Altered Host-Microbe Interaction in HIV: A Target for Intervention with Pro- and Prebiotics

Abstract

The intestinal immune system is severely affected by HIV and circulating microbial products from the intestinal tract that provide an ongoing source of systemic inflammation and concomitant viral replication. In addition, HIV-infected individuals can have a deregulated immune response that may hamper the anti-viral capacity of the host. Various probiotic organisms and prebiotic agents have been shown to enhance intestinal epithelial barrier functions, reduce inflammation, and support effective Th-1 responses. As these characteristics may benefit HIV patients, this review aims to provide a theoretical framework for the development of probiotic and prebiotic interventions specifically for this population.

Keywords :

• HIV
• AIDS
• gastrointestinal tract
• bacterial translocation
• probiotics
• prebiotics

INTESTINAL DEFENSES AND HOMEOSTASIS

The gastrointestinal tract provides a range of habitats for microbes that have either co-evolved with the human species as symbionts or as potential pathogens. The different compartments of the tract host approximately 500 to 1000 bacterial species [1], totaling 10¹³–10¹⁴ cells. Collectively, these organisms represent at least 100 times more genes than the human genome. This complex microbial population influences an estimated 10% of all metabolites in our body [2], and could be regarded as “the neglected organ.” In various eukaryotic species, including humans, the relation between bacterial communities and their host is mutualistic and symbiotic in nature [3]. The symbiotic benefits in humans include energy supply, nutrient metabolism, and prevention of colonization by opportunistic pathogens [4].

In order to benefit from this symbiotic relationship, the immune system has to balance permissive, tolerogenic responses to food antigens and commensal microbes with potentially damaging, inflammatory responses to ward off pathogens. This delicate balance is maintained by the constant interplay between the microbiota, the intestinal barrier, and the mucosal immune system and is a prerequisite for normal gut homeostasis (Figure 1). Imbalance of this system may lead to autoimmune inflammation or infectious pathology.

FIGURE 1  Normal mucosal defenses and homeostasis: The commensal microbiota induces a state of non-responsiveness through interaction with dendritic cells (DC) and subsequent induction of the T-regulatory phenotype and secretion of IL-10 and TGF-β. There is a limited uptake of bacterial antigens, such as lipopolysaccharide (LPS), polysaccharide-A (PSA), and DNA, that induce intestinal defense systems, such as the excretion of β-defensin and secretory IgA.

The first barrier against pathogenic infection and damaging inflammatory responses against commensal bacteria is a degree of physical separation between the intestinal bacteria and the host. Important components of this barrier are intestinal epithelial cells (IEC) that form a physical barrier on the body's largest surface area for interaction with microbes. The epithelium is also home to mucus-producing goblet cells and antimicrobial-peptide-producing Paneth cells [1]. Collectively, these cells produce a mucus layer that selectively limits the contact between bacteria and host cells, a mechanism that is thought to limit damaging inflammatory responses [5].

Despite this physical barrier, sampling and recognition of the intestinal content is a crucial function of the intestinal immune system that is necessary to mount appropriate immune responses. An important mechanism for IEC and immune cells to interact with commensal and pathogenic bacteria is the recognition of microbe-associated molecular patterns by germline-encoded pattern recognition receptors (PRR). The best described PRR are Toll-like receptors (TLR), which have been found on a wide range of cell types. The TLR detect various conserved microbial structures, such as lipoteichoic acid (TLR-2), lipopolysaccharide (LPS) (TLR-4), flagellin (TLR-5), and CpG DNA (TLR-9) (reviewed in [6]). Interaction of commensal microbes with TLRs appears to be essential for IEC integrity [7]. Other important PRR groups include the sugar-binding lectins and NOD protein families. NOD proteins are located in the cytoplasm of IEC and are activated upon invasion. While NOD-1 is located within all IEC, another variant, NOD-2, is only expressed in Paneth cells and plays a role in the synthesis of cryptidin and defensin [8].

Specialized structures for sampling intestinal content are present in the gut-associated lymphoid tissues (GALT). These include Peyer's patches (PP) in the small intestine and lymphoid follicles in the colon, which are covered by follicle-associated epithelium containing non-mucus producing microfold (M-) cells. These cells are devoid of microvilli and are specialized in antigen transport into the PP, where the antigens are taken up by antigen presenting cells (APC) [9]. Dendritic cells (DC), the main APCs in PP, interact with T- and B-lymphocytes to induce suitable adaptive immune responses, depending on the type of stimulus. Therefore, PPs are major inductive sites of mucosal adaptive immune responses (reviewed in [10]). After activation, lymphocytes home to the lamina propria (LP) or intestinal epithelium to perform effector functions. The heterogeneous population of intestinal intra-epithelial lymphocytes (iIEL) is thought to regulate the intestinal epithelial barrier integrity and regeneration, and reduce damage due to local immune responses [11]. Furthermore, the LP contains DCs that can sample luminal content by extending dendrites through the intracellular epithelial tight junctions, providing a mechanism to sample intestinal content outside of PP as well [12]. DCs are central regulators of adaptive immune responses, initiating either effector responses or inducing tolerance. The many different DC subpopulations that are present within the mucosal immune system each have different functional characteristics (reviewed in [13]).

