pathogens Communication Dolosigranulum pigrum Modulates Immunity against SARS-CoV-2 in Respiratory Epithelial Cells Md. Aminul Islam 1,2 , Leonardo Albarracin 1,3 , Vyacheslav Melnikov 4 , Bruno G. N. Andrade 5 , Rafael R. C. Cuadrat 6,7 , Haruki Kitazawa 1,8,* and Julio Villena 1,3,* 1 Food and Feed Immunology Group, Laboratory of Animal Food Function, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan; [email protected] (M.A.I.); [email protected] (L.A.) 2 Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh 3 Laboratory of Immunobiotechnology, Reference Centre for Lactobacilli (CERELA-CONICET), Tucumán 4000, Argentina 4 Gabrichevsky Research Institute for Epidemiology and Microbiology, 125212 Moscow, Russia; [email protected] 5 AdaptCentre, Munster Technological University (MTU), T12 P928 Cork, Ireland; [email protected] 6 Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin Institute for Medical Systems Biology (BIMSB), 13125 Berlin, Germany; [email protected] 7 Department of Molecular Epidemiology, German Institute of Human Nutrition Potsdam-Rehbrücke, 14558 Nuthetal, Germany 8 Livestock Immunology Unit, International Education and Research Center for Food and Agricultural Immunology (CFAI), Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan * Correspondence: [email protected] (H.K.); [email protected] (J.V.) Citation: Islam, M..A.; Albarracin, L.; Melnikov, V.; Andrade, B.G.N.; Abstract: In a previous work, we demonstrated that nasally administered Dolosigranulum pigrum Cuadrat, R.R.C.; Kitazawa, H.; 040417 beneficially modulated the respiratory innate immune response triggered by the activation Villena, J. Dolosigranulum pigrum of Toll-like receptor 3 (TLR3) and improved protection against Respiratory Syncytial Virus (RSV) Modulates Immunity against in mice. In this work, we aimed to evaluate the immunomodulatory effects of D. pigrum 040417 in SARS-CoV-2 in Respiratory Epithelial human respiratory epithelial cells and the potential ability of this immunobiotic bacterium to increase Cells. Pathogens 2021, 10, 634. the protection against Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The respiratory https://doi.org/10.3390/ commensal bacterium D. pigrum 040417 differentially modulated the production of IFN-β, IL-6, pathogens10060634 CXCL8, CCL5 and CXCL10 in the culture supernatants of Calu-3 cells stimulated with poly(I:C) or challenged with SARS-CoV-2. The differential cytokine profile induced by the 040417 strain was Academic Editor: Luis Martinez-Sobrido associated with a significant reduction in viral replication and cellular damage after coronavirus infection. Of note, D. pigrum 030918 was not able to modify the resistance of Calu-3 cells to SARS- Received: 14 April 2021 CoV-2 infection, indicating a strain-specific immunomodulatory effect for respiratory commensal Accepted: 19 May 2021 bacteria. The findings of this work improve our understanding of the immunological mechanisms Published: 21 May 2021 involved in the modulation of respiratory immunity induced by respiratory commensal bacteria, by demonstrating their specific effect on respiratory epithelial cells. In addition, the results suggest that Publisher’s Note: MDPI stays neutral particular strains such as D. pigrum 040417 could be used as a promising alternative for combating with regard to jurisdictional claims in SARS-CoV-2 and reducing the severity of COVID-19. published maps and institutional affil- iations. Keywords: respiratory commensal bacteria; respiratory epithelial cells; next generation probiotics; Dolosigranulum pigrum; SARS-CoV-2; COVID-19; coronavirus Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) is the etiological agent distributed under the terms and of the coronavirus disease 19 (COVID-19) pandemic. SARS-CoV-2 receptor recognition conditions of the Creative Commons and attachment are initiated via interactions between the viral S protein and the human Attribution (CC BY) license (https:// angiotensin-converting enzyme 2 (ACE2) expressed by susceptible cells such as respiratory creativecommons.org/licenses/by/ epithelial cells of the airways and the lung parenchyma [1]. Variable clinical manifestations 4.0/). Pathogens 2021, 10, 634. https://doi.org/10.3390/pathogens10060634 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 634 2 of 11 have been reported for COVID-19, ranging from mild respiratory symptoms to acute lung injury and severe acute respiratory distress syndrome [2]. It was shown that in addition to the cytopathic effect induced by the virus, the respiratory alterations caused by SARS-CoV-2 are mediated by an aggressive