Histochemical Evidence for Reduced Immune Response in Nasal Mucosa of Patients with COVID-19.

The primary entry point of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nasal mucosa, where viral-induced inflammation occurs. When the immune response fails against SARS-CoV-2, understanding the altered response becomes crucial. This study aimed to compare SARS-CoV-2 immunological responses in the olfactory and respiratory mucosa by focusing on epithelia and nerves. Between 2020 and 2022, we obtained post mortem tissues from the olfactory cleft from 10 patients with histologically intact olfactory epithelia (OE) who died with or from COVID-19, along with four age-matched controls. These tissues were subjected to immunohistochemical reactions using antibodies against T cell antigens CD3, CD8, CD68, and SARS spike protein for viral evidence. Deceased patients with COVID-19 exhibited peripheral lymphopenia accompanied by a local decrease in CD3+ cells in the OE. However, SARS-CoV-2 spike protein was sparsely detectable in the OE. With regard to the involvement of nerve fibers, the present analysis suggested that SARS-CoV-2 did not significantly alter the immune response in olfactory or trigeminal fibers. On the other hand, SARS spike protein was detectable in both nerves. In summary, the post mortem investigation demonstrated a decreased T cell response in patients with COVID-19 and signs of SARS-CoV-2 presence in olfactory and trigeminal fibers.

Modulation of olfactory signal detection in the olfactory epithelium: focus on the internal and external environment, and the emerging role of the immune system.

Detection and discrimination of odorants by the olfactory system plays a pivotal role in animal survival. Olfactory-based behaviors must be adapted to an ever-changing environment. Part of these adaptations includes changes of odorant detection by olfactory sensory neurons localized in the olfactory epithelium. It is now well established that internal signals such as hormones, neurotransmitters, or paracrine signals directly affect the electric activity of olfactory neurons. Furthermore, recent data have shown that activity-dependent survival of olfactory neurons is important in the olfactory epithelium. Finally, as olfactory neurons are directly exposed to environmental toxicants and pathogens, the olfactory epithelium also interacts closely with the immune system leading to neuroimmune modulations. Here, we review how detection of odorants can be modulated in the vertebrate olfactory epithelium. We choose to focus on three cellular types of the olfactory epithelium (the olfactory sensory neuron, the sustentacular and microvillar cells) to present the diversity of modulation of the detection of odorant in the olfactory epithelium. We also present some of the growing literature on the importance of immune cells in the functioning of the olfactory epithelium, although their impact on odorant detection is only just beginning to be unravelled.

Acute inflammation regulates neuroregeneration through the NF-κB pathway in olfactory epithelium.

Adult neural stem cells/progenitor cells residing in the basal layer of the olfactory epithelium are capable of reconstituting the neuroepithelium even after severe damage. The molecular events underlying this regenerative capacity remain elusive. Here we show that the repair of neuroepithelium after lesioning is accompanied by an acute, but self-limited, inflammatory process. Attenuation of inflammatory cell recruitment and cytokine production by dexamethasone impairs proliferation of progenitor horizontal basal cells (HBCs) and subsequent neuronal differentiation. Using TNF-α receptor-deficient mice, we identify TNF-α signaling as an important contributor to this inflammatory and reparative process, mainly through TNF-α receptor 1. HBC-selective genetic ablation of RelA (p65), the transcriptional activator of the NF-κB pathway, retards inflammation and impedes proliferation at the early stages of regeneration and suggests HBCs directly participate in cross-talk between immune response and neurogenesis. Loss of RelA in the regenerating neuroepithelium perturbs the homeostasis between proliferation and apoptosis while enhancing JNK signaling. Together, our results support a model in which acute inflammation after injury initiates important regenerative signals in part through NF-κB-mediated signaling that activates neural stem cells to reconstitute the olfactory epithelium.

Antibody arrests γ-herpesvirus olfactory super-infection independently of neutralization.

