Figure 1. Probiotic bacteria can modulate cytokine release, thus influencing both innate and adaptive immune response. Part of this figure was created with BioRender (https://biorender.com, accessed on 2 October 2021).

Figure 2. Canonical and Non-Canonical activation of NLRP3 inflammasome. The macrophage in an animal model (transgenic mice-expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2)) [21,22]. (i) The canonical pathway of NLRP3 requires a two-step process: priming and activation. Priming (signal 1) is the upregulation of the NLRP3 inflammasome components, NLRP3, pro-ILβ, and pro-caspase. This transcriptional upregulation is stimulated by PAMPs (bacterial, fungal, viral) or DAMPs which are recognized by PRRs such as TLRs. Additionally, cytokines such as TNF or IL-1 stimulate this upregulation by engaging TNFR and IL1-R, respectively. The activation (signal 2) is provided by various stress signals such as ion or plasma perturbations, ATP, lysosomal rupture, particulate matter, mitochondrial antiviral-signaling protein, mitochondrial reactive oxygen species, ox-mtDNA, etc. Oligomerization of NLRP3 inflammasome activates caspase-1, which in turn cleaves pro-ILβ, pro-IL18, and GSDMD and induces pyroptosis and inflammation [23]. (ii) Gram-negative bacteria (e.g., lipopolysaccharide (LPS) or outer membrane vesicle from bacteria) can activate the non-canonical inflammasome pathway that involves NLRP3-dependent caspase-4/5 in humans (known as caspase-11 in murine models) [24]. Caspase-11, when it is secreted at normal rates, protects against bacterial infections, but its excessive activation can cause tissue damage and pyroptosis which is activated in both pathways through cytosolic gasdermin (GSDMD), a member of the gasdermin family [25]. GSDMD cleavage generates a N-terminal domain that is capable of forming plasma membrane pores and a C-terminal domain that acts as an inhibitor of cytolysis [26]. The cleaved N-terminal domain of GSDMD oligomerizes and forms pores on the host cell membrane, leading to pyroptosis and further activation of NLRP3 by triggering K⁺ efflux [27,28]. This figure was created with BioRender (https://biorender.com, accessed on 2 October 2021).

2. The Antiviral Activity of Probiotic-Produced Metabolites

2.1. Probiotics Targeting Angiotensin-Converting Enzyme

In animal models experimentally infected with SARS-CoV-2, the protein sequence and structure of ACE2 are conserved across mammalians [60]. However, until now, there has been no published study in which NLRP3 activation was investigated following SARS-CoV-2 infection in animals. LAB fractions (mainly belonging to Lactococcus lactis and Lactobacillus helveticus), in in vitro experiments, showed inhibitory activity towards ACE enzymes by blocking the active sites [24,25]. Moreover, the debris of the dead probiotic cells acts as ACE inhibitors too, suggesting that PB could be a potential blocker of ACE and ACE2 receptors which act as a gateway for SARS-CoV-2 to attack GI cells (Figure 3) [22,61]. Recently, another in vitro trial in this field has confirmed that Lactobacillus plantarum bacteriocins (Plantaricin W, D, and JLA-9) inhibit the entry of SARS-CoV-2 by blocking ACE2 receptors and virus transcription (targeting on the S protein and blocking RNA polymerase (RdRp)) [21]