In the absence of inflammatory signals, commensal microorganisms induce tolerogenic maturation of DCs, leading to the induction of various types of regulatory T-cells (Treg), including CD4⁺CD25⁺Foxp3⁺ lymphocytes [14], or hyporesponsive T-cells [15]. The maintenance of the Treg population are dependent on IL-2, IL-10, and TGF-β levels, which in turn are dependent on continuous background activation by commensal micro-organisms [16]. In addition, intestinal DCs are potent inducers of IgA synthesis in B cells, which has anti-pathogenic effects but also prevents commensal bacteria from penetrating the host [17]. IgA accounts for >70% of our body's total immunoglobulin production. Several grams of secretory IgA (sIgA) are secreted in the intestinal lumen daily, which exerts considerable immunological pressure on the intestinal microbiota [18].

Reciprocally, the intestinal bacteria have a major influence on the immune system as well. Studies comparing germ-free mice with microbially-colonized mice have shown that the presence of microbes is crucial for the normal development of GALT as well as other secondary immune organs. In the GALT, the absence of bacteria leads to a multitude of effects, including limited development of Peyer's patches, reduced B-cell activation and IgA production, and reduced numbers of iIEL. Moreover, epithelial function is also affected, as evidenced by reductions in IEC turnover and changes in mucus production [19].

The effects of commensal bacteria on the immune system are dualistic in nature. On the one hand, mechanisms are induced that maintain tolerance and/or prevent inflammation. This includes IgA production, β-defensin production in the epithelium [20, 21], enhanced epithelial barrier integrity through TLR signaling [7], Treg induction and even immunosuppressive effects [22–24]. On the other hand, exposure to commensal bacteria induces the expansion of inflammatory lymphocyte populations, including cytotoxic iIEL and IL-17-producing CD4⁺ T-helper (Th17) cells [25, 26]. Th17 and other IL-17 producing cells have been implicated in many inflammatory and autoimmune conditions [27–29], however, they have also been shown to play important roles in protective mucosal responses against extra-cellular bacteria and fungi (reviewed in [30]). It is interesting to note that the induction of Treg and Th17 populations share a dependency on TGF-β signaling. Furthermore, both populations are relatively abundant in the LP. Therefore, it is thought that both types of T-cells are induced by signals from the intestinal bacteria and the balance between these opposing cell types is determined by the specific host-microbe interactions [31].

Commensal bacteria are able to influence the mucosal immune system, not only through cell-cell interactions, but also through the secretion of immune-modulatory molecules. These include: adenosine triphosphate (ATP), which enhances the polarization of Th-17 T-lymphocytes [32]; polysaccharide A (PSA), which induces maturation of Th-17 cell populations [33]; and DNA, which induces IFN-α syntheses and favors IEC integrity [34]. Furthermore, the intestinal epithelium is also constantly exposed to inflammatory molecules, such as LPS and peptidoglycans. Despite this continuous exposure, the intestinal immune system is unique in its ability to maintain tolerance in the presence of a multitude of immune triggers while minimizing the risk of systemic infection.

In summary, the net effects of the interplay between the commensal microbiota and the mucosal immune system are enhanced mucosal defense mechanisms, balanced by an inhibition of potentially damaging, inflammatory immune responses. In the absence of pathogenic stimuli, virulence factors or chronic inflammation, the host-microbe interaction leads to a predominance of tolerogenic mechanisms and intestinal homeostasis.

THE RESULT OF HIV INFECTION ON THE HOST-MICROBE INTERFACE

HIV infection has a disruptive impact on the physiological interplay between the commensal microbiota and immune system: CD4⁺ cells associated with the mucosal immune system are rapidly depleted after HIV infection [35, 36], including reduced numbers of DCs [37], a change in the composition of iIEL [38], and depletion and anergy of gamma-delta T-lymphocytes [39]. These detrimental changes on the mucosal immune system have severe consequences for the (immunological) function of the intestine and are associated with compromised epithelial repair mechanisms and enhanced epithelial permeability [40, 41]. The net result is an increased risk of gastrointestinal infections at all stages of HIV infection [42] and a high prevalence of gastrointestinal disorders with unknown etiology [43].

