Systematic Network and Meta Analysis On The Antiviral Mechanisms of Probiotics A Preventive and Treatment Strategy To Mitigate SARS CoV 2 Infection
Systematic Network and Meta Analysis On The Antiviral Mechanisms of Probiotics A Preventive and Treatment Strategy To Mitigate SARS CoV 2 Infection
Systematic Network and Meta Analysis On The Antiviral Mechanisms of Probiotics A Preventive and Treatment Strategy To Mitigate SARS CoV 2 Infection
https://doi.org/10.1007/s12602-021-09748-w
Abstract
With the alarming rise of infected cases and deaths, COVID-19 is a pandemic, affecting 220 countries worldwide. Until
now, no specific treatment is available against SARS-CoV-2. The causal virus SARS-CoV-2 primarily infects lung cells,
leading to respiratory illness ranging in severity from the common cold to deadly pneumonia. This, with comorbidities,
worsens the clinical outcome, particularly for immunosuppressed individuals with COVID-19. Interestingly, the commensal
gut microbiota has been shown to improve lung infections by modulating the immune system. Therefore, fine-tuning of
the gut microbiome with probiotics could be an alternative strategy for boosting immunity and treating COVID-19. Here,
we present a systematic biological network and meta-analysis to provide a rationale for the implementation of probiotics
in preventing and/or treating COVID-19. We have identified 90 training genes from the literature analysis (according to
PRISMA guidelines) and generated an association network concerning the candidate genes linked with COVID-19 and
probiotic treatment. The functional modules and pathway enrichment analysis of the association network clearly show
that the application of probiotics could have therapeutic effects on ACE2-mediated virus entry, activation of the systemic
immune response, nlrp3-mediated immunomodulatory pathways, immune cell migration resulting in lung tissue damage
and cardiovascular difficulties, and altered glucose/lipid metabolic pathways in the disease prognosis. We also demonstrate
the potential mechanistic domains as molecular targets for probiotic applications to combat the viral infection. Our study,
therefore, offers probiotics-mediated novel preventive and therapeutic strategies for COVID-19 warfare.
Keywords COVID-19 · SARS-CoV-2 · Probiotics · Gut-lung axis · Biological network analysis · Meta-analysis
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degree of severity, ranging from upper respiratory tract Network analysis offers an effective approach to identify
infection (URTI) to severe interstitial pneumonia and acute molecular mechanisms and connections between genes and
respiratory distress syndrome (ARDS) [3]. Older people their pathways from dynamic networks [12]. Therefore, in
and immunocompromised individuals with existing medical the present network and meta-analysis study, a biological
conditions like diabetes, hypertension, and cardiovascular association network is generated concerning training
complications are critically affected by the disease [4]. genes of SARS-CoV-2 infection and probiotic treatment.
Interestingly, respiratory infections, sepsis, and ARDS The meta-analysis shows the rational justification for the
are associated with a change in gut microbiota composition, implementation of the probiotics approach in respiratory
indicating their possible role in pulmonary health [5]. infections. The analysis of functional modules, pathway
It is also shown that the macrophage response to the enrichment, and topological network parameters reveal
respiratory viruses during the viral infection depends on the that probiotics could have tremendous therapeutic potential
composition of gut microbes [6]. Therefore, the gut and lung during the pathophysiological events of COVID-19. The
are interconnected organs and influence their homeostasis comprehensive network analysis study indicates that the
through immunological communication [7]. Strikingly, application of probiotics at the significant domains of the
similar cross-talk among gut and lungs appears in COVID- infection mechanism could be beneficial in the prevention
19 instances as well [7]. A clear decrease in cell number of and treatment of SARS-CoV-2 infection.
two common gut bacteria, Lactobacillus and Bifidobacterium
spp., has also been found in COVID-19 patients [8]. Next,
it is also reported that bronchoalveolar lavage fluid samples Materials and Methods
of COVID-19 and community-acquired-pneumonia patients
are dominated by bacteria that are common in the oral cavity Despite the genomic similarity with the SARS-CoV, high-
and upper respiratory tract (URT) [7]. Hence, the cross- throughput data and an unambiguous model for SARS-CoV-2
talk between the gut and the lung through the gut-lung axis immunopathology are unavailable. The literature search for
may impart a significant role in SARS-CoV-2 infection [5]. the study was performed according to the PRISMA guideline
Therefore, manipulation of the intestinal microbiota by (Fig. 1). The rationale for probiotics as a cure for the global
application of probiotics could be a potential therapeutic pandemic was justified by the meta-analysis study (Fig. 2).
strategy or adjuvant therapeutic option for maintaining health Only a few clinical trials concerning the direct application
and preventing and/or treating the disease. Consumption of other probiotics like yeast (Saccharomyces boulardii),
of probiotics has also been shown to improve the URTI in Bacilli spp. (Bacillus coagulans), and Enterococcus spp.
numerous human clinical trials [9] (Table 1). (Enterococcus faecalis) on URTI patients are available.
Since the specific strains of probiotics, applied with Therefore, the study was restricted to the most common
appropriate dosage and mode of administration, can probiotic strains Lactobacillus spp. and Bifidobacterium spp.
efficiently treat respiratory complications, we hypothesize to increase the robustness and accuracy of the network meta-
that the application of probiotics could be effective against analysis. Further, a network-analysis study was considered to
SARS-CoV-2 infection. Probiotics build up a protective evaluate the probable bioprotective mechanisms of probiotics
mucus barrier, impart healthy immune response to all age against SARS-CoV-2 infection. The network module analysis
group individuals minimizing the probability of infectious revealed functionally related genes and provided key domains
disease occurrence [10]. Probiotics also maintain the for the potential mechanism of probiotics against SARS-CoV-2
balance of the gut microbial community and protect the infection. The entire methodology is summarized in Fig. S1.