Protecting against persistent viruses is an unsolved challenge. The clearest example for a gamma-herpesvirus is resistance to super-infection by Murid herpesvirus-4 (MuHV-4). Most experimental infections have delivered MuHV-4 into the lungs. A more likely natural entry site is the olfactory epithelium. Its protection remains unexplored. Here, prior exposure to olfactory MuHV-4 gave good protection against super-infection. The protection was upstream of B cell infection, which occurs in lymph nodes, and showed redundancy between antibody and T cells. Adding antibody to virions that blocked heparan binding strongly reduced olfactory host entry - unlike in the lungs, opsonized virions did not reach IgG Fc receptor+ myeloid cells. However, the nasal antibody response to primary infection was too low to reduce host entry. Instead, the antibody acted downstream, reducing viral replication in the olfactory epithelium. This depended on IgG Fc receptor engagement rather than virion neutralization. Thus antibody can protect against natural γ-herpesvirus infection before it reaches B cells and independently of neutralization.

IL-17c is involved in olfactory mucosa responses to Poly(I:C) mimicking virus presence.

At the interface of the environment and the nervous system, the olfactory mucosa (OM) is a privileged pathway for environmental toxicants and pathogens towards the central nervous system. The OM is known to produce antimicrobial and immunological components but the mechanisms of action of the immune system on the OM remain poorly explored. IL-17c is a potent mediator of respiratory epithelial innate immune responses, whose receptors are highly expressed in the OM of mice. We first characterized the presence of the IL-17c and its receptors in the OM. While IL-17c was weakly expressed in the control condition, it was strongly expressed in vivo after intranasal administration of polyinosinic-polycytidylic (Poly I:C), a Toll Like Receptor 3 agonist, mimicking a viral infection. Using calcium imaging and electrophysiological recordings, we found that IL-17c can effectively activate OM cells through the release of ATP. In the longer term, intranasal chronic instillations of IL-17c increased the cellular dynamics of the epithelium and promoted immune cells infiltrations. Finally, IL-17c decreased cell death induced by Poly(I:C) in an OM primary culture. The OM is thus a tissue highly responsive to immune mediators, proving its central role as a barrier against airway pathogens.

Subchronic inhalation exposure to 2-ethyl-1-hexanol impairs the mouse olfactory bulb via injury and subsequent repair of the nasal olfactory epithelium.

The olfactory system can be a toxicological target of volatile organic compounds present in indoor air. Recently, 2-ethyl-1-hexanol (2E1H) emitted from adhesives and carpeting materials has been postulated to cause "sick building syndrome." Patients' symptoms are associated with an increased sense of smell. This investigation aimed to characterize the histopathological changes of the olfactory epithelium (OE) of the nasal cavity and the olfactory bulb (OB) in the brain, due to subchronic exposure to 2E1H. Male ICR mice were exposed to 0, 20, 60, or 150 ppm 2E1H for 8 h every day for 1 week, or 5 days per week for 1 or 3 months. After a 1-week exposure, the OE showed inflammation and degeneration, with a significant concentration-dependent reduction in the staining of olfactory receptor neurons and in the numbers of globose basal cells at ≥20 ppm. Regeneration occurred at 1 month along with an increase in the basal cells, but lymphocytic infiltration, expanded Bowman's glands, and a decrease in the olfactory receptor neurons were observed at 3 months. Intriguingly, the OB at 3 months showed a reduction in the diameters of the glomeruli and in the number of olfactory nerves and tyrosine hydroxylase-positive neurons, but an increased number of ionized calcium-binding adaptor molecule 1-positive microglia in glomeruli. Accordingly, 2E1H inhalation induced degeneration of the OE with the lowest-observed-adverse-effect level of 20 ppm. The altered number of functional cell components in the OB suggests that effects on olfactory sensation persist after subchronic exposure to 2E1H.

Transolfactory neuroinvasion by viruses threatens the human brain.

Viral neuroinvasion via the olfactory system has been investigated in a variety of virus-animal models by scientists in many fields including virologists, pathologists, and neurologists. In humans, herpes simplex virus type 1 (HSV-1), human herpesvirus 6 (HHV-6), Borna disease virus, rabies virus, and influenza A virus have been shown to take the olfactory route for neuroinvasion based on forensic and post-mortem specimens. This article briefly summarizes the anatomy, physiology, and immunology of the olfactory system and presents a battery of neurovirulent viruses that may threaten the human brain by invading through this peripheral pathway, especially focusing on two of the most intensively studied viruses--HSV-1 and influenza A virus. Viruses may insidiously invade the olfactory neural network not only to precipitate encephalitis/encephalopathy but also to promote the development of neurodegenerative and demyelinating disorders. Substantial information obtained by analyzing human specimens is required to argue for or against this hypothesis.