inflammatory response that significantly contributes to damage in the respiratory tissues. The impaired responses of type I and type III interferons (IFNs) and antiviral factors have been associated with more severe COVID-19 cases [3–5]. In addition, it was reported that the replication of SARS-CoV-2 induces extensive death in epithelial cells, and causes the release of pro-inflammatory cytokines/chemokines and the recruitment of inflammatory cells into the respiratory tract [6]. Of note, it was observed that the reduced production of IFN-β in patients with COVID-19 is accompanied by elevated secretion of interleukin (IL)-6, chemokine (C-C motif) ligand 2 (CCL2), CCL5, CCL8, chemokine (C-X-C motif) ligand 8 (CXCL8), CXCL9, CXCL16, and CXCL2, which contribute to aggravating COVID-19 pathology [7,8] through the recruitment of inflammatory monocytes and neutrophils into the respiratory tract [6]. The clinical characteristics and the cellular and molecular events involved in the pathophysiology of COVID-19 have been extensively investigated, and as was mentioned above, great importance has been given to the interaction between SARS-CoV-2 and the host’s immune system in the outcome of the disease. However, recent studies indicate that a third actor could be important in the progression and severity of COVID-19: the respiratory microbiota. In this regard, some works have studied the potential impact of the respiratory microbiota on the severity and outcome of SARS-CoV-2 infection [9–11]. A study investigated the nasopharynx microbial community of patients that developed different severity levels of COVID-19, including mild, moderate and critical cases [10]. The work found that Prevotella spp. and Veillonella spp. were significantly more abundant in patients that presented more severe COVID-19. Interestingly, the work also suggested that Dolosigranulum spp. might protect against the development of severe COVID-19. These findings have been recently supported by a report describing lower abundances of Dolosigranulum pigrum among young adults that developed symptoms in the context of SARS-CoV-2 infection [11]. Although this preliminary evidence does not allow us to conclusively state that differences in microbiota composition are the cause of the different COVID-19 severities, the data suggest that the role of the respiratory microbiota in SARS- CoV-2 infection warrants further investigation. The potential beneficial effect of D. pigrum against respiratory infections was recently reported by our group using mice models of pneumococcal and Respiratory Syncytial Virus (RSV) infections [12]. In our hands, the nasal administration of D. pigrum 040417 to mice prior to the challenge with the Toll-like receptor 3 (TLR3) agonist polyinosinic:polycytidylic acid poly(I:C) or RSV differentially modulated the respiratory innate immune response, allowing improved viral clearance and reduced inflammatory-mediated lung damage. Of note, the ability of D. pigrum to beneficially modulate the respiratory innate immune response was a strain-dependent characteristic, since other strains such as D. pigrum 030918 were not able to achieve this benefit [12]. All in all, these data indicate that immunomodulatory members of the respiratory microbiota could be capable of beneficially regulating the immune response against SARS- CoV-2. In this work, we aimed to evaluate whether the immunomodulatory strain D. pigrum 040417 was able to differentially modulate cytokine production in the culture supernatants of Calu-3 cells stimulated with poly(I:C) or challenged with SARS-CoV-2, and in this way, to reduce viral replication. 2. Materials and Methods 2.1. Respiratory Commensal Bacteria Dolosigranulum pigrum 040417 and D. pigrum 030918 were cultured at 37 ◦C for 18 h (late log phase) in trypticase soy broth. Bacteria suspensions were prepared as previ- ously described [12]. Briefly, the bacteria were harvested by centrifugation at 3000× g for Pathogens 2021, 10, 634 3 of 11 10 min, washed three times with sterile 0.01 M phosphate buffer saline (PBS, pH 7.2), and suspended in sterile PBS. 2.2. Cell Cultures Calu-3 cells, a human lung epithelial cell line originating from a human pulmonary adenocarcinoma (HTB-55, American Type Culture Collection (ATCC), Manassas, VA, USA), was grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum (FBS) and 1% non-essential amino acid solution (Gibco, Grand Island, NY, United States). For antiviral assays, the FBS was reduced to 10% and 1% peni- cillin/streptomycin (Gibco) was added to the medium. Vero 76 cells, from an African green monkey kidney epithelial