respiratory system by preventing secondary bacterial
infections [7]. Recently, Tiwari et al. (2020) have reviewed Rational for Application of Probiotics as Potential
the beneficial strains of lactic acid bacteria (LAB) and their Preventive and an Alternative Treatment Strategy
potential antiviral properties [11]. Metabolites like short- for SARS‑CoV‑2 Infection: A Meta‑analysis
chain fatty acids (SCFAs), lactic acid, hydrogen peroxide,
and antimicrobial peptides (AMPs) like bacteriocins The meta-analysis was conducted to analyze similar
produced by the LAB have been suggested to be beneficial studies that are already available in the literature. In our
in restricting the viral entry and/or reducing the viral load study concerning probiotic treatment on URTI patients,
[11]. Additionally, such AMPs are also important in the Q-statistics is used to assess whether all effect sizes in
colonization of probiotics in the GI tract, cell signaling, the sample of single studies are homogeneous (belong to
and host defense [11]. The activities of bacteriocins in the same population). The I2 index indicates the degree
reinforcing the immunity with their immunomodulatory of heterogeneity. Further, the heterogeneity on the pooled
effects further suggest the prophylactic use of probiotics estimates of the individual outcomes of the meta-analysis
against viral infections, including COVID-19 [11]. was assessed by the forest plot. The effects of probiotics
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Table 1 The probiotic products with formulation supplied in human clinical trials in the prevention of upper respiratory tract infection (URTI)
References Probiotic strain Formulation Age group Dosage Mode of Function
administration
Laursen et al. [13] Bifidobacterium animalis subsp. B. animalis subsp. lactis Infants 1 g of maltodextrin pow- Oral No significant effect
lactis (BB-12) (BB-12) and L. rhamnosus 8‒14 months (healthy) der with 1 × 109 cfu each
(LGG) of BB-12 and LGG for
6 months
Leyer et al. [14] Bifidobacterium animalis subsp. B. animalis subsp. lactis Children 3‒5 years (healthy) 1 g sachet with 1 × 1010 cfu of Oral Reduced fever, rhinorrhoea,
lactis Bi-07 (ATCC PTA- Bi-07 (ATCC PTA-4802), each bacterium with 120 ml cough incidence
4802) L. acidophilus NCFM 1% fat milk twice daily for and antibiotic requirement
Probiotics and Antimicrobial Proteins
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Table 1 (continued)
References Probiotic strain Formulation Age group Dosage Mode of Function
administration
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Strasser et al. [22] Lactobacillus acidophilus W22 B. bifidum W23, B. lactis Adults 20‒35 years 4 g sachet with 1 × 1010 cfu Oral Reduced the incidence of
W51, E. faecium W54, L. (healthy) of each bacterium daily for URTI
acidophilus W22, L. brevis 12 weeks
W63, and Lactococcus
lactis W58
Strasser et al. [22] Lactococcus lactis W58 B. bifidum W23, B. lactis Adults 20‒35 years 4 g sachet with 1 × 1010 cfu Oral Reduced the incidence of
W51, E. faecium W54, L. (healthy) of each bacterium daily for URTI
acidophilus W22, L. brevis 12 weeks
W63, and L. lactis W58
Jespersen et al. Lactobacillus paracasei subsp. Individual Person 18‒60 years 100 ml milk with 1 × 109 cfu Oral Reduced the duration of
[23] paracasei, L. casei 431 (healthy) live cells once daily for the common cold and
6 weeks influenza-like illness
(ILI) episodes in healthy
adults
Pu et al. [24] Lactobacillus paracasei Individual Older person ≥ 45 (healthy) 3.6 × 107 cfu/mL live cells for Oral Reduced the risk of acute
(N1115) 12 weeks upper tract infections in
the elderly. Enhanced
T-cell-mediated natural
immune defense
Corsello et al. Lactobacillus paracasei CBA Individual Children 12‒48 months 150 ml of milk or water with Oral Reduced the risk of acute
[25] L74 (healthy) 5.9 × 109 cfu/g live cells for upper tract infections in
3 months the elderly. Enhanced
T-cell-mediated natural
immune defense
Nocerino et al. Lactobacillus paracasei CBA Individual Children 12‒48 months 150 ml of milk or water with Oral Reduced the risk of acute
[26] L74 (healthy) 5.9 × 109 cfu/g live cells for upper tract infections in
3 months the elderly. Enhanced
T-cell-mediated natural
immune defense
Berggren et al. Lactobacillus paracasei 8700:2 L. paracasei 8700:2 (DSM Person 18‒65 years 1 g maltodextrin and Oral Reduced frequency and
[27] (DSM 13434) 13434) and L. plantarum (healthy) lyophilized bacteria with duration of common cold,
HEAL 9 (DSM 15312) 1 × 109 cfu/day live cells for URTI
12 weeks
Szymanski et al. Lactobacillus plantarum PL02 B. longum PL03 (33%), L. Children 5 months to 1 tablet with 1 × 108 cfu cells Oral No significant function was
[28] rhamnosus KL53A (33%), 16 years (with respiratory twice daily for 4 weeks observed
and L. plantarum PL02 tract infection)
(34%)
Hirose et al. [29] Lactobacillus plantarum L-137 heat-killed L. plantarum Older person 40‒64 years 1 tablet with 50 mg of bacte- Oral The decreased URTI inci-
L-137 (HK L-137) (healthy) ria daily for 12 weeks dence in healthy subjects
through augmentation of
immune functions
Probiotics and Antimicrobial Proteins
Table 1 (continued)
References Probiotic strain Formulation Age group Dosage Mode of Function
administration
Tubelius et al. Lactobacillus reuteri protectis Individual Person 18‒65 years 100 ml liquid with 1 × 108 cfu Oral Shortened duration of
[30] (ATCC 55730) (healthy) live cells for 80 days respiratory diseases
Pregliasco et al. Lactobacillus rhamnosus (Pro- L. plantarum (Probial LP Person 15‒ 62 years 1 capsule (5 g) with Oral Improved health by reduc-
[15] bial LR 04-DSM 16605) 02-LMG P-21020), L. (healthy) 0.1 g = 10 × 109 cfu L. plan- ing the incidence and
rhamnosus (Probial LR tarum; 0.1 g = 10 × 109 cfu severity of respiratory
04-DSM 16605), and B. L. rhamnosus; diseases
lactis (Probial BS 01-LMG 0.1 g = 10 × 109 cfu B.
