Translation of Immunomodulatory Effects of Probiotics into Clinical Practice

Abstract

Probiotics have emerged as an in-demand and highly marketed commodity in the healthcare space. In 2021, the global market valued the probiotic industry at USD 58.17 billion in 2021. It is expected to have a compound annual growth rate of 7.5% yearly from 2021 to 2030. The inclusion of probiotics in various products has become synonymous with health benefits despite limited understanding of mechanism of action or benefit. This chapter will survey the state of our understanding of the interactions between probiotics with the innate immunity, adaptive immunity, and the host gut microbiome. Additionally, we will also highlight the theorized beneficial and possible detrimental immunomodulatory effects of probiotics on human health.

1. Introduction

The word probiotic comes from the Latin word pro and the Greek word bios, which when joined together, literally means “for life.” The concept of probiotics has evolved over the many millennia but modern scientific theory of it only began in the twentieth century. The term probiotics was first used in 1953 by Werner Kollath, a German scientist who defined it as an “active substance that is essential for a healthy development of life.” The definition of the term changed multiple times through the century but the most accepted one comes from the Food and Agriculture Organization of the United Nations and the World Health Organization (FAO/WHO). They define probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host [1, 2, 3].”

Although probiotics are believed to confer important health benefits, including amelioration of C. diff colitis, inflammatory bowel disease, metabolic syndrome, etc., the understanding of the mechanisms of action of probiotics is limited. Thus, the aim of the present chapter is to review the immune modulatory effects of probiotics and how it interacts with the host gut microbiome. We will also highlight the practical clinical uses of probiotics on human health and disease. Lastly, we will speculate on the future direction on the use of probiotics.

2. Modulation of innate immunity by probiotics

Innate immunity is one of the major arms in our immune system and consists of a complex complement cascade that acts as a physical and chemical barrier. It works to protect against infectious agents by recognizing conserved features of pathogens that become quickly activated to help destroy microbial invaders and to produce factors such as cytokines to activate adaptive immune response.

The most recognized innate mechanism comes from the concept of a barrier. This intrinsic wall helps evade foreign microbe penetration and prevents all the deleterious effects of colonization. The three major components that have been studied in barrier protection are mucin production, reinforcement of tight junctions in the epithelial layer, and enzyme regulation. Mucin production made by epithelial cells helps deter pathogen attachment. The permeable gel-like layer offers innate immunity by helping release secretory IgA, which prevents invading pathogen adherence. Additionally, the mucin layer helps identify self with nonself and can activate the immune system against invaders. Pathogen-associated molecular patterns are embedded in commensal microbiome organisms and these are recognized by Toll-like receptors to be noninvasive microbiota. The mechanisms of mucin for barrier enforcement are well known but studies now are starting to show how probiotics may help with boosting this barrier production [4]. In vivo rat studies have shown that administration of a specific probiotic mixture named VSL#3, which included strains of Lactobacilli, Bifidobacteria, and Streptococci, was associated with increased mucin gene expression and secretion. Increased mucin production theoretically leads to a more robust barrier [5]. The third barrier mechanism includes enzyme activity modification. Foreign microbes can also invade by activating destructive enzymatic processes. Pathogens secrete enzymes like Β-glucuronidase that result in toxic metabolites and can be pre-carcinogenic in the intestines. Bifidobacterium longum is a probiotic that was used in animal studies and showed a decrease in fecal B-glucuronidase activity and abnormal intestinal crypt structure by about 53% [6].

Tight junctions between epithelial cells help create a firm seal and prevent invasion. Lactiplantibacillus plantarum WCFS1, a probiotic, was noted to increase proteins like zonula occludens-1 (ZO-1) and transmembrane occludins near tight junctions, which help promote a good seal and ensure integrity of the epithelial barrier [7]. Another probiotic, Lactobacillus rhamnosus, was used to pretreat intestinal epithelial cells of pigs and was later exposed to enterotoxigenic Escherichia coli (ETEC). Pretreated cells have less TNF-α inflammatory response, higher ZO-1/occludin levels, and helped deter pathogenic adhesion to the epithelium. TNF-α activity correlates with an inflammatory cytokine response, which can lead to cell injury, so a subdued response helps taper these detrimental events [8]. In addition to the formation of a strong barrier, the components of the epithelial layer also add to its fortitude. Basolateral cells in the intestine have B cells that secrete sIgA, a secretory IgA transporter, which helps build a robust innate immunity. This secretory immunoglobulin works through a process called immune exclusion, which is when sIgA recognizes surface molecules on pathogens and prevents adherence. Probiotics like Bifidobacterium breve, L. rhamnosus, and Lactobacillus casei have shown to increase the sIgA production and thus prevent colonization [9, 10].

Competitive exclusion is another important innate mechanism used to prevent pathogenic growth. The general concept here is that one microbe outcompetes, through various mechanisms, another and dominates the microbiome. Probiotics take advantage of this principle by creating toxic environments, competitively taking over resources, and producing antimicrobial bacteriocins to overtake pathogens [9].