Chronic immune activation and inflammation have long been described as characteristic features of progressive HIV disease, while the source of inflammation has remained unidentified. Indeed, increased B-cell activation, increased T-cell turnover, and increased pro-inflammatory cytokines are observed with HIV infection. In this pro-inflammatory state, the replication of HIV is markedly enhanced [44] and activation of the nuclear factor (NF) κB transcription factor plays a crucial role in this phenomenon [45]. Strikingly, the degree of systemic immune activation, indicated by the expression of the immune activation marker CD38 on CD8⁺ cells, is a better predictor of HIV progression than viral load or CD4⁺ count alone [46]. Recently, it has been suggested that the gut might be a source of chronic inflammation. The hypothesis is that dysfunction of the mucosal immune response due to preferential depletion of intestinal mucosal immune cells, including effector CD4⁺ cells and DCs [36, 37, 47], may affect systemic immune activation through the increased translocation of microbes and bacterial products from the intestinal tract [48]. The resultant pro-inflammatory environment [40] may then cause further damage to the gut barrier function, augmenting bacterial translocation and subsequently fueling systemic inflammation. Indeed, evidence suggests that bacterial translocation affects the activation state of the immune system, and in turn HIV progression (Table 1).

Some HIV-infected individuals, termed “non-progressors” have a low HIV viral load even without treatment and maintain a low degree of systemic inflammation [49, 50]. A mechanism that appears to contribute to the control of the virus is a capacity to maintain the integrity of the gut barrier and to mount an attenuated response to bacterial products and, thus, potentially reduce bacterial translocation [40, 51]. In non-progressors, serum LPS has been shown to be lower than those with progressive HIV infection [48]. One week of treatment with a “gut sterilizing” antibiotic regimen markedly reduced serum LPS levels in macaques, concomitant with a reduction of fecal Gram-negative bacteria and inflammatory markers. However, after two weeks of antibiotics, plasma LPS had increased again, apparently due to the growth of other bacterial species in the gut [48]. Although anti-retroviral treatment (ART) has been shown to enhance epithelial barrier functions [52], the efficiency of CD4⁺ recovery may still be compromised by bacterial translocation [53]. Future studies will need to focus on the role of the epithelial barrier and the microbiota composition along with translocation in the progression of HIV.

TABLE 1  Evidence Implicating the Intestinal Microbiota and Epithelial Cell Barrier as a Factor in HIV Progression

• Aberrant intestinal microbiota among HIV patients with fewer lactobacilli and bifidobacteria, and higher numbers of pathogenic P. aeruginosa, and C. albicans present [62, 63]

• Antibiotics can temporarily reduce bacterial translocation in SIV infected macaques [48]

• Increased bacterial translocation among HIV progressors and those not effectively responding to ARV [48, 53]

• Enhanced epithelial inflammation and scarring of GALT among HIV progressors [40]

• Increased epithelial permeability with HIV at all stages of infection [41]

The intestinal microbiota has been shown to play important roles in other disease conditions. For example, bacterial translocation occurs during surgery [54], and plays a role in alcohol-induced liver cirrhosis [55], in exacerbation of graft versus host disease [56] and in inflammatory bowel disease (IBD) [57, 58]. Furthermore, correlations between the microbiota composition and disease have been shown for IBD [59] and obesity [60]. Strikingly, the transplantation of gut microbes from obese mice (ob-/ob-) to bacteria-free mice resulted in obesity in the recipients [61], suggesting that the microbiota may be a mediator of specific conditions. Lessons from such studies that focus on microbiota-disease interactions may help to provide more insight in the role of an aberrant microbiota in HIV-infected subjects.

The intestinal microbiota of HIV patients appears to contain higher levels of pathogens, such as Pseudomonas aeruginosa and Candida albicans [62], and reduced or undetectable levels of Bifidobacterium and Lactobacillus species [63]. In the macaque model, colitis is very common after simian immunodeficiency virus (SIV) infection, resulting in a reduced microbial diversity and an increased proportion of Campylobacteriaceae [64]. If the aberrant microbiota among HIV patients more easily translocates and provides an inflammatory stimulus for HIV replication, therapeutic modification of the gut microbiota might have a beneficial impact on HIV progression.

Several lines of evidence suggest that HIV modulates systemic immunity by skewing the Th1/Th2 balance towards Th2 responses. Recently, it was suggested that the induction of T helper-2 (Th-2) cytokine synthesis [65, 66] and the gradual increase of IL-4 and IgE [67, 68] observed after HIV infection, might be due to a Th-2 response to viral proteins, such as gp-120, p24, and p17 [69]. It is well known that HIV patients suffer from high rates of allergies [70] and the ability of subgroups of HIV patients to maintain a vigorous Th-1 response and higher levels of IFN-γ is associated with increased survival [71, 72]. An HIV-triggered Th-2-skewed state of the immune system could compromise immunological control of HIV replication and lead to reduced protection to opportunistic infections. This immune imbalance may also aggravate inflammation and barrier dysfunction in the gut, as the increase in IL-4 production can compromise the antimicrobial function of Th-17 cells [73] that line the intestine.

PROBIOTICS

Probiotics, defined as “live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host” [74], have been studied in a myriad of conditions related to intestinal dysbiosis, including IBD, infectious diarrhea, allergy, and surgery. Relevant research from these fields and studies on probiotic interventions among people living with HIV and safety considerations are now discussed.