Innate immune responses and neuroepithelial degeneration and regeneration in the mouse olfactory mucosa induced by intranasal administration of Poly(I:C).

The pathogenesis of postviral olfactory disorder (PVOD) has not been fully elucidated. We investigated morphological changes and innate immune responses in the mouse olfactory mucosa induced by intranasal administration of polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA. Mice received three administrations of saline with or without Poly(I:C), once every 24 h. The olfactory mucosa was harvested at various intervals after the first administration (8 h, 3, 9 and 24 days). In the Poly(I:C) group, the number of apoptotic cells in the olfactory neuroepithelium had increased at 8 h. At 9 days, the olfactory neuroepithelium had severely degenerated and behavioral tests demonstrated that the mice showed signs of olfactory deterioration. At 24 days, the structure of the neuroepithelium had regenerated almost completely. Regarding the innate immune responses, many neutrophils had infiltrated the olfactory neuroepithelium at 8 h and had exuded into the nasal cavity by 3 days. Macrophages had also infiltrated the olfactory neuroepithelium at 8 h although to a lesser extent, but they still remained in the neuroepithelium at 24 days. Poly(I:C)-induced neuroepithelial damage was significantly inhibited by a neutrophil elastase inhibitor and was suppressed in neutropenic model mice. These findings suggest that the secondary damage caused by the neutrophil-mediated innate immune response plays an important role in the pathogenesis of PVOD.

Olfactory immunology: the missing piece in airway and CNS defence.

The olfactory mucosa is a component of the nasal airway that mediates the sense of smell. Recent studies point to an important role for the olfactory mucosa as a barrier to both respiratory pathogens and to neuroinvasive pathogens that hijack the olfactory nerve and invade the CNS. In particular, the COVID-19 pandemic has demonstrated that the olfactory mucosa is an integral part of a heterogeneous nasal mucosal barrier critical to upper airway immunity. However, our insufficient knowledge of olfactory mucosal immunity hinders attempts to protect this tissue from infection and other diseases. This Review summarizes the state of olfactory immunology by highlighting the unique immunologically relevant anatomy of the olfactory mucosa, describing what is known of olfactory immune cells, and considering the impact of common infectious diseases and inflammatory disorders at this site. We will offer our perspective on the future of the field and the many unresolved questions pertaining to olfactory immunity.

Olfactory ensheathing cells: the primary innate immunocytes in the olfactory pathway to engulf apoptotic olfactory nerve debris.

The olfactory system is an unusual tissue in which olfactory receptor neurons (ORNs) are continuously replaced throughout the life of mammals. Clearance of the apoptotic ORNs corpses is a fundamental process serving important functions in the regulation of olfactory nerve turnover and regeneration. However, little is known about the underlying mechanisms. Olfactory ensheathing cells (OECs) are a unique type of glial cells that wrap olfactory axons and support their continual regeneration from the olfactory epithelium to the bulb. In the present study, OECs were identified to exist in two different states, resting and reactive, in which resting OECs could be activated by LPS stimulation and functioned as phagocytes for cleaning apoptotic ORNs corpses. Confocal analysis revealed that dead ORNs debris were engulfed by OECs and co-localized with lysosome associated membrane protein 1. Moreover, phosphatidylserine (PS) receptor was identified to express on OECs, which allowed OECs to recognize apoptotic ORNs by binding to PS. Importantly, engulfment of olfactory nerve debris by OECs was found in olfactory mucosa under normal turnover and was significantly increased in the animal model of olfactory bulbectomy, while little phagocytosis by Iba-1-positive microglia/macrophages was observed. Together, these results implicate OEC as a primary innate immunocyte in the olfactory pathway, and suggest a cellular and molecular mechanism by which ORNs corpses are removed during olfactory nerve turnover and regeneration.

Cytokines and olfactory bulb microglia in response to bacterial challenge in the compromised primary olfactory pathway.