Another key area of probiotic function comes from the cytokine cascade that leads to immune activity. Several examples exist but to understand their function, a brief review of immune cells and cytokines will help showcase the various mechanisms. Natural killer (NK) cells are lymphocytes that work to kill foreign pathogens with their cytotoxic proteases. Monocytes include macrophages and dendritic cells, which work by phagocytosis and present antigens to adaptive immune cells, respectively. IL-10 and IL-4 are anti-inflammatory interleukins that can prevent cell damage [11]. A plethora of studies exists to showcase how particular strains evoke a complex cytokine pathway. Daily consumption of Lactobacillus salivarius, a probiotic from breast milk, increased production of natural killer cells, monocytes, immunoglobulins, and IL-10 [12]. The SETOPROB study showed that probiotics like L. rhamnosus, L. casei, and B. breve increased IL-4, IL-10 and fecal secretory IgA. These cytokine and cell activations lead to downregulation of inflammation and prompt the activation of the adaptive immune system [9].

Finally, probiotics help maintain homeostasis by way of pathogen recognition and T cell regulation. Pattern recognition receptors (PRRs) bind to pathogen-associated molecular patterns (PAMPs), or damage-associated molecular patterns (DAMPs), which are expressed on most pathogens. PRRs are made up of toll-like receptors (TLRs) and NOD-like receptors (NODLRs), which function to activate immune activation and protect the cytoplasm. Additionally, TLR activation by PAMPs or DAMPs on monocytes triggers T cell activation and naïve T cells are prompted to differentiate. Activation of TLRs and NODLRs prompts cytokine cascade activation and the resulting inflammation could facilitate cell damage. Probiotics, however, regulate nuclear factor-κB (NFκB) and dampen the inflammatory response [9, 13, 14, 15].

The innate immunity is the body’s initial defense mechanism and is made up of a variety of pathways to fortify the barriers and activate immune cascades. Probiotics assist in this pathway in many ways as outlined above. This initial response lends itself to initiate the acquired immunity discussed below, which goes on to form a more long-lasting immune response.

3. Modulation of adaptive immunity by probiotics

Adaptive immunity is responsible for identifying and destroying individual invading microbes in mammalian hosts. The cells that carry out the adaptive immune response are B and T cells, which produce a cascade of immune responses upon recognition of foreign antigens interacting with their specific toll-like receptors (TLRs) [16]. Unlike the innate immune system, which is preprogrammed to react to common broad categories of pathogens, the development of adaptive immune responses to new pathogens is slower. Due to its highly specific antigen receptors, specifically the B cell receptor (BCR) and T cell receptor (TCR), to the pathogen, the body has encountered. Adaptive immunity creates immunological memory after an initial response to a specific pathogen and leads to a robust response to encounter with pathogens in the future. B cells act via humoral immunity by secreting antibodies while T cells work via T helper cells (CD4+) and cytotoxic T cells (CD8+) to either expand or suppress downstream immune activation [17]. CD4 T cells can be broken down into 5 major subsets: Th1, Th2, Th17, T regulatory (Treg), and follicular T helper (Tfh). This categorization is determined based on the expression of specific cytokines and lineage-specific transcription factors. Th1 cells activate macrophages to help protect against intracellular pathogens such as bacteria and viruses. Th2 cells recruit eosinophils, basophils, and mast cells to sites of infections caused by parasites. Th17 cells aid in the clearance of extracellular bacteria by stimulating continuous neutrophil recruitment and the creation of antimicrobial peptides by epithelial cells. Treg cells contribute to the maintenance of immune tolerance and the prevention of autoimmune diseases. Tfh cells support B cells in the production of antibody formation by aiding in germinal center formation and immunoglobulin class switching [18]. The gastrointestinal tract is the largest immune organ in the human body and comprises the epithelial layer, lamina propria, and mucosal-associated lymphoid tissue (MALT). Adaptive immunity plays a vital role in the development and maintenance of the mucosal immune system (MIS) [16, 19]. Peyer’s patches are aggregates of lymphoid follicles found throughout the intestinal mucosal cells [20]. They are the main site for B cell activation and class-switch recombination from IgM to IgA. These cells also aid the immune system in discriminating between pathogenic and commensal bacteria. Their function is imperative to maintaining the integrity of the gut mucosal barrier, protecting the host from infections, and maintaining homeostasis with the native microbiota [21]. At the mucosal level, antigen-presenting cells (APCs) present in Peyer’s patches will retrieve immunoglobulin A antigen from mucosal folds and communicate with T cells resulting in different T cell activation, which then ensures mucosal barrier integrity [16, 22].

Numerous studies have demonstrated a variety of molecular pathways where probiotics appear to have influence, such as the production of cytokines, IgA secretion, formation of antibacterial compounds, mucosal cellular integrity, and competition with opportunistic pathogens for enterocyte adherence. A proposed probiotic immunomodulation works by antigenic proteins native to the probiotic microorganism crossing epithelial cells and interacting with the innate and adaptive immune system that resides in Peyer’s patches [23]. In turn, this interaction produces a cascading effect resulting in the release of cytokines, such as tumor necrosis factor (TNF), interferons (IFN), interleukins (IL), and chemokines. This interaction between probiotics and the host suggests probiotics play an important role in the production and deployment of a more robust immune response by the host when faced with pathogenic organisms. Cellular wall compounds, such as lipoteichoic acid, which is found in Bifidobacteria and Lactobacilli, are known to stimulate nitric oxide (NO) synthase. The production of NO is a critical component in the cell death mechanism carried out by macrophages when dealing with pathogen-infected cells [24]. B. longum is considered one of the first immune-priming probiotics. Known as the “maternal probiotic”, most of the inoculation comes via the mode of vaginal birth. Studies have demonstrated that B. longum plays a crucial role in immune system priming, Peyer’s patch development, and IgA production [25]. Lactic acid bacteria such as L. casei, Lactobacillus acidophilus, L. rhamnosus, Lactococcus lactis, and Streptococcus thermophilus have been shown to play a role in maintaining the intestinal barrier by stimulating B cells to produce IgA. Lai, Hung-Hsiang, et al. administered L. casei and L. rhamnosus to children with acute diarrheal illness. When compared to the control group, the children who received the probiotics had higher total fecal IgA levels and significantly lower concentrations of fecal lactoferrin and calprotectin. This study suggests that the probiotics L. casei and L. rhamnosus may be useful supplements during acute diarrhea to reduce clinical severity and intestinal inflammatory reaction [26].