Effects on Gut Barrier Function

Supplementation of probiotic strains may enhance or restore the beneficial interactions between the commensal enteric flora and the host in both healthy and disease conditions, leading to an enhanced barrier function and reduced bacterial translocation (Figure 2). Effects have been described in animal and human studies that may have relevance for HIV-infected subjects. For example, the Gram-negative probiotic strain Escherichia coli Nissle 1917 was shown to enhance the intestinal epithelial integrity via the induction of epithelial tight junctions proteins (ZO-1 and ZO-2) [75, 76]. Prior administration of candidate probiotic strains Lactobacillus acidophilus (ATCC19258) and Streptococcus thermophilus (ATCC4356) to IEC has been shown to reduce the epithelial permeability induced by TNF-α and IFN-γ, suggesting that the impact of inflammation can be reduced [77]. It appears that these various protective mechanisms occur through the production of as yet uncharacterized proteins [78] or through direct interaction of microbes with IEC through TLR-4, TLR-5, and TLR-9 [34]. That the enhancement of the epithelial barrier might also translate to a reduction in bacterial translocation was shown in a murine model of enterohemorrhagic shock. In this model, prior challenge with L. rhamnosus LMG P-22799 reduced bacterial translocation and systemic inflammation [79], as did use of Bifidobacterium adolescentis in a murine model of burn wounds [80]. Furthermore, allergy-associated intestinal hyper-permeability [81] as well as alcohol-induced loss of gut barrier function have been found to be reversed by application of L. rhamnosus GG, resulting in less intestinal and liver inflammation in the latter [82].

FIGURE 2  Potential benefits of probiotics and prebiotics in HIV-induced intestinal pathogenesis: HIV infection induces effects and positive feedback mechanisms that induce a loss of intestinal homeostasis and promote replication of the virus (triangles). Pro- and prebiotics may ameliorate the HIV-induced intestinal problems through effects on the microbiota and its metabolism, on various cells of the immune system (as represented by the arrow pointing at the sampling DC), and on intestinal epithelial cells.

In human clinical studies, probiotics have been applied to reduce bacterial translocation among different patient populations with varying degrees of success. A randomized controlled trial (RCT) among 65 critically-ill patients showed reduced rates of infections, sepsis, and mortality with a combination of probiotics and prebiotics (Synbiotic 2000 Forte) [83]. In consecutive trials, the same product was shown to increase fecal IgA [84], reduce the incidence of bacteremia [85], and lower the rate of post-operative infections [86]. Two other RCTs using different synbiotic preparations showed similar results. L. casei Shirota and L. breve Yakult given with galacto-oligosaccharides (GOS) supplementation (15 gr/day) before biliary cancer surgery, resulted in reduced inflammatory markers and post-operative complications [87]. Another RCT with L. acidophilus La-5 and B. lactis Bb-12 combined with oligofructose (15 gr/day) resulted in a reduced incidence of bacterial translocation [88]. However, the evidence is not conclusive as three other RCTs using L. acidophilus La-5 and B. lactis Bb-12 [89, 90] or L. plantarum 299v [91] reported no reduction in bacterial translocation. This suggests that there may be strain-specific effects or that prebiotics are needed for efficacy.

Effects on Mucosal and Systemic Immunity

The loss of tolerance in the intestine due to a defect in GALT homeostasis can have a detrimental impact on the gut barrier function, and specific probiotic strains have been shown to enhance the recovery of GALT homeostasis. For example, administration of Escherichia coli Nissle 1917 in wild type mice, but not in TLR-2 knock-out mice, ameliorated experimental colitis and reduced pro-inflammatory cytokine expression, suggesting a TLR-2-dependent pathway [92]. This response can be mediated by specific components of probiotic organisms. DNA from probiotic organisms modulates TLR-9 and elicits a different response from immune and epithelial cells than DNA from pathogenic organisms [93]. In HT-29 cells subjected to pro-inflammatory cytokines, challenge with DNA from the VSL#3 probiotic mixture could reduce the expression of the pro-inflammatory IL-8 cytokine and delayed NF-κB activation [94]. In IL-10 deficient mice, the administration of the probiotic mixture VSL#3 led to a reduction in mucosal TNF-α and IFN-γ release and improved the histological disease in a TLR-9 dependent manner [94].