BACKGROUND: The primary olfactory pathway is a potential route through which microorganisms from the periphery could potentially access the central nervous system. Our previous studies demonstrated that if the olfactory epithelium was damaged, bacteria administered into the nasal cavity induced nitric oxide production in olfactory ensheathing cells. This study investigates the cytokine profile of olfactory tissues as a consequence of bacterial challenge and establishes whether or not the bacteria are able to reach the olfactory bulb in the central nervous system. METHODS: The olfactory epithelium of C57BL/6 mice was damaged by unilateral Triton X-100 nasal washing, and Staphylococcus aureus was administered ipsilaterally 4 days later. Olfactory mucosa and bulb were harvested 6 h, 24 h and 5 days after inoculation and their cytokine profile compared to control tissues. The fate of S. aureus and the response of bulbar microglia were examined using fluorescence microscopy and transmission electron microscopy. RESULTS: In the olfactory mucosa, administered S. aureus was present in supporting cells of the olfactory epithelium, and macrophages and olfactory nerve bundles in the lamina propria. Fluorescein isothiocyanate-conjugated S. aureus was observed within the olfactory mucosa and bulb 6 h after inoculation, but remained restricted to the peripheral layers up to 5 days later. At the 24-h time point, the level of interleukin-6 (IL-6) and tumour necrosis factor-α in the compromised olfactory tissues challenged with bacteria (12,466 ± 956 pg/ml and 552 ± 193 pg/ml, respectively) was significantly higher than that in compromised olfactory tissues alone (6,092 ± 1,403 pg/ml and 80 ± 2 pg/ml, respectively). Immunohistochemistry confirmed that IL-6 was present in several cell types including olfactory ensheathing cells and mitral cells of the olfactory bulb. Concurrently, there was a 4.4-, 4.5- and 2.8-fold increase in the density of iNOS-expressing cells in the olfactory mucosa, olfactory nerve and glomerular layers combined, and granule layer of the olfactory bulb, respectively. CONCLUSIONS: Bacteria are able to penetrate the immunological defence of the compromised olfactory mucosa and infiltrate the olfactory bulb within 6 h even though a proinflammatory profile is mounted. Activated microglia may have a role in restricting bacteria to the outer layers of the olfactory bulb.

A genetic model of chronic rhinosinusitis-associated olfactory inflammation reveals reversible functional impairment and dramatic neuroepithelial reorganization.

Inflammatory sinus and nasal disease is a common cause of human olfactory loss. To explore the mechanisms underlying rhinosinusitis-associated olfactory loss, we have generated a transgenic mouse model of olfactory inflammation, in which tumor necrosis factor alpha (TNF-alpha) expression is induced in a temporally controlled manner specifically within the olfactory epithelium (OE). Like the human disease, TNF-alpha expression leads to a progressive infiltration of inflammatory cells into the OE. Using this model, we have defined specific phases of the pathologic process. An initial loss of sensation without significant disruption is observed, followed by a striking reorganization of the sensory neuroepithelium. An inflamed and disrupted state is sustained chronically by continued induction of cytokine expression. After prolonged maintenance in a deficient state, there is a dramatic recovery of function and a normal histologic appearance when TNF-alpha expression is extinguished. Although obstruction of airflow is also a contributing factor in human rhinosinusitis, this in vivo model demonstrates for the first time that direct effects of inflammation on OE structure and function are important mechanisms of olfactory dysfunction. These features mimic essential aspects of chronic rhinosinusitis-associated olfactory loss, and illuminate underlying cellular and molecular aspects of the disease. This manipulable model also serves as a platform for developing novel therapeutic interventions.

PACAP protects against TNFα-induced cell death in olfactory epithelium and olfactory placodal cell lines.

In mouse olfactory epithelium (OE), pituitary adenylate cyclase-activating peptide (PACAP) protects against axotomy-induced apoptosis. We used mouse OE to determine whether PACAP protects neurons during exposure to the inflammatory cytokine TNFα. Live slices of neonatal mouse OE were treated with 40 ng/ml TNFα ± 40nM PACAP for 6h and dying cells were live-labeled with 0.5% propidium iodide. TNFα significantly increased the percentage of dying cells while co-incubation with PACAP prevented cell death. PACAP also prevented TNFα-mediated cell death in the olfactory placodal (OP) cell lines, OP6 and OP27. Although OP cell lines express all three PACAP receptors (PAC1, VPAC1,VPAC2), PACAP's protection of these cells from TNFα was mimicked by the specific PAC1 receptor agonist maxadilan and abolished by the PAC1 antagonist PACAP6-38. Treatment of OP cell lines with blockers or activators of the PLC and AC/MAPKK pathways revealed that PACAP-mediated protection from TNFα involved both pathways. PACAP may therefore function through PAC1 receptors to protect neurons from cell death during inflammatory cytokine release in vivo as would occur upon viral infection or allergic rhinitis-associated injury.

