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It is known that statins, cholesterol-lowering drugs have been shown to reduce aldosterone levels in humans (Baudrand et al., 2015; Hornik et al., 2020). Moreover, in vitro studies have demonstrated that cholesterol supplementation boosts the production of aldosterone from cultured cells (Simpson et al., 1989; Cherradi et al., 2001; Kopprasch et al., 2009). Given that the adrenal-derived mouse corticosteroids, most notably corticosterone and aldosterone, derive from cholesterol as the common precursor (Figure 1A), we hypothesised that cholesterol binding scavenger receptors could modulate adrenal corticosteroid output by regulating the availability of cholesterol that could feed into the steroid hormone biosynthetic pathway. To test this hypothesis, we measured the concentrations of aldosterone and corticosterone in the plasma of Marco^−/− and wild-type (WT) mice. We found that male Marco^−/− mice had significantly elevated levels of plasma aldosterone relative to WT mice (Figure 1B). In contrast, plasma corticosterone levels were not significantly altered in male mice lacking Marco (Figure 1C). Marco-deficient female mice did not have altered levels of aldosterone relative to WT, but plasma corticosterone was increased relative to WT counterparts (Figure 1D, E). We observed that the adrenal glands from Marco-deficient male mice were significantly lighter than WT controls in male but not female mice (Figure 1F, G). To establish whether cholesterol could explain the elevated plasma aldosterone we observe in Marco-deficient male mice, we measured the levels of total serum cholesterol and intra-adrenal cholesterol in males. We found that Marco^−/− mice had reduced serum cholesterol relative to WT controls (Figure 1H), while the normalised levels of intra-adrenal cholesterol were similar between both mouse strains (Figure 1I). Taken collectively, these findings suggest that, while Marco-deficient male mice have elevated plasma aldosterone concentrations, this is not dependent on systemic or intra-adrenal cholesterol availability. For the purposes of this paper, we focused on the phenotype evident in male mice, namely the increase in plasma aldosterone concentrations.

We next hypothesised that adrenal gland-derived Marco could play a role in modulating aldosterone output. Analysis of publicly available single-cell sequencing data from murine adrenal glands shows that adrenals do contain a substantial population of macrophages expressing Ptprc (CD45), Adgre1 (F4/80), and Cd68 (CD68). However, we did not detect Marco expression in this cluster of cells, nor any other cluster identified in our analyses (Figure 2A). This finding was corroborated by immunostaining of male murine adrenal glands, which showed CD68⁺ macrophages in the adrenal zona fasciculata and zona glomerulosa that did not stain positively for MARCO (Figure 2B). Given that the lung is another site in the RAAS axis, we postulated that Marco-expressing cells in the lung could be involved in mediating the aldosterone phenotype we observed in Figure 1B. Indeed, single-cell RNA-seq analysis of the murine lung shows that Ptprc (CD45), Adgre1 (F4/80), and Cd68 (CD68) expressing cells (alveolar macrophages) also express Marco (Figure 2C), a finding corroborated by immunostaining in the lung (Figure 2C). To further validate our single-cell sequencing and immunofluorescence data, we carried out qPCR for Marco in the lungs and adrenal glands from male WT and Marco^−/− mice, which further demonstrated that the lung is the primary site of Marco expression in the RAAS (Figure 2E).

Aldosterone is a potent blood pressure-regulating hormone, the dysregulation of which can cause severe hypertension and increased cardiovascular risk. It therefore follows that its production is tightly regulated. Aldosterone biosynthesis is fundamentally regulated intra-adrenally by cytochrome P450 family members in the corticosteroid biosynthetic pathway (Figure 1A). We therefore tested whether altered expression of enzymes in this pathway could explain the hyperaldosteronism observed in Marco-deficient mice. Marco^−/− male and female mice showed similar expression of aldosterone biosynthetic enzymes (Star, Cyp11a1, Hsd3b1, Cyp11b1, and Cyp11b2) as WT mice (Figure 3A, B). While Cyp11b2 (aldosterone synthase) is only expressed in the adrenal zona glomerulosa, other biosynthetic enzymes essential for aldosterone production are expressed in the zona fasciculata.