Another way of restoring GALT homeostasis is through the induction of regulatory mechanisms to down-regulate inflammation. Induction of regulatory mechanisms by specific probiotics appears to be partly dependent on modulation of DCs [95]. Some probiotic organisms induce regulatory T-cells (CD4⁺FoxP3⁺), mediated by DCs [16, 96]. This induces tolerance that is partly mediated through IL-10 and TGF-β production. Recently, it was demonstrated that ingestion of a specific probiotic mixture (IRT-5) could induce CD4⁺FoxP3⁺ cells in mesenteric lymph nodes. Interestingly, probiotics alone, without the presence of DCs, could not induce this effect. Administration of the probiotic mixture also induced both T-cell and B-cell hypo responsiveness, down-regulated both Th-1 and Th-2 functions, and reduced the secretion of pro-inflammatory cytokines in GALT [97]. The biological relevance of these changes was verified in an IBD model in which administration of probiotics was shown to enhance GALT homeostasis and reduce the severity of the disease. The therapeutic effects were associated with an enrichment of CD4⁺Foxp3⁺ T-cells in inflamed regions [97]. O’Mahony et al. demonstrated that challenge with Lactobacillus salivarius or Bifidobacterium infantis of DCs from mesenteric lymph nodes induced secretion of IL-10. This was in contrast to a challenge with Salmonella strains, which induced the secretion of pro-inflammatory IL-12. Strikingly, DCs from peripheral blood did not show a differential response to lactobacilli or Salmonella strains [98], suggesting that the response of DCs depends on their immunological compartment. In the intestine, tolerogenic effects of T-regulatory cells and anti-inflammatory effects may improve barrier function and intestinal homeostasis. Furthermore, a reduction in the chronic inflammatory state may reduce immune activation and potentially affect disease progression [48].

Systemically, specific probiotics have also been shown to induce a T-regulatory phenotype and counter-balance a Th-1 or Th-2 dominant state in vivo and in vitro [95, 99, 100]. The induction of a T-regulatory phenotype frequently occurred together with increased levels of anti-inflammatory IL-10 [101, 102]. A prime example of the clinical effects of IL-10 induction comes from an RCT of 77 adults with an abnormal IL-10/IL-12 ratio and concurrent IBS. B. infantis 35624 was shown to normalize the IL-10/IL-12 ratio in parallel with a reduction in clinical symptoms [103]. In Crohn's disease, a Th-1 mediated condition, a strain of L. rhamnosus was able to decrease both the syntheses of the Th-1 parameter IFN-γ, and IL-2, which is an essential survival and proliferation factor for effector T-cells [104]. Remarkably, IL-4, a potent Th-2 cytokine, was also reduced with probiotic supplementation. Hence, the effects of probiotic supplementation were unlikely due to mere Th-1/Th-2 skewing and are best explained by the induction of a regulatory DC phenotype with the ability to induce a general hypo-responsiveness. In a study of children with atopic dermatitis, a Th-2-dominant condition, an up-regulation of IL-10 was noted after supplementation with L. rhamnosus GG [105]. These findings might be valuable in relation to HIV management, as both the induction of T-regulatory cells and anti-inflammatory effects can potentially be of benefit to HIV patients.

In vitro evidence indicates that several probiotic candidate strains can down-regulate the production of Th-2 cytokines and chemokines [106, 107] and modulate DCs to skew T-cell polarization toward a Th1-response [108]. The ability of probiotics to skew the immune system away from a Th-2 dominant state has been studied to some extent in humans, albeit in HIV uninfected subjects. An RCT of 230 children with a cow's milk allergy showed that L. rhamnosus GG ingestion could reduce symptoms of atopic eczema and dermatitis [109] and up-regulate IFN-γ, indicative of a more pronounced Th-1 response [110, 111]. Moreover, L. rhamnosus GG was shown to up-regulate IL-6, involved in the mucosal response to stress [112] but also epithelial IgA production and mucosal protein syntheses [113], suggesting a direct effect of L. rhamnosus GG on IECs. Two other RCTs have shown similar results [114, 115] but one study in children did not confirm this outcome [116]. In addition to the ability of probiotics to improve barrier function and aspects of intestinal homeostasis, specific probiotic strains may, therefore, be able to skew away from an HIV-induced Th-2 predominance.

Effects on Intestinal Microbiota and Infections

Probiotics can interfere with the function and proliferation of pathogens in the gastrointestinal tract in various ways. They can enhance the secretion of pathogen-specific IgA [117], induce β-defensin secretion [118] or secrete bactericidal proteins [119], and reduce the adhesion and invasion of pathogens [120, 121]. Antibiotic-like compounds, such as reuterin produced by L. reuteri, exhibit broad spectrum effects against Gram-positive, Gram-negative bacteria as well as fungi, yeast, and protozoa [119], while non-reuterin producing strains, such as L. reuteri RC-14, produce signaling molecules that down-regulate Staphylococcus aureus toxin production [122]. These characteristics could be beneficial for acquired immune deficiency syndrome (AIDS) as L. reuteri was shown to prevent cryptosporidiosis in a murine AIDS model [123].

Use of probiotics can lead to at least temporary modification of the intestinal microbiota. For example, in an RCT of 69 preterm babies, B. lactis Bb-12 significantly increased the levels of bifidobacteria and lactobacilli while reducing the numbers of enterobacteria and clostridia [124]. In 36 adults receiving triple therapy for Helicobacter pylori infection, the addition of L. acidophilus CUL60 and CUL21 and Bifidobacterium spp. decreased the intestinal load of C. albicans, facultative anaerobes, and enterobacteria [125]. Moreover, genomic and metabolic studies suggest that probiotic microbes change the behavior of the intestinal microbiota [126].