Smell and taste disorders in COVID-19: From pathogenesis to clinical features and outcomes.

Patients with COVID-19 often complain of smell and taste disorders (STD). STD emerge early in the course of the disease, seem to be more common in SARS-CoV-2 infection than in other upper respiratory tract infections, and could in some cases persist for long after resolution of respiratory symptoms. Current evidence suggests that STD probably result from a loss of function of olfactory sensory neurons and taste buds, mainly caused by infection, inflammation, and subsequent dysfunction of supporting non-neuronal cells in the mucosa. However, the possible occurrence of other mechanisms leading to chemosensory dysfunction has also been hypothesized, and contrasting data have been reported regarding the direct infection of sensory neurons by SARS-CoV-2. In this mini-review, we summarize the currently available literature on pathogenesis, clinical manifestations, diagnosis, and outcomes of STD in COVID-19 and discuss possible future directions of research on this topic.

Anosmia and hyposmia.

There has been some renewed interest in recent years in disorders of olfaction. Decreased sense of smell can lead to significant impairment of quality of life, including taste disturbance and loss of pleasure from eating with resulting changes in weight and difficulty in avoiding health risks such as spoiled food or leaking natural gas. Recent epidemiological reports have shown that despite fairly low self-reported prevalence of these disorders in large population studies, when validated smell identification or threshold tests are used, they reveal quite a high prevalence of hyposmia and anosmia in certain groups, especially the elderly. Several different pathophysiologic processes, such as head trauma, aging, autoimmunity, and toxic exposures, can contribute to smell impairment, with distinct implications concerning prognosis and possible treatment. As allergists, we are most likely to see this symptom in patients with chronic rhinosinusitis, and this now appears to be due more to the mucosal inflammation than to physical airway obstruction.

Anosmia: a missing link in the neuroimmunology of coronavirus disease 2019 (COVID-19).

Just before 2020 began, a novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), brought for humans a potentially fatal disease known as coronavirus disease 2019 (COVID-19). The world has thoroughly been affected by COVID-19, while there has been little progress towards understanding the pathogenesis of COVID-19. Patients with a severe phenotype of disease and those who died from the disease have shown hyperinflammation and were more likely to develop neurological manifestations, linking the clinical disease with neuroimmunological features. Anosmia frequently occurs early in the course of COVID-19. The prevalence of anosmia would be influenced by self-diagnosis as well as self-misdiagnosis in patients with COVID-19. Despite this, the association between anosmia and COVID-19 has been a hope for research, aiming to understand the pathogenesis of COVID-19. Studies have suggested differently probable mechanisms for the development of anosmia in COVID-19, including olfactory cleft syndrome, postviral anosmia syndrome, cytokine storm, direct damage of olfactory sensory neurons, and impairment of the olfactory perception center in the brain. Thus, the observation of anosmia would direct us to find the pathogenesis of COVID-19 in the central nervous system, and this is consistent with numerous neurological manifestations related to COVID-19. Like other neurotropic viruses, SARS-CoV-2 might be able to enter the central nervous system via the olfactory epithelium and induce innate immune responses at the site of entry. Viral replication in the nonneural olfactory cells indirectly causes damage to the olfactory receptor nerves, and as a consequence, anosmia occurs. Further studies are required to investigate the neuroimmunology of COVID-19 in relation to anosmia.

Olfactory Dysfunction and Chronic Rhinosinusitis.

