Altered olfactory function is a common symptom of COVID-19, but its etiology is unknown. A key question is whether SARS-CoV-2 (CoV-2) – the causal agent in COVID-19 – affects olfaction directly, by infecting olfactory sensory neurons or their targets in the olfactory bulb, or indirectly, through perturbation of supporting cells. Here we identify cell types in the olfactory epithelium and olfactory bulb that express SARS-CoV-2 cell entry molecules. Bulk sequencing demonstrated that mouse, non-human primate and human olfactory mucosa expresses two key genes involved in CoV-2 entry, ACE2 and TMPRSS2. However, single cell sequencing revealed that ACE2 is expressed in support cells, stem cells, and perivascular cells, rather than in neurons. Immunostaining confirmed these results and revealed pervasive expression of ACE2 protein in dorsally-located olfactory epithelial sustentacular cells and olfactory bulb pericytes in the mouse. These findings suggest that CoV-2 infection of non-neuronal cell types leads to anosmia and related disturbances in odor perception in COVID-19 patients.
Here we show that subsets of OE sustentacular cells, HBCs, and Bowman’s gland cells in both mouse and human samples express the CoV-2 receptor ACE2 and the spike protein protease TMPRSS2. Human OE sustentacular cells express these genes at levels comparable to those observed in lung cells. In contrast, we failed to detect ACE2 expression in mature OSNs at either the transcript or protein levels. Similarly, mouse vascular pericytes in the OB express ACE2, while we did not detect ACE2 in OB neurons. Thus primary infection of non-neuronal cell types — rather than sensory or bulb neurons — may be responsible for anosmia and related disturbances in odor perception in COVID-19 patients.
The identification of non-neuronal cell types in the OE and OB susceptible to CoV-2 infection suggests four possible, non-mutually-exclusive mechanisms for the acute loss of smell reported in COVID-19 patients. First, local infection of support and vascular cells in the nose and bulb could cause significant inflammatory responses (including cytokine release) whose downstream effects could block effective odor conduction, or alter the function of OSNs or OB neurons. Second, damage to support cells (which are responsible for local water and ion balance) could indirectly influence signaling from OSNs to the brain (55). Third, damage to sustentacular cells and Bowman’s gland cells in mouse models can lead to OSN death, which in turn could abrogate smell perception . Finally, vascular damage could lead to hypoperfusion and inflammation leading to changes in OB function.
Although scSeq revealed ACE2 transcripts in only a subset of OE cells, this low level of observed expression matches or exceeds that observed in respiratory cells types that are infected by CoV-2 in COVID-19 patients. Critically, immunostaining in the mouse suggests that ACE2 protein is (nearly) ubiquitously expressed in sustentacular cells in the dorsal OE, despite sparse detection of Ace2 transcripts using scSeq. Similarly, nearly all vascular pericytes also expressed ACE2 protein, although only a fraction of OB pericytes were positive for Ace2 transcripts when assessed using scSeq. Although Ace2 transcripts were more rarely detected than protein, there was a clear concordance at the cell type level: expression of Ace2 mRNA in a particular cell type accurately predicted the presence of ACE2 protein, while Ace2 transcript-negative cell types (including OSNs) did not express ACE2 protein. These observations are consistent with recent findings in the respiratory epithelium suggesting that scSeq substantially underestimates the fraction of a given cell type that expresses the Ace2 transcript, but that “new” Ace2-expressing cell types are not discovered with more sensitive forms of analysis. If our findings in the mouse OE translate to the human (a reasonable possibility given the precise match in olfactory cell types that express CoV-2 cell entry genes between the two species), then ACE2 protein is likely to be expressed in a significant subset of human sustentacular cells. Thus, there may be many olfactory support cells available for CoV-2 infection in the human epithelium, which in turn could recruit a diffuse inflammatory process. However, it remains possible that damage to the OE could be caused by more limited cell infection. For example, infection of subsets of sustentacular cells by the SDAV coronavirus in rats ultimately leads to disruption of the global architecture of the OE, suggesting that focal coronavirus infection may be sufficient to cause diffuse epithelial damage.
We observe that activated HBCs, which are recruited after injury, express Ace2 at higher levels than those apparent in resting stem cells. The natural history of CoV2-induced anosmia is only now being defined; while recovery of smell on timescales of weeks in many patients has been reported, it remains unclear whether in a subset of patients smell disturbances will be long-lasting or permanent. While on its own it is unlikely that infection of stem cells would cause acute smell deficits, the capacity of CoV-2 to infect stem cells may play an important role in those cases in which COVID-19-associated anosmia is persistent, a context in which infection of stem cells could inhibit OE regeneration and repair over time.