The application of probiotics for the prevention and treatment of gastrointestinal infections has been well established and might be especially useful among people living with HIV. A Cochrane review concluded that probiotics are a useful adjunct to lower the occurrence and reduce the length of episodes of infectious diarrhea (reviewed in [127]). Synbiotics (probiotics combined with prebiotics) have also been shown to reduce diarrhea associated with ART use [128] but these findings could not be confirmed by a cross-over study [129]. Although the application of probiotics to prevent gastrointestinal infections and the concomitant inflammatory state among HIV patients bears promise, no studies have so far been conducted to assess its potential.

Probiotic Interventions in HIV

A limited number of studies suggest that the probiotic benefits could be translated to people living with HIV. One RCT of 77 children in Brazil showed an increase of 118 CD4⁺ cells/μl among those receiving B. bifidum and S. thermophilus for two months compared to a decrease of 42 CD4⁺ cells/μl among the placebo group (p = 0.05) [130]. An RCT of 24 HIV patients in Nigeria showed after four weeks of L. rhamnosus GR-1 and L. reuteri RC-14 an increase of 6.7 CD4⁺ cells/μl compared to a decrease of 2.2 CD4⁺ cells/μl among the placebo group (p < 0.05) [131]. A large RCT in Malawi (n = 795) testing the effect of Synbiotic 2000 Forte on malnutrition also included a proportion of HIV-infected children (n = 361) [132]. Although there was no improvement in nutritional cure, there was an overall reduction in outpatient mortality, including a trend towards a reduced mortality among the subgroup of HIV-infected children.

Safety

Among HIV patients, several studies have been conducted to assess the safety of probiotic interventions. When treated with L. reuteri SD2112, no safety concerns arose among moderately immune-compromised HIV patients (>350 CD4⁺ cells/μl) [63]. In another study of severe immune compromised HIV patients (<200 CD4⁺ cells/μl), no safety concerns were detected with use of L. rhamnosus [129]. To date, five case studies of lactobacillemia have been reported in end-stage AIDS patients. Of these, three patients were reported to have central venous catheters and one patient to have pneumonia, all of whom had extremely low CD4 counts (<55 CD4⁺ cells/μl) [133–135]. Currently, no indication exists to avoid oral probiotic use in HIV populations, but close monitoring of safety parameters is recommended.

PREBIOTICS

Altered Microbe-Host Interaction

Prebiotics were defined most recently as “a non-viable food component that confers a health benefit on the host associated with modulation of the microbiota” [136], although the term is often used less strictly for components that modify the composition or metabolism of the intestinal microbiota. Prebiotics can modify host-microbe interactions via the microbiota and its metabolism, host epithelial and other cells, as well as by modifying receptor expression and bacterial adhesion. As alluded to earlier, prebiotics are candidate agents to improve the intestinal homeostasis in HIV-infected individuals. Since prebiotics do not contain bacteria but provide substrate for the intestinal microbiota, their fermentation depends on the organisms present in the host. Prebiotic fructans and galacto-oligosaccharides (GOS) increase the percentage of “beneficial bacteria” through selective fermentation as shown in a variety of human target groups, including infants [137, 138], healthy volunteers [139], and seniors [140]. The fermentation of fructans and GOS increases the production of short-chain fatty acids (SCFA), lactate, and other bacterial metabolites [141, 142]. SCFA are known to have a plethora of effects on the intestinal milieu, epithelial cells as well as local immune cells. Various prebiotics induce differential effects on SCFA production and the ratios of butyrate, acetate, and propionate [143]. The degree of specificity of prebiotic agents enables the potential development of specific prebiotics optimized to target HIV-specific issues. Relevant potential benefits for HIV patients are discussed in the next sections, focusing both on the effects of prebiotic intervention and on the effects of purified bacterial metabolites, such as SCFA.

Effects on Barrier Function

As described previously, decreased barrier function and increased bacterial translocation is observed in HIV-infected subjects. Prebiotics have been shown to influence barrier function via various mechanisms. A combination of fermentable fibers has been shown to significantly reduce endotoxemia over a 30-day intervention period in an RCT of 55 cirrhosis patients [144]. Animal studies using alcohol-induced liver damage showed similar beneficial results preventing intestinal dysbiosis with an oat-based prebiotic intervention [145].

There appear to be several mechanisms whereby prebiotics can enhance intestinal barrier function. Recently, Cani and coworkers [146] have shown that a prebiotic-induced increase in glucagon-like peptide-2 (GLP-2) played an important role in the beneficial effects of prebiotic intervention in ob/ob mice on a high-fat diet. Increased intestinal barrier function and expression of tight-junction proteins were observed, leading to reductions of hepatic markers of oxidative stress and inflammation, as well as reduced levels of systemic inflammatory mediators and endotoxemia [146]. The production of SCFA during fermentation of prebiotic agents can also lead to an improved barrier function. Butyrate, in particular, is an energy source for intestinal epithelial cells and, through the modulation of intestinal prostaglandins, it stimulates mucus production [147]. Recently, butyrate was shown to enhance intestinal barrier function in vitro by regulating the assembly of tight junctions in Caco-2 cells [148].