Additionally, probiotics have been observed to modulate pro−/or anti-inflammatory responses by the adaptive immune system via interaction with dendritic, Th1, Th2, and Treg cells at the intestinal mucosal surface [24]. In Celiac disease (CD) patients, dysbiosis is thought to play a primary role in its pathogenesis [27]. Numerous studies have demonstrated a significant difference between intestinal microbial populations in healthy children and children with CD [28]. With a gluten-free diet, many of these microbial differences dissipate, except for persistently reduced levels of Bifidobacterium in the CD subjects [29]. This finding is particularly important as Bifidobacteria has been shown to protect human intestinal cells from the noxious effects of gliadin peptides by altering their molecular structure. Unmodified gliadin peptides result in an adaptive immune response leading to the development of anti-tissue transglutaminase antibodies (anti-tTG), which cause a local inflammation destroying microvilli responsible for nutrient absorption and disrupting the intestinal mucosal barrier [30]. Current murine model studies suggest that the immunomodulatory effects of probiotics are strain specific. Borruel et al. studied ileal mucosal samples from patients with active Crohn’s disease and cultured them with either Escherichia coli, Lactobacillus casei, Lactobacillus bulgaricus or Lactobacillus crispatus. The probiotic Lactobacillus bulgaricus and Lactobacillus casei cultured samples demonstrated a significant reduction in TNF-α, a known proinflammatory cytokine. The most robust effect on the downregulation of TNF-α came from viable bacteria, while heat-killed bacteria did not produce a statistically significant change. This finding suggests that cellular products manufactured by viable bacteria play an important role in the suppression of TNF-α production in inflamed tissue [31]. Livingston et al. explored the immunoregulatory response of bone marrow-derived dendritic cells to Lactobacillus reuteri 100–23, as previous studies suggested that this bacterial strain had modulatory effects on proinflammatory cytokines in murine models. They found that exposure to L. reuteri increased the production of IL-10 suggesting an induction of a regulatory dendritic cell phenotype. This resulted in lower IL-2 production while increasing TGF-𝛽 output [32]. This is important as IL-10 and TGF-𝛽 are immunoregulatory cytokines and the overall suppression of the murine immune response directed at L. reuteri allows the bacteria to colonize and have a commensalistic relationship with the host. Moreover, various probiotic strains have demonstrated the ability to stimulate immunoglobulin receptors in intestinal epithelial cells [33].

4. Modulation of the gut microbiome by probiotics

Probiotic mechanisms resulting in human gut microbiome alteration include effects on the microbial composition and function of these native organisms. More recent studies have utilized culture-dependent methods and metagenomic sequencing techniques to evaluate probiotic effects on changes in microbiome composition, diversity, and function. Certain strains of probiotics have been shown to release antimicrobial proteins or metabolic waste products that suppress the growth of other bacteria in the local vicinity. Others have been shown to compete with local bacterial populations for receptors and binding sites on the intestinal epithelial cells [34, 35, 36]. Lactobacillus reuteri is an anaerobic probiotic that converts glycerol into reuterin, a potent antimicrobial compound that inhibits the growth of pathogenic gram-negative and gram-positive bacteria. Agar spot testing has demonstrated these inhibitory effects on enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), Salmonella enterica, Shigella sonnei, and Vibrio cholerae [34]. Gut microbiota growth and metabolism are heavily dependent on the supply of dietary carbohydrates. The probiotic Bifidobacterium has been observed to contribute to interspecies cross-feeding resulting in an increase in beneficial microorganisms, including Firmicutes bacteria. This occurs as Bifidobacterium can utilize starch and fructo-oligosaccharides for energy and release lactate as a metabolic byproduct. The lactate is then used by local Firmicutes bacteria for energy. This relationship is important for the host as Firmicutes bacteria produce butyrate, a beneficial short-chain fatty acid [37, 38]. Interestingly, cross-feeding between different Bifidobacterial strains has been shown to upregulate the transcription and expression of various genes resulting in metabolic profile changes, primarily genes that play a role in carbohydrate metabolism [38]. Shifts in metabolic gene expression have also been observed in murine models when supplemented with fermented milk products that harbored a variety of probiotic bacteria. Results of metatranscriptomic analysis on fecal samples revealed a significant change in carbohydrate enzyme gene expression, further strengthening the proposed relationship between probiotic bacteria supplementation and shifts in the metabolic function of the gut microbiome [39].

