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Clinical Infection & Immunity

Short Communication

Volume 9, Number 1, March 2024, pages 16-19

Immunomodulation of Human Alveolar Macrophage Response to the SARS-CoV-2 S Protein by Oral Microbiota

The lung was believed to be a sterile environment for many years, mainly because of the presence of alveolar macrophages (AMs) whose function is clearing the lung alveoli of invading microorganisms. However, studies have shown that the lungs are inhabited by different species of microorganisms, specifically at the alveoli where gas exchange takes place [1]. The microorganisms that form a symbiotic relationship with their host are called microbiota.

The respiratory tract is classically separated into two parts at the level of the vocal cords, the upper respiratory tract consisting of the nasal and oral cavities, and the lower respiratory tract consisting of the trachea and lungs. Although the upper and lower tracts are continuous, their individual bacterial burdens differ drastically. The bacterial burden of the lower respiratory tract can be 100 to 10,000 times less than that of the upper respiratory tract [2]. This phenomenon is most likely due to the mucosal seal created by the vocal cords, diminishing bacterial introduction into the lower respiratory tract. However, there is still the opportunity of the bacteria to travel from the oral cavity to the lungs through the respiratory tract via migration or microaspiration [1, 3]. In fact, the microbiota inhabiting the oral cavity is relatively identical to the microbiota that inhabits the lungs [1].

The recent coronavirus disease 2019 (COVID-19) pandemic was caused by a respiratory virus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Patients with severe COVID-19 have frequently reported cariogenic and periodontopathic bacteria, suggesting a relation between the oral microbiome and COVID-19 complications [4-6]. Recent studies have suggested that there is an association between the oral lung microbiota and the pathophysiology of SARS-CoV-2 infection [7]. Furthermore, a decrease in the abundance of certain commensals in the lung, like Collinsella spp. can be associated with higher COVID-19 mortality [8].

Studies on the pathogenesis of SARS-CoV-2 infection have shown that the spike S glycoprotein of the virus is a major player in the inflammatory process of COVID-19 [9, 10]. Macrophages are key players in the innate immunity response, which is the first line of defense against an infection. Severe COVID-19 is associated with a maladapted induction of an immune response to the viral infection leading to a “cytokine storm” or cytokine release syndrome [11]. The excessive inflammatory process causes damage to the lungs, which causes the respiratory symptoms that are characteristic of COVID-19.

As the oral microbiota closely resembles such on in the lungs, and it is closely associated with SARS-CoV-2 co-infections in the lungs, it is, therefore, critical to understand the role of the oral microbiota in the inflammatory process that leads to the severity in diseases like COVID-19. With this in mind, we aimed to study if exposure to members of the oral microbiota would affect the inflammatory response of human AMs to SARS-CoV-2 S protein.

The following major oral microbiota members Corynebacterium spp., Prevotella oralis, Streptococcus viridans, Veillonella spp., and Fusobacterium spp. were used in this study. Bacteria were cultured on specific agar media and anaerobic enrichment broth (Anaerobe Systems), under anaerobic conditions (BD GasPak).

Human AMs, Daisy cells, a gift from Dr. L. Sadofsky (University of Hull, United Kingdom), were transfected with the pSIRV-nuclear factor kappa B (NF-κB)-eGFP plasmid (Addgene #118093), which contains the NF-κB gene along with an eGFP reporter.

To maintain uniformity and low abundance of each species, similar to what happens in the lung niche, AMs were exposed to bacteria at a multiplicity of infection (MOI) of 1:1 for 4 h. Cells were then exposed to the SARS-CoV-2 spike protein trimer (BEI resources) for 14 h. NF-κB activity was measured through eGFP expression detection 24 h after stimulation in a microplate reader. A response ratio of the different groups compared to the untreated/unstimulated condition was calculated. Values of three independent experiments, using technical replicates were compared statistically using the t-test after evaluating that data passed normality tests using GraphPad 9.5.

We observed that, in general, the inflammatory response of human AMs to SARS-CoV-2 S protein was diminished if cells had been first incubated with individual species of oral commensal bacteria (Fig. 1). The effect was more pronounced with exposure to certain genera, such as Prevotella, Veillonella, or Streptococcus.