Effects on Gastrointestinal Infections

A few human studies have shown that prebiotics can reduce gastrointestinal infections, a functional characteristic that may potentially be used to counteract the HIV-increased prevalence of gastrointestinal infections [42]. Fructan supplementation for 30 days reduced diarrhea relapse rates in an RCT among 140 patients with Clostridium difficile-associated diarrhea [149]. In addition, fructan supplementation showed a tendency to reduce traveler's diarrhea in an RCT of 244 healthy volunteers [150]. In a 12-month open-label intervention trial among 342 infants, a formula containing a specific combination of GOS and a long-chain fructan induced significant reductions in gastroenteritis and acute diarrhea [151]. The same combination of oligosaccharides was shown to reduce the number of fecal pathogens and increase intestinal IgA production in infants—two mechanisms by which prebiotics could reduce intestinal infections [152, 153].

SCFAs can contribute through acidification of the intestinal content and growth inhibition of acid-sensitive pathogens [154]. Butyrate stimulates the production of antimicrobial peptides, such as cathelicidins, which are able to kill a variety of potential pathogenic bacteria [155].

Prebiotic oligosaccharides can have anti-pathogenic effects that are independent of the intestinal microbiota and its metabolism. Human milk oligosaccharides are known to exhibit receptor-decoy functionality based on the molecular structure and sugar moieties of the oligosaccharides [156], leading to binding of oligosaccharides to potential pathogens and preventing their adherence to the intestinal lining. Similarly, GOS and pectin-derived oligosaccharides can inhibit adherence of specific pathogens to epithelial cells in vitro, demonstrating that this mechanism is not limited to oligosaccharides of mammalian origin [157, 158]. Studies on HIV-infected adults and infants are required to better determine the efficacy of prebiotics against diarrhea, especially in developing countries where such infections can be lethal.

Local Anti-Inflammatory and Immunomodulatory Effects

IBD is characterized by intestinal inflammation in which the microbiota plays an important role, a situation not dissimilar to the HIV-induced inflammation in the gut. Small-scale studies using fructan-based pre- and synbiotic intervention in ulcerative colitis patients have shown beneficial effects on histological inflammation scores, and on mRNA expression of inflammatory mediators in biopsy samples [159, 160]. Similarly, a small, open-label study showed anti-inflammatory effects of fructan supplementation in moderate Crohn's disease patients. In that case, lamina propria biopsies showed that the intervention modulated the phenotype of intestinal DCs, enhancing IL-10 production and expression of TLR-2 and TLR-4 [161]. This suggested that the anti-inflammatory effects are related to changes in microbe-host interactions.

Many preclinical data using prebiotics in IBD models show corresponding results (reviewed in [162]). In mechanistic studies using different chemically-induced inflammation models, it was shown that the beneficial effects of prebiotic intervention could be reproduced in part or completely by infusing lactic acid bacteria intragastrically and/or SCFA into distal parts of the large intestine.

Recent animal studies confirm that the expression of PRRs in epithelial and immune cells can be modified by prebiotics and by butyrate in vitro [163, 164]. However, the molecular mechanisms remain to be elucidated. Whereas butyrate was found to reduce LPS and TNF-α-induced NF-κB activation in a colonic epithelial cell line [165, 166], these results were partly contradicted in a different colonic cell line [163]. A recent study indicated that NF-κB may be modulated directly by unfermented oligosaccharides. Pectin-derived acidic oligosaccharides reduced NF-κB in vitro and reduced HIV-1 viral production in vitro [167].

Another molecular target for SCFA-induced modulation of inflammation are the G-protein-coupled receptor 41 and 43 (GPR-43), that are most efficiently activated by acetate and propionate [168–170]. GPR-43 is expressed mainly in innate immune cells and is critically important in the resolution or reduction of inflammation in a variety of mouse models. These mechanisms highlight the potential of prebiotic anti-inflammatory capacities, which could lead to an amelioration of chronic inflammation and possibly immune activation in HIV-infected subjects [48].

Modulation of Systemic Immunity

In addition to local effects of prebiotics in the gut, systemic immunomodulatory effects of prebiotics have been described that are relevant for HIV-infected individuals. A 10-week cross-over study with GOS in 44 healthy, elderly subjects showed simultaneous bifidogenic and systemic immunomodulatory effects. The phagocytosis capacity and natural killer cell activity of circulating white blood cells was increased, whereas the production of inflammatory cytokines was reduced [140]. Furthermore, specific prebiotic interventions have been shown to modulate the immunological balance, consistent with a shift away from a Th2-dominant state. For example, a specific combination of GOS and short-chain fructans was shown to reduce the incidence of atopic dermatitis and allergy-related symptoms in infants at risk for allergy [171]. Correspondingly, changes in the antibody class and isotype ratios suggestive of a Th1 shift were detected [172]. The ability to induce this shift is thought to be beneficial in HIV patients, as it might result in more effective anti-viral control and a better immunological defense against opportunistic pathogens [69, 173].