A study analyzing the fecal microbiota of 6-month-old infants explored the changes in intestinal microbiota communities when supplemented with L. rhamnosus. Their results showed an abundance of L. rhamnosus and an increased microbial species evenness index suggesting ecological stability and diversity [40]. In murine models, supplementation of L. reuteri resulted in an increase in microbial community evenness and diversity when compared to vehicle-treated mice [41]. These findings are notable as maintaining diversity in microbial communities is associated with ecological stability [42]. Interestingly, insults such as infections or antibiotic therapy that result in a decline in microbial diversity have been associated with autoimmune diseases such as Crohn’s disease and eczema [43, 44]. These findings suggest that probiotics may induce local changes in the gut microbiota and directly contribute to healthy diversity and stabilization of microbial communities.

5. Clinical uses of probiotics

Probiotics are live bacteria meant to inoculate the gut of the host and incorporate into an already diverse microbiota. Probiotics are broadly used in three categories: immunomodulation, normalization of intestinal microbiota, and metabolic effects [45]. In general, the quality of evidence for use in clinical conditions remains low. The literature to support their use has been most clear in necrotizing enterocolitis (NEC) in neonates and pouchitis in ulcerative colitis (UC) patients. However, the use of probiotics well beyond the gastrointestinal tract is ongoing. We will review the studies about the current state of probiotics used in various disease states in this section (Table 1).

Probiotic	Human health condition	Proposed mechanism	References
Saccharomyces cerevisiae variant boulardii	Antibiotic-associated diarrhea	Interference with cell signaling, direct production of bacteriocins, and augmentation of the systemic immune response of the host.	[46]
Lactobacillus GG, E. faecium (SF68 strain) and S. boulardii			[47, 48]
S. boulardii	Clostridioides difficile infection	Protease that inhibits Clostridioides difficile toxin A and B activity.	[1, 49]
Lactobacillus spp.		Inhibit toxin A/B in human enterocytes Caco-2 and HT-29 cells.	[50]
Lactobacillus GG	Inflammatory bowel	To inhibit pro-inflammatory cytokines such as NF-Kb potentially providing anti-inflammatory properties to the host.	[51, 52]
S. boulardii			[46]
Lactobacillus reuteri		Tumor Necrosis Factor (TNF) a common target of biologics often used in IBD treatment was reduced.	[53]
VSL#3 containing Lactobacillus, Bifidobacterium, and Streptococcus			[54]
Bifidobacterium infantis and Lactobacillus acidophilus	Necrotizing enterocolitis	Unclear	[55, 56]
Bifidobacterium spp	Irritable bowel syndrome	Unclear	[57]

Table 1.

Summarization of known effects of probiotics in specific disease states.

5.1 Antibiotic-associated diarrhea

Antibiotic-associated diarrhea (AAD) is a common side-effect of the antibiotics that can affect up to a third of patients receiving antibiotics [58]. Broad-spectrum antibiotics with activity against anaerobes are associated with higher rates of the AAD [47]. AAD may last two months after the onset of antibiotic therapy resulting in significant morbidity [47]. Several randomized-controlled trials and meta-analyses, including bacterial strain-specific trials, have shown that the use of Lactobacillus and Saccharomyces has shown potential benefits of probiotics in addressing AAD [59, 60]. It is postulated that probiotics could antagonize the pathogenic microorganisms in the human flora when the host has been exposed to antibiotics [61]. The mechanism of their interference involves interference with cell signaling, direct production of bacteriocins, and augmentation of the systemic immune response of the host [62, 63, 64, 65, 66]. Most studies in AAD have focused on inpatients who were on intravenous antibiotics at higher concentrations where concurrent administration of probiotics has conferred a protective effect in some instances [67]. Probiotics have also been shown in meta-analyses to have a protective effect in outpatients receiving antibiotics without adverse side effects [68]. However, there remains a dearth of direct comparisons between specific strains and their effectiveness when used in conjunction with specific antibiotics. In addition, in the studies finding evidence of benefit, there are inconsistent definitions of diarrhea, specific infections treated, and the types of antibiotics being used [59]. This makes it challenging for clinicians to target probiotic treatment regiments to specific diseases. Thus, clinicians are not able to make specific recommendations to patients despite the strong interest and high prevalence of AAD.

To date, there is no global consensus on the use of the probiotics for AAD. The World Gastroenterology Organization (WGO) has supported their use of AAD in both adults and pediatrics. The use of L. rhamnosus GG and Saccharomyces boulardii was recommended by the Canadian Agency for Drugs and Technologies (CADTH) [69]. However, this recommendation is not shared by AGA or the IDSA (Infectious Disease Society of America) [49]. While there are certainly benefits to using probiotics in patients with AAD, the recommendations have not been able to clearly define the most appropriate patient or context.

5.2 Probiotics in Clostridioides difficile infections (CDI)

C. difficile is the most prevalent AAD for inpatients and outpatients leading to significant morbidity and mortality [70]. CDI is often associated with exposure to anaerobic coverage and antibiotics such as clindamycin, fluoroquinolones, or cephalosporins. Strategies to prevent C. difficile spread have typically involved patient segregation and hygiene measures. However, attempts to alter the host microbiota with fecal transplant or probiotics have become mainstream and shown themselves to be conclusive. In hospitalized patients receiving antibiotics, prophylactic administration of probiotics has been shown to significantly reduce the risk of developing C. difficile-associated diarrhea [71, 72, 73, 74, 75].