CYP11B1 catalyses the conversion of 11-deoxycorticosterone to corticosterone. Corticosterone can be catalysed to aldosterone by CYP11B2. In this sense, the route via CYP11B1 is a bona fide route for the generation of aldosterone, as evidenced by the fact that Cyp11b1 deletion in mice results in a significant reduction in aldosterone production (Mullins et al., 2009). This route is one that is suppressible via the suppressive action of dexamethasone on ACTH and Cyp11b1 expression. Moreover, ACTH is a known stimulator of aldosterone (Seely et al., 1989; Daidoh et al., 1995). Dexamethasone-mediated suppression of the zona fasciculata (Figure 3C; Finco et al., 2018) was used to test whether the elevated aldosterone phenotype was zona fasciculata-dependent. Marco-deficient mice fed dexamethasone-supplemented drinking water had plasma aldosterone concentrations comparable to vehicle-treated mice (Figure 3D), indicating zona fasciculata activity does not contribute to elevated aldosterone levels in Marco^−/− mice. Taken collectively, these findings indicate that elevated aldosterone observed in Marco-deficient mice arises extra-adrenally and can therefore be considered a form of secondary hyperaldosteronism. Kidney-derived renin is the initiating hormone in the enzymatic cascade that generates Angiotensin II, a potent stimulator of adrenal aldosterone production. We therefore compared plasma renin activity between male WT and Marco^−/− mice, finding no significant differences between the two strains (Figure 3E).

Next, we investigated whether lung-derived angiotensin-converting enzyme (Ace) could explain the elevated aldosterone levels observed in Marco^−/− mice. ACE in the lung catalyses the conversion of Angiotensin I to the aldosterone-stimulating peptide Angiotensin II. We carried out a qPCR test for Ace in the lungs of WT and Marco^−/− mice in both sexes, finding that Marco-deficient male animals had elevated levels of lung Ace relative to WT controls in male mice only (Figure 4A). Immunofluorescent staining of WT and Marco^−/− lungs revealed a substantially higher level of ACE protein in Marco-deficient male mice, while myeloid presence, as measured by CD68 staining, remained unchanged (Figure 4B). While low levels of ACE expression could be detected in CD68⁺ cells (data not shown), the vast majority of ACE was outside of monocytes and macrophages. We used image analysis software to quantify these changes, finding that ACE median fluorescence intensity (MFI) was significantly increased in male Marco-deficient lungs, while CD68⁺ myeloid cells were present in WT and knock-out animals at similar levels (Figure 4C, D). Myeloid cell numbers and lung ACE expression were similar in WT and Marco-deficient female lungs (Figure 4E, F). We also measured the levels of plasma potassium and sodium levels in male and female WT and Marco-deficient animals, but observed no differences between the genotypes (Figure 4G–J). Since aldosterone is a known regulator of blood pressure via regulation of blood fluid balance, we also measured blood pressure using the tail-cuff method. We observed that Marco-deficient male mice had marginally reduced diastolic blood pressures, but systolic and mean blood pressure were no different between the two strains (Figure 4K–M).

We then turned back to analysis of single-cell RNA-seq data to identify the cells in the lung that could be mediating this effect. In the lung, unsupervised cluster analysis revealed a total of 12 cell clusters with distinct gene expression signatures (Figure 5A). We determined the identity of each cell cluster based on the expression of established cell type-specific marker genes, aided by the marker genes identified and outlined in Figure 5B. We next used dot plots to visualise expression of Marco and Ace across the different cell clusters. We observed notable Marco expression only in alveolar macrophages amongst the different cell clusters (Figure 5C). Ace was shown to be primarily expressed by lung endothelial clusters 1 and 2 (Figure 5C), in agreement with what is known about lung Ace expression. To test whether alveolar macrophages are capable of suppressing endothelial cell Ace expression, we co-cultured the MPI alveolar macrophage cell line (Fejer et al., 2013), with or without deletion of Marco, with HUVECs endothelial cells for 24 hr, and measured Ace expression via qPCR. We observed an increase in Ace expression in the Marco-deficient MPI co-cocultures but not WT, though this did not reach statistical significance. Taken collectively, these data suggest a model whereby Marco-expressing alveolar macrophages may, in responses to an as-of-yet unidentified factor, inhibit Ace expression at the gene and protein level, and thereby negatively regulate the cleavage of Angiotensin I to form Angiotensin II, and thereby aldosterone production (Figure 5E).

In conclusion, we hereby demonstrate that Marco is a negative regulator of aldosterone production, associated with a suppression of angiotensin-converting enzyme expression in the lungs of male mice. We propose a model in which extra-adrenal Marco expressing alveolar macrophages, through tissue crosstalk with lung endothelial cells, actively inhibit Ace expression and thereby inhibit the production of aldosterone from the adrenal glands.