During the course of HIV infection, the epithelial barrier of the gastrointestinal (GI) tract is damaged, and the deteriorated gut-associated lymphoid tissue leads to massive loss of intestinal T-CD4+ cells and an anatomical and functional alteration of the intestinal epithelium. This results in increased intestinal permeability1–3 and leads to leakage into the bloodstream of intestinal bacteria and bacterial products such as lipopolysaccharide (LPS), a component of Gram-negative bacterial cell membranes. This induces the production of molecules from the host innate immune system in response to bacterial products; for example, LPS-stimulated hepatocytes release lipopolysaccharide-binding protein (LBP).4,5
It has been demonstrated that microbial translocation in the gut can lead to chronic immune activation and also to disease progression.1,6–8 This immune hyperactivation or chronic inflammation generates a steady release of proinflammatory cytokines and procoagulant substances. These mediators persist throughout the chronic phase of HIV infection, are intrinsically related to the increased cardiovascular risk and progression of atherosclerosis, non-AIDS defining malignancies, early senescence, and neurocognitive deficits, and even accelerate the progression of hepatitis C virus infection.9–12 A recently published study showed that microbial translocation is also associated with the highest prevalence of HIV-associated lymphomas in virologically suppressed patients.13 Likewise, microbial translocation is associated with sustained failure in T-CD4+ cell reconstitution and contributes to the pathogenesis of discordance,14,15 including in infected patients on long-term highly active antiretroviral therapy (HAART).16,17
Therefore, mucosal damage and microbial translocation in the gut lead to increased morbidity and mortality in HIV-treated patients, and even antiretroviral therapy is not sufficient to alleviate these complications.18,19 For this reason, recent studies have focused on developing new therapeutic targets to control bacterial translocation by decreasing intestinal permeability. Previous studies have noted that probiotics and prebiotics improve the symptoms of several GI-related diseases.20,21 Moreover, probiotics safely administered to HIV-1–infected patients have increased T-CD4+ cell counts and improved clinical GI symptoms, even in the absence of HAART.22–24
The properties of probiotics are strain-dependent, and the use of Saccharomyces boulardii as a therapeutic probiotic is supported by its mechanisms of action, pharmacokinetics, and efficacy data provided by studies in animal models and clinical trials. Other Saccharomyces strains may also have probiotic properties, but clinical evidence for this is unclear.25,26 Several clinical trials and experimental studies have shown that S. boulardii is a beneficial biotherapeutic agent that plays a role in preventing and/or treating several diseases, and no adverse effects have been observed in any of the clinical trials.27S. boulardii has an effect on inflammation by impairing T-cell migration, leading to an accumulation of these T cells in mesenteric lymph nodes. Thus, S. boulardii may have a limiting effect on both the infiltration of T-helper-1 (Th1) cells in the inflamed colon and the amplification of inflammation induced by proinflammatory cytokine production,28 resembling the protective effect of the normal colonic flora; moreover, S. boulardii can have a further anti-inflammatory effect by inhibiting LPS-induced activation of human dendritic cells.29,30
Recent advances in understanding GI mucosal immunology and the evidence of the immunomodulatory and anti-inflammatory effects of S. boulardii open promising new perspectives.25,31 These results suggest that S. boulardii administration may have a beneficial effect in the treatment of inflammatory bowel diseases characterized by an increase in gut permeability and bacterial translocation, which can improve intestinal epithelial cell restitution and the inflammatory component of these diseases.32,33 Therefore, it may also be able to reduce bacterial translocation and the consequent chronic inflammation that characterizes HIV infection, a problem that remains in HIV-1–infected patients despite the virologic suppression achieved using an effective HAART regimen.
This is the first randomized, placebo-controlled study in HIV-1–infected patients under an effective HAART regimen. The end point is to decrease bacterial translocation and inflammation. The aim of this study was to assess the impact of treatment with a probiotic agent on markers of microbial translocation and inflammation.
SUBJECTS, MATERIALS, AND METHODS
Population and Study Design
A single-center, randomized, double-blind, placebo-controlled pilot study was conducted in nonconsecutive HIV-1–infected patients aged 18 years or older. Patients were recruited at a tertiary care hospital (Hospital Universitari del Mar) in Barcelona, Spain, which provides outpatient care to more than 1700 HIV-1–infected patients. All patients had chronic HIV infection, undetectable plasma viral load (<20 copies per milliliter) for at least 2 years, and a stable HAART regimen. Half of the patients had T-CD4+ cell counts >400 cells per milliliter, and the other half were discordant patients, defined by an unfavorable persistent immunologic response (T-CD4+ cells <350 cells per microliter) despite long-term suppressed viral load. Clinical and demographic data of the study patients were collected from the medical records.
Exclusion criteria were as follows: exposure to food supplements containing probiotics in the previous 6 months, changes in HAART regimen in the previous 2 years, and presence of a serious concomitant disease or substantial abnormalities in the laboratory tests that prevented study participation. Previous use of antibiotics or current antibiotic treatment during the study was allowed.
At baseline, eligible patients were randomized (1:1) to receive treatment with S. boulardii (2 capsules 3 times a day or 6 × 107 living bacteria) or placebo (2 capsules 3 times a day) for 12 weeks.
