Robust ACE2 protein expression localizes to the motile cilia of the respiratory tract epithelia and is not increased by ACE inhibitors or angiotensin receptor blockers

Abstract

We investigated the expression and subcellular localization of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), within the upper (nasal) and lower (pulmonary) respiratory tracts of healthy human donors. We detected ACE2 protein expression within the cilia organelle of ciliated airway epithelial cells, which likely represents the initial or early subcellular site of SARS-CoV-2 viral entry during respiratory transmission. We further determined whether ACE2 expression in the cilia of upper respiratory cells was influenced by patient demographics, clinical characteristics, co-morbidities, or medication use, and found no evidence that the use of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARBs) increases ACE2 protein expression.

Extended Data Fig. 1 |. Immunohistochemical analysis of ACE2 protein localization across human tissues using multiple anti-ACE2 antibodies.

Representative images of human tissues stained by chromogenic immunohistochemistry using antibodies targeting the ACE2 protein (brown) and counterstained with hematoxylin (blue). Highest ACE2 expression was observed in the villi of the intestinal tract (jejunum), renal tubules, testis, and glandular cells in the seminal vesicle. Minimal to no/non-specific staining can be seen in the heart, stomach, spleen, skin, and liver. Staining of lung pneumocytes was observed using Abcam ab15348 and Sigma HPA000288 (also see Extended Data Fig. 2b). Scale bars: 100 μm.

Extended Data Fig. 2 |. ACE2 protein expression within human vascular endothelial cells and the lung.

a, Representative double immunofluorescence staining of ACE2 and endothelial cell marker, CD31 in the blood vessel of human nasal turbinate using 5 different anti-ACE2 antibodies and anti-CD31. b, Double immunofluorescence staining of ACE2 and type II pneumocyte marker, mucin 1 (MUC1) in the human lung using 5 different anti-ACE2 antibodies and an anti-MUC1 antibody. The scale bars are 20 μm (top) and 5 μm (bottom).

Extended Data Fig. 3 |. ACE2 expression compared to isotype control in human tracheal tissue.

Representative immunofluorescence double staining of ACE2 and ACTUB using anti-ACE2 and anti-ACTUB, respectively, in the top panels compared to double staining of isotype control and ACTUB using rabbit IgG isotype and anti-ACTUB, respectively, in the bottom panels. The nuclei were stained using DAPI. The scale bars are 20 μm.

Extended Data Fig. 4 |. ACE2 mRNA and protein expression are not found in goblet cells of the respiratory tract.

a, Representative immunofluorescence double staining of ACE2 and mucin 5AC (MUC5AC) reveals the absence of co-localization with goblet cells in the human nasal turbinate, uncinate process, and bronchus. b, Representative in situ hybridization using an ACE2 probe in combination with an anti-MUC5AC antibody. ACE2 mRNA expression (red dots) was not noted within goblet cells marked by MUC5AC in the nasal turbinate, uncinate process, and trachea. The nuclei were stained using DAPI. The scale bars are 20 μm (top) and 5 μm (bottom).

Extended Data Fig. 5 |. Comparison of ciliary ACE2 protein expression by age, sex, smoking status, sinus disease, and anatomical region.

a, No statistical significance for ACE2 expression was seen among patients less than or greater than 65 years of age, males versus females, and patients with varying smoking history. (Student’s t-test or Kruskal-Wallis test, p> 0.05). b, No statistical significance of ACE2 expression was seen between healthy controls and patients with chronic rhinosinusitis with polyps (CRSwNP) or without polyps (CRSsNP). (Kruskal-Wallis test, p> 0.05). c, No statistical significance of ACE2 expression was seen between distinct human nasal tissue sites/regions. (Kruskal-Wallis test, p> 0.05). UNC, uncinate process; Turb, nasal turbinates; Eth, ethmoid sinus; NP, benign nasal polyps.