Probiotics and Antimicrobial Proteins
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diseases (CID)
diseases (CID)
strains were measured as mean difference (MD) or
standardized mean difference (SMD), effects size, 95%
Function
Oral
for 112 days
Dosage
subsp. bulgaricus
001
Guillemard et al.
[40]
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Fig. 1 Systematic literature
search selection process. The
PRISMA diagram details the
applied search and selection
process during this study
eliminating biases toward vigorously investigated disease or known interaction term. The gene names and association
phenotypes. The unstructured biomedical texts were terms represented nodes and edges respectively of the text-
regained with simultaneous construction of interaction mining–based network, generated through Cytoscape. In this
networks by text-mining strategy. Thus, the candidate work, Cytoscape 3.7.1 App and Agilent Literature Search
genes associated with SARS-CoV-2 infection and antiviral 3.1.1 beta (LitSearch version 2.69) were used to analyze the
probiotic treatment were constructed in an association existing data source from the published literature available
network by screening the training genes from literature based in the PubMed database. The training genes were used as
on the text-mining approach. An open-source bioinformatics “search terms”; Max Engine Matches was set at 10; the “use
tool, Cytoscape, and the metasearch plug-in Agilent context” and the “concept lexicon restrict search” options
Literature Search was used to visualize the molecular were selected as search controls; “Homo sapiens” was used
interaction networks, integrating the gene expression as “extraction control”.
profiles of the respective training genes. Agilent Literature
Search software fetched documents based on the entered Functional Module Determination: MCODE Analysis
query using multiple text-based search engines, parsed
into sentences. The software generated the association Any disease pathogenesis manifests the complex
network based on a lexicon set definition which defines interaction of biological events modulated by the
the gene names of the parsed sentences as “concepts” and disease-related association network. Identification
the interaction terms of interests as “verbs.” The network of tightly interconnected nodes or genes from a very
was generated by extracting an association for every parsed densely connected network was useful for understanding
sentence containing at least two “concepts” and one “verb” significant biological events in terms of modules and
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Fig. 2 Meta-analysis study rationalizes the application of probiot- against the effect size to examine publication bias shows the effect
ics as a preventive and treatment strategy in COVID-19. Forest plot of probiotics on upper respiratory tract infection (URTI) in different
showing pooled mean difference and 95% confidence intervals for clinical trials (b). The perpendicular line to the x-axis represents the
the effect of probiotics-based therapy versus placebo controls (after pooled effect size. The studies outside the triangle represent positive
adjustment for heterogeneity) on the patients with upper respiratory or negative bias. The lack of significant asymmetry in the funnel plot
tract infection (URTI) (a). Funnel plot of the standard error plotted suggests the absence of publication bias
their interconnections. The association network generated involvement of a node in the participating clusters. This
with SARS-CoV-2 pathogenesis candidate genes was tool showed the degree of participation of individual
subdivided into modules by MCODE plug-in of Cytoscape genes in the whole network and participating clusters.
according to local neighborhood density. The extracted The functional annotations of the highest MCODE score
modules were graphically displayed as an isolated, more with clustered candidate genes were determined by the
considerable dense region with functionally similar genes. integrative human gene database GeneCards (version
The network view of the clusters enabled us to understand 4.14). It provided gene-centric data of the annotated and
the local topology and functional features concerning the predicted human genes with the functions and pathways
whole network consisting of several other candidate genes associated with the candidate genes.
and edges. Network modules with MCODE score of more
than three and a minimum of four nodes were considered Assessment of Overrepresentation of Gene
as significant and carried forward for further analysis of Ontology Categories by BiNGO
functional annotation. The functional annotations and the
pathways associated with candidate genes of the selected To identify enriched biological processes that are affected
MCODE clusters were determined by GeneCards and by COVID-19 immunopathology, a gene ontology (GO)
NCBI. functional enrichment analysis was performed using the
Biological Networks Gene Ontology tool (BiNGO, version
3.0.3) in Cytoscape 2.8.0 (http://www.cytoscape.org/), with
Quantitative Data Synthesis: Network Analysis a threshold of p < 0.001. BiNGO is a tool to determine
statistically overrepresented GO terms for a set of genes
The network topology parameter details of the association associated with any biological processes as recorded in the
network were obtained by analyzing the association Gene Ontology database. The BiNGO analysis was done
network with the NetworkAnalyzer tool in Cytoscape. with the MCODE-derived gene clusters of the association
The node degree and clustering coefficient of the network. The statistical test was set to “hypergeometric
individual node genes together represented the number test,” multiple testing correction as “Benjamini & Hochberg
of connections associated with a node and the degree of False Discovery Rate (FDR) correction,” the significance
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level was set to 0.05, the categories to be visualized were with strain-specific probiotic treatment on URTI patients was
“overrepresented categories after correction,” reference set symmetrical, which indicated that no significant asymmetry
was “use the whole annotation as reference set,” ontology was detected in any of the analyses.
file was “GO_Biological_Process,” and finally organism/
annotation was selected as “Homo sapiens.” Identification of the Respective Training Genes
Associated with COVID‑19 Pathophysiology
and Antiviral Approaches with Probiotic Treatment
Results
The data-mining exploratory technique found out
Overall Effects of Probiotics on URTI about 36 data, including clinical trials, in vitro
experiments, bioinformatics analysis, reviews, mini-
URTI is one of the principal symptoms of SARS-CoV-2 reviews, and editorial letters associated with COVID-
infection. Therefore, in all the meta-analysis studies, the 19 pathophysiology and probiotic treatment from the
duration and severity of URTI were compared between literature [1, 41–47]. The use of specific MeSH terms
individual probiotic or probiotic formulation (or synbiotics) in EndNote and keywords used in the PubMed database
treatment and placebo-controlled groups, including children excluded non-specific search results. This confirmed the
and adult individuals [13–40]. The forest plot showed the accuracy of the mining study performed with the reports
effect of probiotic treatment on URTI (Fig. 2a). As indicated exclusively on SARS-CoV-2 infection and the application
by an I 2 value of 31.47% and Q (chi-square) statistics of of probiotics. Manual analysis of the literature shortlisted
42.32 (p = 0.05), there was no statistical heterogeneity. The 90 training genes of interest associated with COVID-
overall effect size of the study was 2.75, with p = 0.006. 19 pathogenesis (Table 2). These genes were used for
The effect of probiotic treatment on URTI was plotted the generation of the association network and further
according to specific strains of probiotics. The effects size analysis.