A proposed mechanism of this has been seen in Saccharomyces boulardii, which in murine models was shown to make a 54-kDa serine protease that cleaves toxin A and its intestinal receptor [50, 72, 76]. This has also been replicated in humans where toxin A and B cytotoxic effects in the human colon were attenuated when incubated in purified S. boulardii protease prior to being placed in the human colon [77]. When used in combination with metronidazole or vancomycin it reduced the number or relapses of diarrhea [78]. Efforts made for targeted primary prevention of CDI have typically focused on a multi-modal approach involving hygiene, antibiotics, and probiotics. A specific formulation of probiotics known as Bio-K+, which includes L. acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2, has been marketed in North America since 1996. Mouse models exposed to Bio-K+ have been found to increase concentrations of lactobacilli while decreasing levels of staphylococci [1]. The pathology in the human colon arises from toxins A and B of C. difficile that affects the colonic epithelium, which results in loss of cellular integrity and disruption of the colon mucosal cell cytoskeleton. Bio-K+ strains have been shown to produce supernatants (extracellular products) that inhibit toxin A/B in human enterocytes Caco-2 and HT-29 cells [1].

The American College of Gastroenterology (ACG), ESCMID (European Society of Clinical Microbiology and Infectious Diseases), and IDSA recommend probiotics for prevention or treatment of primary and recurrent C. difficile infections. However, the AGA is in favor of the use of S. boulardii, L. acidophilus CL1285, and L. casei for adults and children who are being treated with antibiotics except in situations of severe illness [79]. The difference among professional organizations comes from lack of clear evidence on the safety profiles and whether there is a true benefit [49]. Given the conclusive evidence on fecal microbiota transplants as a definitive treatment for recurrent CDI, there is no question of the significance that microbiota plays in the development of CDI and the potential manipulating it has in the prophylaxis and treatment of CDI. Furthermore, optimizing probiotic supplementation may have a meaningful role in CDI treatment.

5.3 Inflammatory bowel disease and probiotics

IBD pathophysiology involves a complex interplay between genetics, the host microbiome, environmental conditions, and the individual’s immune response [80, 81]. Changes in the intestinal mucosa and microbiota may disrupt homeostasis between the human immune system and the flora [82]. These changes may then trigger a reaction of the human immune system playing a role in development of the IBD. Indeed, specific intestinal microbiota profiles have been associated with active disease [83]. CD and UC patients have been found to have less Firmicutes and Bacteroidetes and more Proteobacteria and Actinobacteria when compared to healthy controls [84]. In addition, CD patients have been found to have reduced levels of Bacteroides, Eubacterium, Faecalibacterium, and Ruminococcus possibly leading to increased gut permeability [84]. A technology to help distinguish commensal from quiescent pathologic bacteria has been developed known as IgA-SEQ , which combines cell sorting with 16srRNA gene sequencing to quantify the amount of IgA on various taxa of bacteria found in the gastrointestinal tract. By measuring the amount of IgA coating, immunostimulatory and immunoregulatory taxa of the microbiota can be measured more accurately. This can then be used to confer susceptibility to IBD. While IgA itself does not contribute to the inflammatory response in IBD, this technology revealed three potential bacteria, which were associated with disease progression in IBD and three protective taxa. Taxa with relatively low abundance (based on 16S rRNA) were Erysipelotrichaceae sp. and Faecalibacterium prausnitzii, as well as low IgA coating of Oscillospira was associated with less progression to surgery [85]. These studies identify disease-modifying taxa and biomarkers for disease severity and progression. By identifying bacteria taxa as so-called “bad actors” in the human microbiome there may be a framework for the development of more refined biomarkers impacting disease courses and the possibility of microbiome-based therapeutics.

There has also been an association between CD and the colonization of adherent-invasive Escherichia coli (AIEC). AIEC is thought to impair mitochondrial function in epithelial cells of the gastrointestinal mucosa by invading the Peyer’s patches and the lamina propria via M cells [86]. It is thought that AIEC incorporates into macrophages and possibly increases the proinflammatory cytokine TNF-α. Patients with highly expressed CEACAM6 and CHI3L1 receptors, which are often expressed during times of inflammation, have been shown to promote the adhesion of AIEC and consequently bacteria invasion at the ileum [87]. Monocyte-derived macrophages (MDM) taken from patients with CD are unable to restrict AIEC as compared to healthy controls MDM in vitro models leading to pathologic immune response [88]. The overall prevalence of AIEC in healthy individuals is about 0–16% in the colon and 6–19% in ileal samples compared to 21–63% in CD patients suggesting that AIEC may be an additive factor in the pathogenesis of CD [51, 89, 90].

The increasing interest in the immune response to the gut microbiome in IBD has been met with interest in probiotic supplementation in this condition for induction and maintenance of remission. Specifically, it is thought that probiotics might be able to impact IBD pathophysiology by improving epithelium integrity, downregulating inflammatory bacterial byproducts, and reducing mucin production [91, 92]. Certain probiotic strains such as lactobacilli and bifidobacteria produce bacteriocins that act as antimicrobial peptides [54, 93]. Lactobacillus paracasei L74 CBA often found in fermented milk products like Kefir has been shown to inhibit pro-inflammatory cytokines such as NF-Kb potentially providing anti-inflammatory properties to the host. In addition, Duary et al. found that TNF-α was reduced by Lactiplantibacillus plantarum Lp91. While these findings have not been shown in human-based clinic trials, they provide a potential mechanism by which probiotics may have some clinical value in IBD [94].