Figure 3. Probiotic bacteria products blocking ACE2 receptors may attenuate COVID-19 infection. Immune response to SARS-CoV-2. Virus invasion is carried out through ACE2 receptor and viral protein S (Spike S glycoprotein) is activated by TMPRSS2 leading to the proinflammatory cytokine cascade. The massive release of cytokines (IL-1β, IL-2, IL-7, IL-8, IFNγ, TNFα, IP10, etc.) from innate immune cells induces the activation of NF-kΒ signaling pathway that regulates the transcription of NLRP3 inflammasome-related components as well as pro-IL-1β and pro-IL-18. As soon as it is formed, NLRP3 inflammasome cleaves pro-caspase-1 and converts it into its active form, caspase-1 [23,62,63]. Subsequently, caspase-1 proceeds with the cleavage of inactive precursors pro-IL-1β and pro-IL-18 to their mature forms IL-1β and IL-18 respectively; at the same time GSDMD is cleaved by the same protease in two domains, an N-terminal and a C-terminal. Following the N-terminal fragment of GSDMD assembles pores in the plasma membrane, inducing pyroptosis and cell death [23]. Experimental studies have shown that SARS-CoV-2 cell entry could be potentially prevented through the antagonistic effect of specific probiotic strains. PB, except for their ability to inhibit the adhesion of pathogens in the intestinal epithelium, compete with pathogens for nutrients and produce antimicrobial substances (e.g., bacteriocins), providing immunomodulatory action and enhancing epithelial barrier function [64]. Their anti-inflammatory effect suppresses cytokine production. Additionally, SARS-CoV-2 evokes inflammation by infecting host cells through the ACE2 receptor and TMPRSS2 inducing proinflammatory cytokine release. Lactobacillus plantarum metabolic product (Plantaricin) blocks the entry of SARS-CoV-2 by binding with ACE2 [21,23,65,66,67]. Parts of this figure were created with BioRender (https://biorender.com, accessed on 2 October 2021) and MOTIFOLIO Download Free Sample (https://www.motifolio.com/sampleslides.html, accessed on 2 October 2021).

2.2. Research Response to the Pandemic

The ability of probiotics to inhibit the virus replication is more effective in the early onset of disease [14]. At present, there are eleven ongoing in vivo studies in this field whilst none of them have been completed (Table 2, Home—ClinicalTrials.gov, assessed on 31 December 2020); thus, the available evidence comes from the laboratory.

Table 2. Ongoing in vivo studies evaluating probiotic treatment in patients with COVID-19 infection (ClinicalTrials.gov accessed on 31 December 2020).