(ES), confidence level (95% CI), and the weight percentage
of clinical trials concerning the specific strains were also
depicted. Thus, the forest plot of the meta-analysis study Generation of Association Network
showed statistically significant effects of probiotic treatment with the Candidate Genes of COVID‑19
on URTI. Pathological Mechanisms and Antiviral Treatments
with Probiotics
Publication Bias
All the 90 shortlisted training genes were used for
The funnel plot showed minimal evidence of publication the development of a text-mining–based association
bias among the selected studies associated with randomized, network. The primary association network obtained
double-blind, placebo-controlled human clinical trials of through Cytoscape with training genes showed 453 nodes
probiotic application on URTI (Fig. 2b). The funnel plot representing the candidate genes and 1273 edges. Fig. S2
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indicated the topology of the central association network, with MCODE score ≥ 3, nodes ≥ 4, edges ≥ 6 were chosen
highlighting the listed training genes involved in the SARS- for functional annotation (Fig. 3). Module 1 with MCODE
CoV-2 infection. Table 3 shows the network parameter score 16, 16 nodes, and 120 edges was the highest-scoring
statistics. According to the network topology, “the number module, whereas module 2 with MCODE score 14, 14 nodes,
of connected components” represented the network and 91 edges was the second-highest, and module 3 with
connectivity, which reflected a pairwise connection score 8.457, 36 nodes, and 147 edges was the third-highest.
among all the nodes. Hence, a lower number of connected The network matrices like the node degree, the clustering
components of 27, obtained by the network statistics, coefficient of the primary network, and MCODE-derived
suggested robust connectivity of the association network modules provided the topological parameters to understand
(Table 3). The clustering coefficient 0.633 designated the the importance of nodes for pathway analysis. The highest-
average clustering coefficient or degree involvement of scoring module 1 (MCODE score 16) consisted of 16 nodes
respective nodes in the participating cluster of the network, (src, limk1, rps6ka3, aak1, mapk1, mknk2, map3k1, gak,
with 5.620 average number of neighbors. The tendency to map2k1, fgfr1, mapkapk5, map3k7, bmp2k, zak, gsk3, yes1)
form high degree nodes or hub nodes was described by (Fig. 3a). The highest number of nodes or edges containing
high “network heterogeneity” of 1.137 and the absence of module 3 (cxcl5, cxcl12, tnc, has2, cxcl6, mmrn1, mmp13,
any isolated nodes. The presence of hub nodes indicated a smox, rela, ccl2, chi3l1, csf2, il5, il25, klk15, lcn2, il18,
real functional network with multiple biologically relevant vegfa, eng, plau, pgf, prl, angpt2, igfbp1, hgf, erbb2, fgf2,
pathways associated with the novel coronavirus infection. stat3, stat5, ifna1, soat1, cd68, il9, itgam, ptprc, cd14) was
The text-mining approach in conjunction with Cytoscape preferred as the most significant functionally relevant clusters
has been successfully adopted to illustrate actin dynamics for SARS-CoV-2 infection. It is worth mentioning here that
during the post-ejaculatory life of spermatozoa [48], to cluster analysis (using MCODE algorithm) is widely adopted
demonstrate the synergistic mechanisms of therapeutic for biological interpretation and elucidation of complex
herbs for rheumatic arthritis [49], and to find out the effects molecular networks (see Discussion) [52].
of psychological stress on innate immunity and metabolism
in human [50].
Determination of Functional Annotation Profile
Cluster Analysis of the Association Network of the Sub‑clusters to Elucidate the Protective
Identified the Groups of Promising Genes Related Avenues of Probiotics Against SARS‑CoV‑2 Infection
to COVID‑19 Clinical Spectrum and Antiviral
Probiotic Treatment A higher-scoring MCODE module with a high number of
nodes and edges implied a possible multi-functional role. Such
MCODE tool of Cytoscape derived 38 densely bridged modules could be assigned in the essential regulatory functions
modules from the first network (Fig. S3) by an algorithm of the disease pathogenesis and thereby could be the target for
called “top overlap.” It made groups of “genes of interest” the probiotic treatment.
according to their correlation coefficient and highest-scoring The genomic information of the nodes showed that
edge among all other candidate genes of the network. The MCODE cluster 1 candidate genes src, limk1, rps6ka3, aak1,
algorithm also minimized the chances of false-positive mapk1, mknk2, map3k1, gak, map2k1, fgfr1, mapkapk5,
results, increasing the robustness and accuracy of the analysis map3k7, bmp2k, zak, gsk3, yes1 were involved in the receptor-
[51]. The individual clusters were further selected based on mediated endocytosis and phagocytosis, stress-mediated
their scores for functional studies with the removal of lower cellular metabolic pathways (Fig. 3a). Therefore, probiotic
confidence data points. The details of the modules are given in supplementation could heal the disrupted intestinal barrier and
table S1. Among the 38 derived modules, 11 resultant clusters prevent virus entry.
The table contains details of the primary association network obtained by text-mining results using an Agilent Literature search (ALS) plugin.
