number of tissues. These activities are dependent on reciprocal interactions between Mf and their target cells within discrete niches that produce trophic factors to nurture Mf. In return, Mf provide positive feedback signals to their niche, generating mutually beneficial circuits between niche cells and Mf. Interestingly, our preliminary data relying on depletion of adrenal Mf by pharmacological inhibition of CSF1R, suggest that they play a key role in clearance of apoptotic foetal cells and remodelling of the zG to form mature glomeruli. Therefore, we hypothesize that bi-directional interactions between Mf and their niches within the adrenal, play an all-encompassing, yet overlooked role in post-natal adrenal cortex morphogenesis, hormonal response and renewal, allowing for the maintenance of endocrine and body homeostasis.
To test this hypothesis, we aim to generate novel mouse models in which Mf of the adrenal cortex will be depleted via pharmacological inhibition and genetic deletion of Mf trophic factors in their niche. To this aim, we will identify the cells within the cortex that constitute Mf nurturing niches and will delete the corresponding trophic factors. Having developed specific models of Mf depletion, we will use these models to uncover the roles of Mf in the maturation of the post-natal cortex and will evaluate their involvement in modulation and maintenance of the endocrine activity of the fully developed cortex. We will then decipher the molecular dialog between Mf and their niches by single nucleus RNA sequencing of whole adrenals and will genetically inactivate three of the key factors identified by this approach, to evaluate their role in Mf/adrenal communication. Altogether, this innovative proposal should significantly advance our understanding of both adrenal and Mf biology fields and may also uncover deregulation of Mf biology as an underlying cause of adrenal diseases.