Number of Clinical Trial	Study Title	Sample Size	Condition/Disease	Group	Interventions	Duration	Measurements	Outcomes
04621071	Efficacy of probiotics in reducing duration and symptoms of COVID-19	84	COVID-19	Control: Placebo Intervention: 2 probiotic strains	Placebo: (potato starch and magnesium stearate) Probiotic: (2 strains 10 × 109 CFU	25 days	Questionnaires (socio-demographic, medical (weight, height, general health, current medication, symptoms), food intake etc.). Optional collection of saliva and stool samples.	Duration of symptoms. Number of days before symptoms disappear.
04458519	Efficacy of intranasal probiotic treatment to reduce severity of symptoms in COVID-19 infection	40	COVID-19	Control: Placebo Intervention: Probiotics	Placebo: Saline solution Probiotic: Lactococcus lactis W136 2.4 billion CFU given intranasally	4 weeks	Visual Analogue Scale (VAS)	Change in severity of COVID-19 infection. Number of days with any symptom of COVID-19 infection ≥ to 35 as measured on VAS at the 28-day endpoint.
04390477	Study to evaluate the effect of a probiotic in COVID-19	40	COVID-19	Control: No treatment Intervention: Probiotics	Probiotic pill 1 × 109 CFU	30 days	Patients’ status	Subjects discharged from ICU.
4366180	Evaluation of the probiotic Lactobacillus Coryniformis K8 on COVID-19 prevention in healthcare workers	314	COVID-19	Control: Placebo Intervention: Probiotics	Placebo: maltodextrin Probiotic: Lactobacillus K8 per day (3 × 109 CFU/day)	2 months	SARS-CoV-2 detection by PCR or antigen test	Incidence of SARS-CoV-2 infection in healthcare workers
04666116	Changes in viral load in COVID-19 after probiotics	96	COVID-19	Control: No dietary administration (probiotics). Medication agreed by the hospital committee Intervention: Medication agreed by the hospital committee and nutritional supplement (probiotics)	Dietary supplementation with strains from Bifidobacterium longum, Bifidobacterium animalis subsp. Lactis and Lactobacillus rhamnosus	1 year	SARS-CoV-2 detection by PCR	Viral load during the period of admission
04366089	Oxygen-Ozone as adjuvant treatment in early control of COVID-19 progression and modulation of the gut microbial flora	152	COVID-19 SARS-CoV-2 Pneumonia, Viral Coronavirus Infection	Control: Drugs (Standard of care) Intervention: Oxygen-ozone, Probiotics and Drugs (Standard of care)	Drugs: Azithromycin, Hydroxychloroquine Oxygen-ozone therapy + Probiotics Streptococcus thermophilus DSM322245, Bifidobacterium lactis DSM 32246, Bifidobacterium lactis DSM 32247, Lactobacillus acidophilus DSM 32241, Lactobacillus helveticus DSM 32242, Lactobacillus paracasei DSM 32243, Lactobacillus plantarum DSM 32244, Lactobacillus brevis DSM 27961 (200 billion) + Drugs (Azithromycin, Hydroxychloroquine)	21 days	Delta neutrophil index (Reflects the ratio of circulating immature neutrophils)	Delta index in the number of patients requiring orotracheal intubation despite treatment
04517422	Efficacy of L. Plantarum and P. Acidilactisi in adults with SARS-CoV-2 and COVID-19	300	COVID-19	Control: Placebo Intervention: Probiotics	Placebo: Combination of maltodextrin (E1400, qs) in a vegetable hydroxymethylpropyl-cellulose capsule Probiotic: Combination of Lactobacillus plantarum CECT7481, Lactobacillus plantarum CECT 7484, Lactobacillus plantarum CECT 7485, and Pediococcus acidilactici CECT 7483	30 days	WHO Clinical Progression Scale	Severity progression of COVID-19
04462627	Reduction of COVID-19 transmission to health care professionals	500	COVID-19	Intervention 1: COVID-19 positive patients Intervention 2: COVID-19 negative patients Intervention 3: Untested healthy volunteers	Probiotic (intervention 3)	3 weeks	Determination of the blood group (ABO/LE)	Anti-A antibody concentration Anti-B antibody concentration Blood group
04420676	Synbiotic therapy of gastrointestinal symptoms during COVID-19 infection	108	COVID-19	Control: Placebo Intervention: Probiotics	Placebo: matrix containing maize starch, maltodextrin, inulin, potassium chloride, hydrolysed rice protein, magnesium sulphate, fructooligosaccharide (FOS), enzymes (amylases), vanilla flavour and manganese sulphate. Probiotic: Bifidobacterium bifidum W23, Bifidobacterium lactis W51, Enterococcus faecium W54, Lactobacillus acidophilus W37, Lactobacillus acidophilus W55, Lactobacillus paracasei W20, Lactobacillus plantarum W1, Lactobacillus plantarum W62, Lactobacillus rhamnosus W71 and Lactobacillus salivarius W24	30 days	Feces	Duration of diarrhea (defined as days with 3 or more loose stools)
04399252	Effect of Lactobacillus on the microbiome of household contacts exposed to COVID-19	1000	Microbiome/COVID-19	Control: Placebo Intervention: Probiotics	Lactobaciltus rhamnosus GG Placebo Probiotic: Lactobacillus rhamnosus GG	28 days	Fecal samples	Change in Shannon Diversity Index

Research, at the time of the pandemic, poses a lot of challenges for academics. The design of the studies was dramatically rapid and, in some cases, with an impact on quality. The studies below enrolled a small number of participants, whilst heterogeneity was observed in measurements of the outcomes. Another possible drawback is the use of one probiotic strain in some interventions. Multispecies PB seems to be more effective compared to monostrain probiotics. When multispecies PB are combined, they act synergistically and can have a significantly positive effect. Their activity can also be stimulated through symbiosis among different strains [68].