“Network clustering coefficient” is the average of the clustering coefficients for all nodes in the network. The “average number of neighbors”
indicates the average connectivity of a node in the network
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Fig. 3 Network topologies of the most significant eleven MCODE creb1, rps6ka5, jun, ephb2, mapk8, atf2, fos, maa), and edges 44 (d).
clusters derived from association network of SARS-CoV-2 pathogen- MCODE-derived cluster 6: score 5, nodes 5 (dgat2, adipor2, dgat1,
esis and probiotic treatment selected for further GO enrichment and pgc, adipoq), and edges 10 (e). MCODE-derived cluster 9: score
pathway analysis. MCODE derived cluster 1: score 16, nodes 16 (src, 4.818, nodes 23 (il27ra, il27, cxcl10, il17d, il6st, il6r, jak2, ptpn18,
limk1, rps6ka3, aak1, mapk1, mknk2, map3k1, gak, map2k1, fgfr1, mapk3, epo, akt1, egfr, reg1a, ptpn1, cat, frap1, malat1, acan, eif-
mapkapk5, map3k7, bmp2k, zak, gsk3, yes1), and edges 120 (a). 4ebp1, smad4, smad6, smad2, smad7), and edges 53 (f). MCODE-
MCODE-derived cluster 2: score 14, nodes 14 (tnf, il6, map1lc3b, derived cluster 10: score 4.167 and 4, nodes 13 (ace2, mas1, ace,
ros1, cd38, bax, fas, sqstm1, il10, fcgr3a, cybb, il32, bcl2, icam1), ang, agtr1, il22, il2, gli2, cd4, cd40lg, fus, th1l, pdgfb) (g). MCODE-
and edges 91 (b). MCODE-derived cluster 3: score 8.457, nodes 36 derived cluster 11: score 4, nodes 5 (rab18, rab13, mtg1, rce1), edges
(cxcl5, cxcl12, tnc, has2, cxcl6, mmrn1, mmp13, smox, rela, ccl2, 25 (h). MCODE-derived cluster 13: score of 4, nodes 4 (hif1a, dlk1,
chi3l1, csf2, il5, il25, klk15, lcn2, il18, vegfa, eng, plau, pgf, prl, adam17, epas1), and edges 6 (i). MCODE-derived cluster 35: score
angpt2, igfbp1, hgf, erbb2, fgf2, stat3, stat5, ifna1, soat1, cd68, il9, of 3, nodes 7 (pparg, twist1, lpa, slc12a, mbtps1, srebf1, mbtps2), and
itgam, ptprc, cd14), and edges 148 (c). MCODE-derived cluster edges 9 (j). MCODE-derived cluster 38: score of 2.857, nodes 8 (hk2,
5: score 6.769, nodes 14 (tlr2, tlr4, nlrp3, myd88, hmgb1, mapk14, nr1i2, foxa2, pik3ca, f10, inpp5d, hspb1, mcl1), and edges 10 (k)
MCODE cluster 2 consisting of 14 nodes (tnf, il6, the viral infection: (a) activation of principal antiviral
map1lc3b, ros1, cd38, bax, fas, sqstm1, il10, fcgr3a, cybb, interferons (IFNs) and innate immune cells (stat3, stat5,
il32, bcl2, icam1) (Fig. 3b) had functions in the activation ifna1, soat1, cd68, il9, itgam, ptprc, cd14); (b) activation of
of immune cells (B cells, T cells, natural killer cells) in proinflammatory cytokines and their downstream signaling
response to stress stimuli, induction of the NF-κb-mediated through JAK-STAT pathway (ccl2, chi3l1, csf2, il5, il25,
inflammatory pathways, and subsequent apoptosis as klk15, lcn2, il18), (c) activation of proinflammatory cytokines
well as the production of cytokines. Several probiotic and chemokines resulting in the immune cell (neutrophil,
strains are reported to balance the immune activations monocyte) migration and infiltration in lung tissues, and (d)
and inflammatory cytokines, which could have immense synthesis of lung-damaging hyaluronan by has2 or hyaluronan
implications in the SARS-CoV-2-induced immunological synthase-2 (cxcl5, cxcl12, tnc, has2, cxcl6, mmrn1, mmp13,
complications. smox, rela), a proinflammatory cytokine-induced growth
Although MCODE cluster 3 was the third in rank based factor, responsible for vascular permeability and pulmonary
on score, it contained the highest number of nodes and edges dysfunction (vegfa, eng, plau, pgf, prl, angpt2, igfbp1, hgf,
that might indicate multiple cellular pathways (Fig. 3c). The erbb2, fgf2). Although limited studies are available on the
GeneCards-derived functional profile of nodes expectedly ability of probiotics to degrade glycosaminoglycan molecules,
showed the interconnections of four cellular events upon probiotics could have an impact on the degradation of
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hyaluronan substances synthesized by SARS-CoV-2-induced elucidated the promising yet unexplored functional pathways
proinflammatory cytokines. of these genes.
The candidate genes of cluster 5 (tlr2, tlr4, nlrp3, myd88,
hmgb1, mapk14, creb1, rps6ka5, jun, ephb2, mapk8, atf2, GO Enrichment Analysis to Assess
fos, maa) represented the genes responsible for TLR- the Overrepresentation of Gene Ontology
mediated innate immune responses and subsequent activation Categories by the BiNGO
of inflammasome complex, with stress-induced MAPK
signaling pathway (Fig. 3d). The virus entry might activate the The enrichment or overrepresented GO terms associated with
host’s innate immune system through TLR-myd88-mediated the MCODE cluster candidate genes, designated by p values
pathways and eventually activate the cellular inflammasome in BiNGO enrichment analysis, are detailed in table S2. The
complex. The functional annotations of cluster 6 candidate analysis showed that cluster 1, cluster 2, cluster 3, and cluster
genes (dgat2, adipor2, dgat1, pgc, adipoq) indicated glucose 5 involved the pathways related to viral infection, pathogen-
and lipid metabolic pathways and innate immune responses associated molecular patterns (PAMPs)–mediated immune
(Fig. 3e). Host defense mechanisms by the proliferation of response to extracellular signals, stress, leukocyte migration,
Th1 cell suppressing Th2 and Th17 cells, isotype switching activation of cellular oxidative stress and inflammatory
and anti-inflammatory cytokine production, and induction of processes through phosphate metabolic processes, regulation
growth regulatory pathways were represented by MCODE of MAPKKK cascade, and jun kinase activity. Therefore,
cluster 9 candidate genes (il27ra, il27, cxcl10, il17d, il6st, these physiological events associated with SARS-CoV-2
il6r, jak2, ptpn18, mapk3, epo, akt1, egfr, reg1a, ptpn1, infections could be modulated by probiotic supplementation.