Effects of the specific combination of GOS and long-chain fructans in multiple mouse models are also consistent with a shift from Th2 to Th1 responses, as allergic responses were reduced and Th1-dependent vaccine-specific DTH responses were enhanced [174–176]. In addition to modifying the microbiota, prebiotics may also mediate effects via the carbohydrate structures on immune cells. Very low-level systemic bioavailability of short-chain fructans have been described in the urine of healthy volunteers [177], suggesting the potential for direct systemic effects through interactions with lectins, galectins, or other sugar-binding molecules.

Prebiotic Intervention in HIV-Infected Individuals

A limited number of studies that have used prebiotics in HIV-infected individuals indicate that the benefits described above may be relevant for this population. Recently, a prebiotic intervention study was performed to investigate potential microbiological and immunological benefits among 57 HIV patients. A 12-week intervention with a specific mixture of GOS, long-chain fructans and pectin-derived oligosaccharides in ART-naïve HIV-1 infected individuals resulted in increased bifidobacterial levels and reduced numbers of the pathogenic Clostridium histolyticum cluster. In addition, reduced levels in the pathogenic E. rectale and C. coccoides cluster were found [178]. The prebiotic intervention was associated with reduced CD4⁺ T-cell activation, measured as a percentage of CD4⁺/CD25⁺ T-cells. In addition, improved NK-cell cytotoxicity was observed [179]. The beneficial effects of this pilot study were confirmed in a multi-center RCT, in which a one-year intervention was tested in HIV-infected individuals not on ART. A total of 340 participants were included in the trial and received the intervention product or an isocaloric, isonitrogenous control product. The intervention product consisted of the same mixture of prebiotic oligosaccharides in combination with bovine colostrum, omega-3 polyunsaturated fatty acids, and N-acetyl cysteine. The intervention significantly slowed down the decline of the CD4 count as the decrease in the intervention group (-28 cells/μl) was lower than the decrease in the control group (-68 cells/μl) [180]. These findings are very promising and show the potential for nutrition-based strategies to become an integral part of disease management.

CONCLUSION AND FUTURE PROSPECTS

The interaction between the gastrointestinal microbiota and the human host plays a crucial role in intestinal homeostasis and the health status of the host. The gut-associated immune system tightly regulates this interaction and, under normal conditions, prevents damaging inflammatory reactions by maintaining a tolerogenic state. HIV infection has a disruptive impact on the intestinal homeostasis as it directly affects the host and indirectly affects the intestinal microbiota. The loss of intestinal CD4⁺ T-cells, epithelial function, and immune regulation, in combination with a pathogen-enriched microbiota composition, leads to an increase in intestinal permeability, bacterial translocation, and an inflammatory state.

Pro- and prebiotics are modulators of both microbiota and host factors, making them potential agents to ameliorate the intestinal problems induced by HIV. Various beneficial effects of pro- and prebiotic interventions have been demonstrated that may translate to applications in HIV. These effects include improved barrier function, reductions in the translocation of bacterial products, reductions in pathogenic load, local and systemic anti-inflammatory effects, and immunomodulatory effects to restore a proper Th1/Th2 balance.

Various therapeutic applications of pro- and prebiotics are conceivable, such as before initiation of ART, where they could potentially help reduce HIV-induced intestinal inflammation, intestinal infection, or diarrhea. In addition, by reducing systemic inflammation and associated immune activation, disease progression may be slowed down. Obviously, pro- and prebiotics should not be used as alternatives to ART but might have a role as conjoint therapy especially among immunological non-responders to ART.

An additional benefit of the pro- and prebiotic is the possible application in low-cost interventions of limited complexity, which might be especially useful in resource-limited countries [181]. Depending on the target group, the oral application of pro- and prebiotics makes it possible to combine the intervention with specific (micro-) nutrients to prevent specific deficiencies or to aim at multiple targets simultaneously. Applied as interventions to improve intestinal homeostasis in HIV-infected individuals, pro- and prebiotics have potential to contribute beneficially to integrated disease management.

In recent years, promising initial studies encompassing pro- and prebiotic interventions have been performed in HIV-infected individuals, showing the potential for improvement of intestinal homeostasis and potentially a reduction in the decline in CD4 count. However, a clear need remains for additional well-designed double-blind, randomized studies to provide evidence for the efficacy of specific pro- and prebiotic interventions. Figures 1 and 2, Table 1

ACKNOWLEDGMENTS

We are grateful for the careful review of our manuscript and knowledgeable feedback of Prof. Dr. Johan Garssen, Jaimie Hemsworth, and Wayne Miller. Support from NSERC and AFMnet is gratefully appreciated.

Declaration of Interest

As indicated in the affiliations, APV, BL, and KN are employed by Danone Research—Centre for Specialised Nutrition, which is part of the Danone group.