The most used formation of probiotics in IBD patients is known as Visbiome®/VSL #3® (Italian form), which was developed by Sigma-Tau Healthscience/Alfasigma. The original formulation was changed in 2016 and there is now a U.S. version known as Visbiome® and an Italian version known as (VSL3®). In CD, the data has remained mixed on the efficacy of probiotics to induce or retain remission as an adjuvant or stand-alone therapy. The mechanism of action possibly includes improving tight junction protein function, positive composition of the intestinal microbiota, and regulating immune-related cytokine expression. In regard to CD, there was one randomized control (RCT) that evaluated the ability of VSL#3 to prevent human recurrence after surgery. This study looked at early and the late administration of VSL#3 and found that early VSL#3 administration was associated with later recurrence after surgery. While there have been no statistical differences in endoscopic recurrence rates at day 90 between patients who received VSL#3 and patients who received placebo. Levels of inflammatory cytokines and recurrence rates leading to repeat surgery were lower among patients who received early VSL#3 (for the entire 365 days). This indicated that this probiotic should be further investigated for prevention of Crohn’s disease recurrence [94, 95].

While it is understood that there may be a potential for probiotics in UC, there is still no convincing data to constitute a recommendation. In a small cohort of pediatric patients with UC, Lactobacillus reuteri was shown to improve clinical and endoscopic disease activity [96, 97]. This has not been replicated in adults. However, in patients with UC who have undergone total proctocolectomy and ileal pouch-anal anastomosis for UC, a definitive connection to gut microbiota has been made. There has been a potential benefit in VSL#3 containing Lactobacillus, Bifidobacterium, and Streptococcus for prevention of the initial episode of acute pouchitis. To date, there have been four clinical trials showing VSL#3 could prevent or maintain remission in patients with chronic pouchitis [98, 99, 100, 101]. A potential mechanism suggested is the improvement of the intestinal barrier function (IBF). While VSL#3 has shown efficacy in chronic pouchitis, an open-label trial showed that most patients on chronic antibiotics for pouchitis were not able to use VSL#3 for long-term therapy largely due to disease recurrence [102]. This formulation has been demonstrated in preventing future episodes and improving inflammation [96]. However, according to the AGA, this only constitutes a weak recommendation due to the small size of the patient population in which these studies were done.

The next generation of probiotics in IBD may involve the use of genetically engineered bacteria that could release therapeutically operative molecules in the intestine. This will involve organisms that could sense and respond to intestinal inflammatory cytokines or topically produce molecules to treat the inflammation. Harnessing the power of the biotherapeutics with synthetic biology could provide a future of personalized medicine in the diverse IBD patient population [103].

5.4 Necrotizing enterocolitis

Preterm birth impacts about 10% of newborns born in the US and 15 million pregnancies worldwide. A preterm infant’s gut is exposed to colonization of commensal and pathological bacteria. During this time, their innate immune system is sorting through a constant excess of peptidoglycans and liposaccharides [104]. In this delicate time, NEC inflammation can be driven by Toll-like receptor 4. By influencing the innate and adaptive immune systems, probiotics are thought to aid in the balance of these two systems and prevent the pathogenesis of NEC [104, 105]. NEC is associated with bowel necrosis leading to short bowel syndrome and impaired development, and can be fatal in up to 30% of patients [55]. There have been case-control studies identifying an overpopulation or so-called “bloom” of Gammaproteobacteria tending to precede NEC in many preterm infants [56, 106]. In contrast, commensal bacteria such as bifidobacterial are found to be protective of NEC and plentiful in breastfed infants likely due to the breast milk-specific oligosaccharides that this preferentially consumes [107].

A Cochrane review article found probiotics were superior to placebo in reducing the risk of severe necrotizing enterocolitis (RR = 0.43; 95% CI, 0.33–0.56; 20 studies with 5529 infants) and mortality (RR = 0.65; 95% CI, 0.52–0.81; 17 studies with 5112 infants) [108]. Combinations of certain probiotics containing Bifidobacterium infantis and L. acidophilus have shown strong association with preventing NEC and reducing need for abdominal surgery and all-cause mortality [109, 110].

There are numerous hypotheses on the mechanism of how they might protect against NEC in infants. One such proposition involves the production of butyrate and other short-chain fatty acids that could supply nutrition to the colonocytes thereby lowering the pH and decreasing the oxygen tension within the intestinal lumen. This ultimately is thought to suppress the growth of Enterobacteriaceae (phylum Proteobacteria), which is well known to be pathologic in NEC [111, 112]. Other proposed mechanisms include supporting the maturation and functions of the infants’ bowels by regulating the Th1:Th2 balance [57, 113]. Specifically, it is known that an imbalance of Th2 levels greater than Th1 levels can predispose to autoimmune disease and gut inflammation by lack of regulation of the gut immune response [57]. According to the AGA, in babies less than 37 weeks of gestational age and low-birth-weight infants, it is recommended to use a combination of probiotics containing Lactobacillus spp. and Bifidobacterium spp. over no probiotics to prevent the development of NEC in this population. This constitutes a conditional recommendation with a moderate to high level of evidence in this population.