cat, frap1, malat1, acan, eif4ebp1, smad4, smad6, smad2, Probiotics could heal the intestinal barriers and minimize
smad7) (Fig. 3f). The genomic functional annotations of viral entry, as well as balance the immune responses and
cluster 10 (ace2, mas1, ace, ang, agtr1, il22, il2, gli2, cd4, inflammatory conditions. Cluster 6, cluster 9, cluster 10,
cd40lg, fus, th1l, pdgfb) and cluster 11 (rab18, rab13, mtg1, cluster 11, and cluster 13 were involved in the regulation
rce1) candidate genes showed the reported ACE2-ADAM17- of innate immune cell differentiation, regulation of
mediated entry of SARS-CoV-2 in the host cell with the glucose and lipid transport, cholesterol efflux, glycerol,
resulting dysregulation of the renin-angiotensin system glycerolipid, acylglycerol, triglyceride, TNF, and cytokine
(RAS) involving ace2, ace, mas1, ang genes (Fig. 3g and h). signaling by JAK-STAT pathway in addition to regulation
Moreover, cluster 13 candidate genes (hif1a, dlk1, adam17, of renin-angiotensin–mediated blood pressure. Probiotic
epas1) indicated the receptor-mediated endocytosis of the supplementation could play an immense role in immune cell
virus entry with the consequent activation of oxidative stress- development and differentiation, regulation of inflammatory
responsive pathways mediated by hif1a (Fig. 3i). Probiotic processes, metabolic activity, and the regulation of blood
supplementation could alter the adverse conditions of the pressure through RAS. Cluster 35 and cluster 38 genes
infection by regulating host metabolic pathways through were involved in lipid, cholesterol, steroid, glucose, and
the secretion of SCFAs. Additionally, probiotics could monosaccharide metabolism, xenobiotic drug transport,
balance the inflammatory cytokines and chemokine levels regulation of monocyte and neutrophil differentiation, and
in the body by regulating oxidative stress, altering the response to cytokine IL6. This enrichment analysis of the
SARS-CoV-2 infection condition. The effects of probiotics GO terms of candidate genes enlightened us on the major
in controlling blood pressure may significantly alter ace2- biological processes associated with SARS-CoV-2 infection
mediated dysregulation of RAS. Finally, the candidate genes pathogenesis. It provided the cellular domains for antiviral
of MCODE cluster 35 (pparg, twist1, lpa, slc12a, mbtps1, probiotic mechanisms to defeat the viral infection. Several
srebf1, mbtps2) and cluster 38 (hk2, nr1i2, foxa2, pik3ca, f10, research groups have used similar BiNGO enrichment
inpp5d, hspb1, mcl1) designated the involvement of glucose analysis to understand breast cancer susceptibility or to
and lipid metabolic pathways, adipocyte differentiation, detect key pathways responsible for gastric cancer or analyze
VEGF signaling pathway, and induction of atherosclerosis the protein-protein network [12].
(Fig. 3j and k). Although the MCODE score of cluster 38
was <3, the module was selected for analysis due to the 8
nodes and 10 edges that might reflect significant function Discussion
and pathways in the disease. Treatment with probiotic
formulations might reverse any metabolic abnormality upon The successful application of probiotics in respiratory
SARS-CoV-2 infections by releasing SCFAs. The molecular infections of human clinical trials encourages us to study
events and pathways indicated by MCODE clusters were the probiotics application to alleviate SARS-CoV-2
supported by the published scientific shreds of evidence infection. The meta-analysis is an epidemiological study
available for SARS-CoV-2 infection and pathogenesis, which design that quantitatively examines the available outcomes
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Probiotics and Antimicrobial Proteins
and estimates the effect of treatment much precisely than Probiotics Could Play a Protective Role in Cytokine
a single study contributing to the pooled analysis. In this Storm and Lung Injury Caused by SARS‑CoV‑2
study, by using a systematic network and meta-analysis, we Infection
provide a promising probiotic mechanism that can reinforce
the immunity and mitigate the SARS-CoV-2 infection. Frequent interaction with the external environment in terms
of food and air alters the microbial composition of intestinal
Probiotics Could Confine the Virus Entry and Reduce microbiota as well as lungs. Several GI disorders also have
the Adverse Effects Caused by Antiviral Immune manifestations of respiratory complications. The high
Activation and Dysregulation of RAS expression of ACE2 receptor in gut and lung epithelial cells
with the prevalence of respiratory and GI tract infections
ACE2 receptor is a master regulator of RAS and blood in COVID-19 patients strongly suggests the bi-directional
pressure. However, SARS-CoV-2 enters the host enterocytes cross-talk of the “gut-lung axis” [7]. After entry, the viral
and lung tissues through ADAM17-mediated proteolytic RNAs activate the cellular innate immune system (TLRs)
cleavage of the ACE2 receptor [53]. The disruption of the [62] and inflammatory pathways (NLRP3 and NF-κB) as
protective “gut-lung axis” increases the propensity of the a protective mechanism. Activated TLRs promote first-line
infection in the underlying ACE2 containing epithelial cells. antiviral response through MYD88 and IRF3/7-mediated
SARS-CoV-2 infection may downregulate cellular ACE2 type-I IFN production [63, 64]. The NLRP3 inflammasome
expression with the resulting accumulation of angiotensin II complex assists in the secretion of proinflammatory IL1b
(Ang II). Therefore, infected lung alveolar and small intestinal and IL18, which activates T-cells or macrophages to secrete
epithelial cells, heart, kidney, vascular endothelial, and smooth IL6 and TNFα. The released proinflammatory cytokines
muscle cells (highly expressing ACE2) cause vasoconstriction, (IL1B, IL18, IL6, TNFα) further convert naive T-cells
tissue inflammation, and oxidative stress, which worsen the to Th1/CTLs/CD8+ or Th17 and triggers the secretion
health conditions of COVID-19 patients [53]. of proinflammatory IFNγ and IL17. Therefore, the gut