Extended Data Fig. 6 |. Subgroup analysis of ciliary ACE2 expression among patients taking ARBs or ACEI.

a, In the Stanford cohort, when including only controls with hypertension (HTN) on other medications (“HTN w/o ARBs/ACEI”), ACE2 expression was statistically different between the groups (Kruskal-Wallis test, p=0.048) but Dunn’s multiple comparison post-hoc test did not reveal any statistical significance between the three groups. b, When cohorts from all three institutions were normalized by Z score and integrated, patients taking ACEI (−0.71 ± 0.46) had a lower ACE2 expression compared to controls with hypertension (0.40 ± 1.09). (Kruskal-Wallis test, p=0.035; Dunn’s multiple comparison post-hoc test, *p<0.05). Patients taking ARBs (−0.16 ± 0.99) showed a trend towards lower ACE2 compared to controls with hypertension, but this was not statistically significant. c, ACE2 expression among patients of older age (≥65 years old) and patients <65 years old taking ARBs and ACEI was not statistically divergent from control patients of the same age group. (Kruskal-Wallis test, p>0.05). d, ACE2 expression among male and female patients on ARBs or ACEI trended lower than same-sex controls except for males taking ARBs in the CMU group who showed a trend towards higher ACE2 expression. No statistically significant differences were observed. (Kruskal-Wallis test, p>0.05). e, Among non-smokers, there was a statistically significant trend towards lower ACE2 expression in patients taking ACEI compared to controls in the Stanford group (Kruskal-Wallis test, p=0.005; Dunn’s multiple comparison post-hoc test, *p<0.05). No statistical significance was observed with the non-smokers on ARBs.

Fig. 1 |. ACE2 protein expression in the cilia organelle of ciliated epithelial cells in the upper and lower respiratory tract and in a ciliated kidney epithelial cell line.

a, Representative double immunofluorescence staining of ACE2 and acetylated α-tubulin (ACTUB) on normal human nasal turbinate, ethmoid sinus, uncinate process (sinus), trachea, and bronchus, using anti-ACE2 and anti-ACTUB antibodies, respectively. b, Representative double immunofluorescence staining of ACE2 and ACTUB on normal C57BL/6J mouse nasal turbinate and trachea. c, Immunofluorescent staining of (top panel) ACE2, cilia marker ADP-ribosylation factor-like protein 13B (ARL13B), and cilia centrosome marker FGFR1 Oncogene Partner (FOP); (bottom panel) ACE2, and cilia markers ACTUB and ARL13B in a ciliated mouse cell line, IMCD3. d, Immunofluorescent staining of ACE2 in the primary cilia of IMCD3 cells transiently transfected with human ACE2 (yellow outline) compared to endogenous mouse ACE2 (blue outline). e, Quantified percentages of endogenous ACE2-positive cilia (34.67 ± 13.58%; control (Ctrl)) versus cilia with overexpressed human ACE2 (82.67 ± 4.73%). Ciliated cells were identified by staining of ARL13B. Error bars represent mean ± SD. (n = 3 independent experiments with 100 cells scored per experiment). (Student’s t test, **p<0.01). The nuclei were stained using DAPI (blue) as a counterstain. Scale bars: 20 μm (a,b, top); 5 μm (a,b, bottom); 2 μm (c,d).

Fig. 2 |. ACE2 expression in the nasal cilia is not increased in patients taking angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARBs), but potentially decreased in ACEI patients.

a, Quantification of ACE2 in controls and patients taking ARB and ACEI. In the Stanford cohort, ACE2 is slightly but statistically significantly lower in patients taking ACEI (0.19 ± 0.03) compared to controls (0.26 ± 0.06). (Kruskal-Wallis test p=0.012; Dunn’s multiple comparison post-hoc test, *p< 0.05). There were no statistically significant differences in ACE2 expression between patients taking ARB and controls in the Stanford, National Taiwan University (NTU), and China Medical University (CMU) cohorts. All data are noted as mean ± SD. b, Representative images displaying the slight but significantly decreased ACE2 in patients taking ACEI, and the non-significant trend towards lower ACE2 expression in the cilia (marked by ACTUB) of patients taking ARB. Scale bars: 20 μm (top); 5 μm (bottom).