5.5 Irritable bowel syndrome

Irritable bowel syndrome (IBS) is classified as a functional gastrointestinal disease [114]. Prevalence rates worldwide are around 11% with impact on younger patients. For this reason, there is a significant economic and sociologic burden associated with this disease. This has amounted to around $20 billion per year in direct and indirect costs to the U.S. Economy [115]. The pathophysiology of IBS involves changes in the gut microbiota, malabsorption of bile acid, and changes to the enteric nervous system. Prior metanalyses have found that probiotics demonstrate improved overall symptom response and pain [116, 117].

One particular strain, Bifidobacterium bifidum MIMBb75, was found in a randomized control study by Guglielmetti et al. to cause a significant reduction in global assessment of IBS by −0.88 points (95% CI: −1.07; −0.69) when compared with only −0.16 (95% CI: −0.32; 0.00) points in the placebo group (P < 0.0001) with excellent tolerability and no difference in adverse events [118]. Andresen et al. replicated this result using a heat-inactivated Bifidobacterium bifidum MIMBb75 (SYN-HI-001) in a high-powered study finding that the beneficial bacterial effects of this strain on IBS were independent of bacteria viability [119].

The metabolites of microbiota often include bile acid (BA), which has been attributed to IBS symptoms. BAs are released in the duodenum after conjugation in the liver, which are then made into secondary BAs by gut bacteria. BAs can have prosecretory effects that can regulate gut motility and impact gut sensitivity [120]. BAs are impacted by bacteria in the gut and impact the gut themselves, thus it is thought they may impact IBS. Patients with IBS have been reported to have changes in their microbial profiles. For example, there has been a significant increase in fecal primary BA and a decrease in secondary BA in patients with IBS-predominant diarrhea. There has also been a direct positive correlation between primary BA and IBS symptoms. In IBS with predominant diarrhea, there has been an observed reduction in bacteria from genera Ruminococcaceae and a negative correlation with primary BAs. There seems to be a definite connection between BAs and IBS, which will need to be further investigated [120].

Overall, the quality of the evidence behind the use in IBS remains weak. Indeed, the ACG states that there is very low evidence for the use of probiotics in IBS, which has resulted in a weak recommendation for their use in IBS. The AGA shares this sentiment and makes no recommendation for the use of probiotics in IBS [121]. This weak recommendation is justified given significant heterogeneity between studies, publication bias, and small sample size studies. This being said, the ACG does acknowledge that when probiotics are studied as a group, they improve bloating and the flatulence in IBS patients [121]. While there has been no broad recommendation for the use of probiotics in IBS. There is evidence that they make a difference and are of continued interest among patients and providers.

5.6 Probiotics in the critically ill

There is growing evidence that probiotics may reduce the rate of the ventilator- associated pneumonia (VAP), overall infection rate, nosocomial pneumonia, duration of mechanical ventilation, and antibiotic use for critically ill patients. VAP is considered the second most common nosocomial infection in the U.S. imposing a significant economic burden. While the American Thoracic Society (ATS) makes recommendations on the prophylaxis of the VAP in patients in the ICU typically involving antibiotics, the prospect of probiotics is compelling [122, 123]. Probiotics have also been used in patients with pancreatitis in the ICU. A meta-analysis analyzing 13 studies with N = 1188 found a statistically significant decrease in the length of ICU stay when probiotics were administered [124]. While no study has been able to find any effect on probiotics and length of hospital stay or mortality, there is convincing evidence that the flora may impact the outcomes of the critically ill patients. Like most areas of probiotics research, more detailed research needs to be done on how specific strains impact specific problems experienced by the patient.

6. Safety of probiotics

Probiotics are often perceived as “natural” and safe alternatives to pharmaceuticals. They are routinely marketed as something which restores or aligns the patient back into a state of health rather than treating a specific disease state. In general, probiotics are considered safe provided the user has a competent immune system. A review by the Agency for Healthcare Research and Quality (AHRQ) looked at 387 studies of which there were 24,615 users and there was no statistically significant increase in the number of adverse events in the probiotic group compared to the control group [125].

Although there have been great strides to incorporate probiotic therapy into modern medicine, researchers have presented concerns about the potential negative effects of probiotic supplementation. Numerous virulent pathways can be expressed and carried out by probiotics that can put the human host at risk as there is a possibility of resistance transfer from the probiotic to pathogenic bacteria. Horizontal gene transfer (HGT) is the movement of genetic code between organisms mediated by transformation, transduction, conjugal transfer, or with specialized gene transfer vehicles such as viruses or other bacteria [126]. Recent literature has suggested the human gut rich in HGT activity and the transfer of genetic code from successfully adapted organisms to recipients provides useful properties resulting in increased fitness and competitiveness in the microbial ecosystem. Examples of HGT among probiotic strains have been documented for Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus reuteri, and Lactobacillus plantarum. Some literature suggests there has been a gene flux from Gram-positive cocci for genes encoding for streptogramin resistance [127]. Tetracycline resistance gene transfer has been reported from L. reuteri to other bacteria native to the human gut microbiota [128].