Our functional annotations and enrichment analysis of microbiome controls innate and adaptive immune responses
candidate genes of MCODE cluster 10 (ace2, mas1, ace, against the respiratory pathogens [7]. However, the NF-κB
ang, agtr1, il22, il2, gli2, cd4, cd40lg, fus, th1l, pdgfb) pathway can be turned on in either of two ways: activated
and MCODE cluster 13 (hif1a, dlk1, adam17, epas1) NLRP3 or TLR4 and stress-induced MAPK signaling
(Fig. 3g and i; Table S3) indicate that probiotics could have (ERK1/2, JNKs, and p38/MAPK14) pathway [65, 66]. The
potential roles in limiting viral entry through ADAM17- activated NF-κB pathway contributes to proinflammatory
ACE2–mediated receptor endocytosis. Further enrichment cytokine secretion and apoptosis in enterocytes and lung
analysis shows that the application of probiotics could tissues. The resulting tissue injury activates local circulatory
mitigate the adverse effects of dysregulated RAS system, innate immune cells and establishes a proinflammatory feed-
hif1a-mediated oxidative stress [54], activated immune cells forward loop of cytokines called cytokine storm [65]. The
(monocytes and NK cells), and elevated proinflammatory surge of cytokines and chemokines induces VEGF, IL8,
cytokines. and additional IL6 and reduces E-cadherin expression on
Probiotics are known to heal the damaged epithelial endothelial cells ensuing vascular permeability, while it also
barrier and thereby protect the underlying ACE2 receptor- elicits leukocyte trafficking and migration of monocytes,
expressing cells [55]. Bacteriocin-producing probiotic strains neutrophils, NK cells, macrophages, and dendritic cells in
can colonize in the GI tract better than bacteriocin non- the lung cells. The resulting immune cells and cytokines
producers. Therefore, bacteriocins also play a crucial role induce hyaluronan synthesis, which participates in the
in microbial colonization [11]. Probiotics have been reported pathophysiology of ARDS, the hallmark of SARS-CoV-2
to reduce nitric oxide (NO) production [56], hypertension infection [67]. The “gut-lung axis” facilitates the migration
[57] and oxidative stress [58], and secrete ACE-inhibitory of immune cells from the gut to the lung through circulation
peptides [59], SCFAs (acetate, propionate, and butyrate) to and enforces immunity in health and disease. Therefore,
induce anti-inflammation and control the blood pressure the immunological coordination between the gut and lung
[60]. Recombinant probiotic L. paracasei in conjugation affects each other’s homeostasis and influences the acquired
with ACE2 has been applied for the cardiovascular protective immunity in health and disease of the host.
role of ACE2 in mice [61]. Additionally, cinnamycin-like Our functional annotations and enrichment analysis of
lantibiotics (bacteriocins) mediate inflammatory responses MCODE cluster 5 candidate genes (tlr2, tlr4, nlrp3, myd88,
by inactivating phospholipase A2. One such lantibiotic hmgb1, mapk14, creb1, rps6ka5, jun, ephb2, mapk8,
ancovenin is reported to inhibit ACE, thereby preventing atf2, fos, maa) indicate that probiotics could be associated
the conversion of Ang I to Ang II and regulate the blood with TLR-mediated innate immune response during SARS-
pressure [11]. CoV-2 infection. TLR-3, 7/8, reported to recognize the
13
Probiotics and Antimicrobial Proteins
SARS-CoV-2 RNA, was also present in the association molecules [71] that may result in reduced expression of has2
network but not in the MCODE cluster (Fig. 3d; Table S4). and hyaluronan breakdown. Probiotics are also proven to
The presence of nlrp3 in cluster 5 hints the activation of downregulate the NF-κB signaling pathway by regulating
inflammatory and antiviral IFNs which is further supported MAPK and ERK pathways reducing systemic inflammation
by cluster 2 (tnf, il6, map1lc3b, ros1, cd38, bax, fas, [72].
sqstm1, il10, fcgr3a, cybb, il32, bcl2, icam1) and cluster 3
(cxcl5, cxcl12, tnc, has2, cxcl6, mmrn1, mmp13, smoxlus, Probiotics Could Improve Cardiovascular
rela, ccl2, chi3l1, csf2, il5, il25, klk15, lcn2, il18, vegfa, Complications and Lipidomic Abnormalities
eng, plau, pgf, prl, angpt2, igfbp1, hgf, erbb2, fgf2, stat3,
stat5, ifna1, soat1, cd68, il9, itgam, ptprc, cd14) candidate COVID-19 comorbidities like obesity, metabolic
genes (Fig. 3b and c; Table S5). The highest node degree of syndrome (e.g., IBD), are associated with an inadequate
IL6 and all the cytokine and chemokine genes confirm their gut-microbial composition linked to disruption of the
significant participation indicating reported elevation in the bi-directional communication network in the gut-heart
immunopathology of COVID-19. Additionally, apoptotic axis. Such dysbiosis reduces gut-barrier integrity,
genes like bax, bcl2, fas, and cybb may indicate TNFα, accelerates intestinal inflammation, and contributes as an
IL6-mediated programmed cell death of T-cells depicting extragenomic factor to cardiovascular complications [73].
the observed global lymphocytopenia in the COVID-19 Dysregulation of RAS due to SARS-CoV-2 entry promotes
patients [68]. However, receptors like cd68 (NK cells), cd14 inflammation, vasoconstriction, hypertrophy, proliferation,
(monocytes), and has2 gene may depict the rapid migration and fibrosis, all factors that contribute to the development
and infiltration of innate immune cells in the lung tissue and progression of cardiopulmonary diseases [74]. Chronic
with injury in response to chemoattractant chemokines. The elevation of IL6 and cytokine storm promotes macrophages
candidate genes of cluster 9 (il27ra, il27, cxcl10, il17d, il6st, to release MCP-1, which aids atherogenesis, expression of
il6r, jak2, ptpn18, mapk3, epo, akt1, egfr, reg1a, ptpn1, cell adhesion molecules, and proliferation and migration
cat, frap1, malat1, acan, eif4ebp1, smad4, smad6, smad2, of vascular smooth muscle cells resulting in cardiovascular
smad7) and cluster 1 (src, limk1, rps6ka3, aak1, mapk1, diseases (CVDs) (e.g., coronary atherosclerosis,
mknk2, map3k1, gak, map2k1, fgfr1, mapkapk5, map3k7, inflammation in the vascular system, and thrombosis) [75].