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Figure 1. Human alveolar macrophages’ response to SARS-CoV-2 S protein upon exposure to individual members of the oral microbiota. Daisy cells expressing an NF-κB reporter were exposed to Prevotella oralis, Fusobacterium spp., Streptococcus viridans, Corynebacterium spp., or Veillonella spp., and then stimulated with SARS-CoV-2 trimer S glycoprotein. NF-κB: nuclear factor kappa B; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

Since these organisms exist as a bacterial community, we then examined if this same phenomenon occurred if AM cells were first incubated with a pool of all the bacteria used in the first experiment. A significant decrease in the inflammatory response of the AMs to the S protein was observed when the cells had been incubated with the pooled bacterial pool (Fig. 2).

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Figure 2. Human alveolar macrophages’ inflammatory response to SARS-CoV-2 S protein upon exposure to pooled members of oral microbiota. Daisy cells expressing an NF-κB reporter were exposed to an artificial bacterial community composed of Corynebacterium spp., Prevotella oralis, Streptococcus viridans, Veillonella spp., and Fusobacterium spp., and then stimulated with SARS-CoV-2 trimer S glycoprotein. *P < 0.05 (t-test). NF-κB: nuclear factor kappa B; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

As part of the innate immune system in the lung, AMs are the first ones facing invading organisms at this site. AMs have formed a symbiotic relationship with commensal microorganisms coexisting in this environment [7]. In healthy individuals, changes in the lung microbiota are associated with subclinical pulmonary inflammation [3].

The oral microbiome could be a driving force in the regulation of immunity in the lung, as bronchoalveolar lavage from healthy individuals is enriched with oral taxa [3]. Our study shows that exposure of AMs to members of the oral microbiota can decrease their inflammatory response to SARS-CoV-2 S protein. This suggests the role of the oral microbiota in modulating AMs inflammatory responses in respiratory infections. A decreased inflammatory response may translate in less severity of COVID-19, and less damage to the lungs in response to SARS-CoV-2.

Since our findings on an intriguing question are limited by the in vitro nature of the study, more work is needed to support the idea that oral microbiota has an immunomodulatory effect in COVID-19. It is also possible that this immunomodulation by oral commensals is expected in other respiratory infections. Our findings could lead to new therapeutic options that could potentially ameliorate the excessive inflammatory manifestations observed in many respiratory infections. This offers the possibility that prophylactic treatment with oral microbiota can be given for respiratory infections. We used a community of major players in the composition of the oral microbiota [7] that also populate the lung prior to stimulation with the S protein. Considering that symbiotic species always exist as a bacterial community, our utilization of pooled bacteria depicts a more accurate picture of how the inflammatory response of AMs to the S protein is modulated in the lung alveolar space.

Alterations of the oral and gut microbiome communities in COVID-19 patients, with some species like Granulicatella and Rothia mucilaginosa being elevated in association with SARS-CoV-2 viral load have been reported [12]. Such alterations may take over a year to start to return to normal [13]. Poor oral hygiene is considered a major ecological driver of oral microbial dysbiosis [4]. A decrease of species richness, and an elevated abundance of certain pathogens observed in COVID-19 patients should point out the importance or good oral hygiene in the outcome of respiratory infections [14].

Besides the role of oral hygiene in the prevention of outcomes steered by oral dysbiosis, oral health care measures may have an impact in reducing lung infections or severity of lung disease. In the future, a combination of bacteria that could exert an immunomodulatory effect could help decrease an excessive and novice inflammatory response to SARS-CoV-2, observed in severe COVID-19. Such prophylactic use of probiotics could potentially be used for other respiratory illnesses, and be used as a public health measure.

Acknowledgments

Special thanks to Dr. Laura Sadofsky (University of Hull, United Kingdom) for providing us with the Daisy cells.

Financial Disclosure

None to declare.

Conflict of Interest

None to declare.

Informed Consent

Not applicable.

Author Contributions

JC helped with the conceptualization, formal analysis, and supervision of the article. YR, JB, and JC helped with the data curation. YR, MM, and JB helped with the experimental investigation. BYH helped with the materials. YR, JG, BYH, and JC helped with the writing.

Date Availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Clinical Infection and Immunity is published by Elmer Press Inc.