The use of probiotics in the processed food industry has increased over the years as some byproducts of these organisms are used as additives. One of the most popular microbial-derived additives is transglutaminase. Interestingly, this catalytic enzyme has been implicated in intestinal tight junction permeability and the increasing incidence of autoimmune diseases [129]. This molecule can be detrimental when crosslinked with gliadin as this complex mimics tissue transglutaminase and is immunogenic in patients with celiac disease [130]. In addition to the potential for immunogenicity, numerous case reports have described systemic infections caused by probiotic strains. Fungemia caused by Saccharomyces cerevisiae and Saccharomyces boulardii is by the far the most reported single event associated with the consumption of S. boulardii [131]. Other complications reported include overt sepsis and endocarditis associated with S. bouldarii, Lactobacillus GG, Bacillus subtilis, Bifidobacterium breve, Lactobacillus, and Streptococcus species [132, 133, 134, 135, 136, 137].

The PROPATRIA trial highlighted a concern surrounding probiotic safety in critically ill patients. Researchers explored the ability of multi-strain probiotics to help prevent infectious complications in patients with severe acute pancreatitis. Patients in the experimental arm that received the probiotic were shown to have a much higher mortality rate. The authors of the study suggested that the increase in mortality was associated with bowel ischemia caused by either increased mucosal oxygen demand by the exogenous bacterial metabolic demand in the setting of decreased blood flow or an inflammatory cascade triggered by the probiotic in the setting of decreased capillary blood flow [138]. Other metabolic derangements such as D-lactic acidemia and acidosis in humans have been associated with Lactobacillus and Bifidobacterium species. Interestingly, these species of bacteria are among the most used in probiotic formulations. D-lactic acidosis has been associated with abdominal bloating, chronic fatigue syndrome, and neurocognitive symptoms such as brain fogginess [139, 140, 141, 142]. These findings have also been implicated in the literature surrounding small bowel intestinal overgrowth (SIBO). The resolution of symptoms after antibiotic therapy, in this population, reinforces the proposed causative association [143]. In general, the current medical literature cautions against the use of probiotic supplementation in patients with immunocompromised states such as those undergoing chemotherapy or immunosuppressive therapy, HIV/AIDS, post-organ transplant, pregnancy, neutropenia, antibiotic-induced diarrhea, and inflammatory bowel disease [144, 145, 146].

To date, there is no data on long-term safety of probiotic usage. This makes meaningful safety recommendations on such a diverse array of bacterial strains and dosages within probiotic formulations a nearly impossible task. For this reason, probiotics tend to fall under the unregulated form of other supplements.

7. Future research and directions

As mentioned earlier, probiotics are living nonpathogenic bacteria or yeast that can potentially be beneficial by restoring the microbial balance in the gut; however, only some probiotic products are backed by evidence-based trials [147, 148, 149, 150]. Probiotics have been extensively utilized in numerous disease states, including gastrointestinal diseases, metabolic syndrome, cardiovascular disease, periodontal disease, and osteoporosis [49]. The hallmark of maintaining a healthy intestinal ecosystem is the integrity of the interstitial barrier [151], and probiotics employ their beneficial effects by modulating immunologic response, strengthening gut barrier function and competing with pathogenic bacteria [152]. Numerous in vitro and animal studies have implied the significance of improving the mucosal barrier function by probiotic treatment [153]; however, extrapolating these studies to humans is challenging. For example, some probiotic species, such as Akkermansia muciniphila, VSL#3 encompassing Lactobacillus and Bifidobacteria strains, and L. plantarum Dad-13, have proven benefits of protecting against obesity, dyslipidemia, insulin resistance, and fat mass development in mice [46, 48, 52, 154]; however, this has not been significantly reproduced in human studies. While multiple clinical trials have attempted to evaluate the prophylactic and therapeutic effect of probiotics in different disease states, the quality of evidence to support clinical use of probiotics is poor. In addition, it is unclear which species and their respective optimal quantity and duration are beneficial for specific disease states. Hence, there is no consensus recommendation for its use. More research is warranted exploring the overall safety of probiotic supplementation. In addition, given the laxity in oversight by the Food and Drug Administration (FDA), allergic reactions and anaphylaxis should be a focus of safety as some probiotic blends can include allergens such as cow milk and chicken egg protein [146]. Systematic reviews published within the last 5 years have highlighted concern about the broad generalization of conclusions, lack of structural classification, variations in bacterial strains and dosages, and incomplete reporting of probiotic supplementation regimens and subject population identification [53, 144, 155]. Moving forward, more randomized controlled trials with larger sample sizes would help strengthen current data surrounding the utility of probiotics and aid in identifying any serious deleterious effects on patients’ health.

8. Conclusion

Over the last 20 years, there has been significant basic science, translational, and clinical research into the use of probiotics in the treatment of disease. There is a widespread belief among patients that probiotics preserve a healthy state and even have curative properties. It is widely believed that the beneficial aspects of probiotics involve antagonism against pathogenic molecules, infections, and augmentation of the gut microbiota, thereby maintaining the host’s immune homeostasis. Clinical applications of probiotics are diverse, branching well beyond the gastrointestinal system. While often used as a panacea of sorts by the public, there remains limited evidence of specific species and dosage of efficacy for specific diseases. There is a growing body of research supporting the clinical use of probiotics for applications well beyond gastrointestinal ailments. More research and collaboration among basic science researchers and clinicians to specifically define appropriate usage of probiotics based on the disease targets, dosage, and specific strain deployed.

Conflict of interest

The authors declare no conflict of interest.