Olfactory dysfunction (OD) is one of the cardinal symptoms of chronic rhinosinusitis (CRS), and its prevalence ranges from 60% to 80% in patients with CRS. It is much more common in CRS with nasal polyposis patients compared to CRS without nasal polyposis. Decreased olfactory function is associated with significant decreases in patient-reported quality of life (QOL), and notably, depression and the enjoyment of food. Objective measures can help detail the degree of OD, whereas subjective measures can help to determine in the impact on patient. There is variable treatment response to OD with both medical and surgical therapies.

SARS-CoV-2: Olfaction, Brain Infection, and the Urgent Need for Clinical Samples Allowing Earlier Virus Detection.

The novel SARS-CoV-2 virus has very high infectivity, which allows it to spread rapidly around the world. Attempts at slowing the pandemic at this stage depend on the number and quality of diagnostic tests performed. We propose that the olfactory epithelium from the nasal cavity may be a more appropriate tissue for detection of SARS-CoV-2 virus at the earliest stages, prior to onset of symptoms or even in asymptomatic people, as compared to commonly used sputum or nasopharyngeal swabs. Here we emphasize that the nasal cavity olfactory epithelium is the likely site of enhanced binding of SARS-CoV-2. Multiple non-neuronal cell types present in the olfactory epithelium express two host receptors, ACE2 and TMPRSS2 proteases, that facilitate SARS-CoV-2 binding, replication, and accumulation. This may be the underlying mechanism for the recently reported cases of smell dysfunction in patients with COVID-19. Moreover, the possibility of subsequent brain infection should be considered which begins in olfactory neurons. In addition, we discuss the possibility that olfactory receptor neurons may initiate rapid immune responses at early stages of the disease. We emphasize the need to undertake research focused on additional aspects of SARS-CoV-2 actions in the nervous system, especially in the olfactory pathway.

Comparative models for human nasal infections and immunity.

The human olfactory system is a mucosal surface and a major portal of entry for respiratory and neurotropic pathogens into the body. Understanding how the human nasopharynx-associated lymphoid tissue (NALT) halts the progression of pathogens into the lower respiratory tract or the central nervous system is key for developing effective cures. Although traditionally mice have been used as the gold-standard model for the study of human nasal diseases, mouse models present important caveats due to major anatomical and functional differences of the human and murine olfactory system and NALT. We summarize the NALT anatomy of different animal groups that have thus far been used to study host-pathogen interactions at the olfactory mucosa and to test nasal vaccines. The goal of this review is to highlight the strengths and limitations of each animal model of nasal immunity and to identify the areas of research that require further investigation to advance human health.

IFN-λ Decreases Murid Herpesvirus-4 Infection of the Olfactory Epithelium but Fails to Prevent Virus Reactivation in the Vaginal Mucosa.

Murid herpesvirus-4 (MuHV-4), a natural gammaherpesvirus of rodents, can infect the mouse through the nasal mucosa, where it targets sustentacular cells and olfactory neurons in the olfactory epithelium before it propagates to myeloid cells and then to B cells in lymphoid tissues. After establishment of latency in B cells, viral reactivation occurs in the genital tract in 80% of female mice, which can lead to spontaneous sexual transmission to co-housed males. Interferon-lambda (IFN-λ) is a key player of the innate immune response at mucosal surfaces and is believed to limit the transmission of numerous viruses by acting on epithelial cells. We used in vivo plasmid-mediated IFN-λ expression to assess whether IFN-λ could prophylactically limit MuHV-4 infection in the olfactory and vaginal mucosae. In vitro, IFN-λ decreased MuHV-4 infection in cells that overexpressed IFN-λ receptor 1 (IFNLR1). In vivo, prophylactic IFN-λ expression decreased infection of the olfactory epithelium but did not prevent virus propagation to downstream organs, such as the spleen where the virus establishes latency. In the olfactory epithelium, sustentacular cells readily responded to IFN-λ. In contrast, olfactory neurons did not respond to IFN-λ, thus, likely allowing viral entry. In the female genital tract, columnar epithelial cells strongly responded to IFN-λ, as did most vaginal epithelial cells, although with some variation from mouse to mouse. IFN-λ expression, however, failed to prevent virus reactivation in the vaginal mucosa. In conclusion, IFN-λ decreased MuHV-4 replication in the upper respiratory epithelium, likely by protecting the sustentacular epithelial cells, but it did not protect olfactory neurons and failed to block virus reactivation in the genital mucosa.