bmp2k, zak, gsk3, yes1) show the involvement of MAPK Enhanced angiotensin II also causes EGFR transactivation-
signaling and proinflammatory cytokine-induced growth induced vascular remodeling [76]. The induction of
regulatory pathways (Figs. 3f and S1; Table S6). Therefore, CVDs further functionally damages intestinal epithelial
our analysis indicates that probiotics could play a protective cells (IECs), which, together with barrier dysfunction,
role in the above-mentioned signaling pathways. allows translocation of gut bacteria–derived endotoxins
Probiotics protect the intestinal barrier by inhibiting (lipopolysaccharide or LPS) and metabolites into the
cytokine-induced intestinal epithelial cell apoptosis [69]. A circulation. The recognition of LPS by receptors like
probiotics mixture consisting of L. acidophilus, L. casei, TLR4 on cardiomyocytes induces tissue inflammation and
L. reuteri, B. bifidium, and Streptococcus thermophilus is increases cytokine secretion (TNFα). The pathophysiology
reported to induce both T-cell and B-cell hyporesponsiveness of coronary atherosclerosis is also associated with aberrant
and downregulate T helper (Th) Th1, Th2, and Th17 production of metabolites like betaine, choline, and TMAO
cytokines without inducing apoptosis [70]. Further, by microbial digestion. These metabolites and LPS affect
probiotic-derived molecules (i.e., bacteriocins) are also cholesterol and lipoprotein (LDL) metabolic pathways,
known to trigger CD4(+) and CD8(+) T cell activation and which amplifies inflammation and worsen SARS-CoV-2
modulate interleukin production [11]. Thus, probiotics could infection [73].
have a promising role against lymphocytopenia or reduction The candidate genes encoding growth factors of
of T cell count, which is observed in COVID-19 patients. MCODE cluster 3 (vegfa, eng, plau, pgf, prl, angpt2,
Moreover, probiotic formulations can balance pro- and anti- igfbp1, hgf, erbb2, fgf2, stat3, stat5) along with
inflammatory cytokine secretion, which is the key factor for overrepresentation of GO terms indicates the role of
robust immune (adaptive and innate) system. Although few probiotics in the mitigation of elevated angiotensin
reports are available on the immunomodulatory effects of II-induced cardiovascular complications (Fig. 3c;
bacteriocins, the activity of nisin, nisaplin, and pediocin Table S5). The functional annotations and enrichment
is well documented [11]. Several clinical trials on URTI analysis of the candidate genes of MCODE cluster 6
individuals have reported the decreased proinflammatory (dgat2, adipor2, dgat1, pgc, adipoq), cluster 35 (pparg,
and increased anti-inflammatory cytokine upon probiotic twist1, lpa, slc12a, mbtps1, srebf1, mbtps2), and cluster
application. Limited search has been done, which reveals 38 (hk2, nr1i2, foxa2, pik3ca, f10, inpp5d, hspb1, mcl1)
the ability of probiotics to degrade glycosaminoglycan indicates that probiotics could have potential roles
13
Probiotics and Antimicrobial Proteins
in glucose and cholesterol or lipoprotein metabolism bacteria and probiotics are reported to regulate blood
(Fig. 3e, j, k; Table S7). The lipidomic and cholesterol pressure (BP). Receptors like GPR41 and Olfr78 are
metabolic abnormalities due to SARS-CoV-2 infection responsible for hypo- and hypertensive effects of SCFAs,
are poorly documented. The limited reports indicate respectively [73]. A number of LAB probiotic strains
enhanced glucose and lipid need for viral replication are reported to exhibit antiviral activity by producing
and metabolism since viruses hijack the host’s metabolic bacteriocins, which mostly inhibit viral replication that
processes. The overrepresentation of GO terms associated indicates their potential role in reducing SARS-CoV-2
with lipid and cholesterol metabolism reflects the lipid- infection [11].
demanding processes (viral replication, endocytosis,
and exocytosis) involved in SARS-CoV-2 infection [77].
They also indicate cardiovascular complications due to Conclusion
gut-dysbiosis and COVID-19.
Probiotic supplementation has been proved to Our systematic network and meta-analysis study aid
reduce total cholesterol, LDL, and triglycerides and us to propose a mechanistic model of probiotic actions
increase HDL count. The antihypertension capability in the alleviation of COVID-19 (Fig. 4). Probiotic
of the probiotics makes them an affordable and consumption could reduce the propensity of viral entry
adjunctive treatment option in hypertension, diabetes, by healing the ACE2 containing epithelial barrier. The
and cardiovascular diseases, and other dyslipidemia- antimicrobial peptides or bacteriocins, SCFAs, and ACE
associated health issues [78]. SCFAs released by gut inhibitory peptides released by beneficial bacteria could
Fig. 4 The mechanistic model of probiotics action against COVID- Th1, Th2, and Th17 cells (v). Which, in turn, help in the production
19. Probiotics induce a stronger epithelial barrier that prevents viral of more anti-inflammatory cytokines (vi). The anti-inflammatory
entry through the gut (i), probiotics modulate gut microbiota and cytokines regulate monocytes, macrophages, dendritic cells, and
induce the synthesis of SCFAs that regulate blood pressure and neutrophils (vii) to downregulate SARS-CoV-2 infection–mediated
inflammation (ii). Probiotics also release ACE-inhibitory peptides cytokine storm (viii), resulting in decreased total cholesterol, LDL,
that could reduce angiotensin II (Ang II) expression, thereby inhibit- triglycerides, VEGF, EGF, PDGF, TNF, and CRP level in the blood
ing viral entry into the cell (iii). Probiotics induce anti-inflammation stream (ix). The reduced cytokine storm and inflammation exerted by
by suppressing NF-κb signaling and reducing the levels of IL1β, probiotics cause the reduction in hyaluronan synthesis, which eventu-
IL18, NO, and TNF (iv). Bacteriocin and other anti-, as well as proin- ally could improve the ARDS condition in SARS-CoV-2 infection (x)
flammatory cytokines produced by the effects of probiotics modulate
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