Two anosmic COVID-19 patients have presented with fMRI-identified hyperintensity in both OBs that reverted to normal after resolution of the anosmia, consistent with central involvement in at least some cases. Many viruses, including coronaviruses, have been shown to propagate from the nasal epithelium past the cribriform plate to infect the OB; this form of central infection has been suggested to mediate olfactory deficits, even in the absence of lasting OE damage. The rodent coronavirus MHV passes from the nose to the bulb, even though rodent OSNs do not express Ceacam1, the main MHV receptor (Figures S4C, S5E, S6A), suggesting that CoVs in the nasal mucosa can reach the brain through mechanisms independent of axonal transport by sensory nerves; interestingly, OB dopaminergic juxtaglomerular cells express Ceacam1, which likely supports the ability of MHV to target the bulb and change odor perception. Although SARS-CoV has been shown to infect the OB in a transgenic mouse model that ectopically expresses human ACE2, it is unclear to what extent similar results will be observed for CoV-2 in these and in recently-developed mouse models expressing human ACE2 that better recapitulate native expression patterns. One speculative possibility is that local seeding of the OE with CoV-2-infected cells can result in OSN-independent transfer of virions from the nose to the bulb, perhaps via the vascular supply shared between the OB and the OSN axons that comprise CN I. Although CN I was not directly queried in our datasets, it is reasonable to infer that vascular pericytes in CN I also express ACE2, which suggests a possible route of entry for CoV-2 from the nose into the brain. Given the absence of ACE2 in mouse OB neurons — and the near-ubiquity of ACE2 expression in OB pericytes — we speculate that any central olfactory dysfunction in COVID-19 is the secondary consequence of inflammation arising locally from pericytes, or in response to diffusable factors arising from more distant sources.
Multiple immunostaining studies reveal that ACE2 protein in the human brain is predominantly or exclusively expressed in vasculature (and specifically expressed within pericytes), and many neurological symptoms associated with CoV-2 infection like stroke or altered consciousness are consistent with an underlying vasculopathy. In addition, human CSF samples have failed thus far to reveal CoV-2 RNA (73, 77), and autopsies from human patients have found that the brain contains the lowest levels of CoV-2 across organs sampled. On the other hand, multiple other studies have suggested that ACE2 may be expressed in human neurons and glia. Additionally, two recent studies in mouse models expressing human ACE2 have found CoV-2 in the brain after intranasal inoculation, although neither specifically queried the OB; this work stands in contrast to results in a non-human primate model of COVID-19, in which nasal infection did not lead to the presence of identifiable CoV-2 antigens in the brain. Further work will be required to resolve these inconsistencies, and to definitively characterize the distribution of ACE2 protein and ultimately CoV2-infected cells in the human OB and brain.
We note several caveats that temper our conclusions. Although current data suggest that ACE2 is the most likely receptor for CoV-2 in vivo, it is possible (although it has not yet been demonstrated) that other molecules such as BSG may enable CoV-2 entry independently of ACE2 (Figures S1E, S4C, S5E, S6A). In addition, it has recently been reported that low level expression of ACE2 can support CoV-2 cell entry; it is possible, therefore, that ACE2 expression beneath the level of detection in our assays may yet enable CoV-2 infection of apparently ACE2 negative cell types. We also propose that damage to the olfactory system is either due to primary infection or secondary inflammation; it is possible (although has not yet been demonstrated) that cells infected with CoV-2 can form syncytia with cells that do not express ACE2. Such a mechanism could damage neurons adjacent to infected cells. Finally, it has recently been reported that inflammation can induce expression of ACE2 in human cells. It is therefore possible that our survey of ACE2 expression, and other recent reports demonstrating expression of ACE2 in OE support and stem cells but not neurons, might under-represent the cell types that express ACE2 under conditions of CoV-2 infection.
Any reasonable pathophysiological mechanism for COVID-19-associated anosmia must account for the high penetrance of smell disorders relative to endemic viruses, the apparent suddenness of smell loss that can precede the development of other symptoms, and the transient nature of dysfunction in many patients; definitive identification of the disease mechanisms underlying COVID-19-mediated anosmia will require additional research. Nonetheless, our identification of cells in the OE and OB expressing molecules known to be involved in CoV-2 entry illuminates a path forward for future studies.
Reference & Source information: https://advances.sciencemag.org/
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