A computer-generated randomization list was used. Randomization was performed by a hospital pharmacist unrelated to the care of the patients, using sequentially numbered containers. The participants and the care providers were blinded after assignment to interventions. The study was approved by the Ethics Committee of Hospital Universitari del Mar-IMIM. Written informed consent was obtained from all participants before enrollment in the study. The primary end point of the study was a change in serum LBP levels as a marker of microbial translocation.
Secondary endpoints were as follows:
- Change in markers of immune activity, including serum concentrations of soluble CD14 (sCD14), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), high-sensitivity C-reactive protein (Hs-CRP), β2 microglobulin, erythrocyte sedimentation rate, and fibrinogen.
- Change in immune status (T-CD4+ and T-CD8+ lymphocyte count).
- Safety and tolerability.
After screening for eligibility, subjects were visited at baseline (start of treatment), at week 12 (treatment discontinuation), and at week 24 (3 months after discontinuation of treatment). At each visit, clinical assessment and adverse events were recorded, and blood samples were collected into 9 mL Vacuette serum separator tubes (Greiner Bio-One GmbH, Kremsmünster, Austria). Within 30 minutes after puncture, blood was centrifuged at 3000 rpm for 15 minutes, and serum samples were aliquoted and stored at −80°C until analysis. For all the tests, thawed serum samples were vortexed and centrifuged at 14,000 rpm for 10 minutes before testing.
For analysis of IL-6 and TNF-α, the Milliplex MAP assay on a fluorescent microsphere-based instrument (Merck Millipore, Merck KGaA, Darmstadt, Germany; Luminex/Bioplex200-Luminex Corp., Austin, TX and Bio-Rad, Hercules, CA) was used. Multianalyte profiling of serum concentrations was performed using a commercially available multiplex immunoassay kit (HSCYTMAG-60K; Millipore, Billerica, MA). Hs-CRP and β2 microglobulin were measured using the Immulite chemiluminescent immunometric assay on its automated analyzer (Immulite One; Siemens Healthcare, Llanberis, United Kingdom). Plasma fibrinogen was measured using the Clauss coagulative method with HemosIL reactives (Instrumentation Laboratory, Bedford, MA).
Microbial Translocation Markers
Serum levels of soluble CD14 were quantified in duplicate with a commercially available enzyme-linked immunosorbent assay kit (Quantikine; R&D Systems, Minneapolis, MN) using a Quanta-Lyser 160 robotic workstation (Inova Diagnostics, San Diego, CA). The lowest sCD14 detectable concentration was 0.25 ng/mL, with an intra-assay and inter-assay variation of <6.5% and <7.5%, respectively. Serum LBP was measured using the Immulite chemiluminescent immunometric assay on its automated analyzer. The analytical sensitivity of the assay was 1.2 μg/mL, and the coefficients of variation (%) were <5.9 within, and <10.7 between, runs. All assays were performed according to the manufacturer's instructions.
No previous data were available to estimate the sample size; thus, for an alpha risk of 0.05 and a beta risk of 0.20 in a bilateral contrast, 22 patients per study group were necessary to detect statistically significant differences between proportions. Based on baseline LBP levels and using a P value of 0.05, 22 subjects in each arm gave 80% power to detect a serum LBP reduction of >50% between groups. Data were analyzed by Intention-To-Treat (ITT), in which all 44 patients were included, and also after excluding those patients who had significant clinical events (as urinary sepsis or pneumonia who needed antimicrobial therapy) or low adherence during the course of the study (On-Treatment [OT] analysis) (Fig. 1). A quantitative analysis was performed; qualitative analysis of the parameters was also performed and defined as a decrease or increase of the values related to baseline data.
Student t test was used to assess differences between 2 mean values, and a Mann–Whitney U test was performed when data were not normally distributed. Either the χ2 test or the Fisher exact test was used to test the degree of association of categorical variables. Statistical significance was set at P < 0.05. Statistical analyses were performed using SPSS software version 20.0 (SPSS, Inc, Chicago, IL).
Between August 2012 and July 2013, 44 patients were included in the study: 22 were defined as immunodiscordant and 22 had good immune responses (patients immunoresponders with T-CD4+ cell counts >400 cells per milliliter). Most of the demographic and clinical parameters were well balanced between the probiotic treatment group (n = 22) and placebo group (n = 22), showed in Table 1. All participants completed the study; none discontinued their participation because of side effects of the treatments. As expected, significantly lower nadir T-CD4+ cell counts were observed in discordant patients compared with concordant patients.
With respect to safety analyses, no significant changes in serum biochemistry or hematology markers were observed in fecal morphology (measured by the Bristol stool scale) or frequency.
Changes in Parameters of Microbial Translocation and Inflammation After Intervention
In an ITT quantitative analysis (n = 44), only β2-microglobulin levels showed a decreasing trend in the probiotic group vs the placebo group (P < 0.06). In the ITT qualitative analysis, the only parameter that was significantly decreased after 12 weeks of treatment was LBP: 13.6% (n = 3) in the placebo group vs 50% (n = 11) in the probiotic group (P = 0.02). β2-microglobulin levels also showed a significant decrease at week 24 (P = 0.01). These data are summarized in Table 2.
When an OT analysis was performed (n = 35), there was a significant quantitative decrease in LBP (P = 0.02) and IL-6 (P = 0.00) as well as a decreasing trend in Hs-CRP (P = 0.07) in the probiotic group vs the placebo group after 12 weeks of treatment. The changes in LBP and IL-6 remained statistically significant at 3 months after treatment discontinuation. The differences in LBP between the 2 groups remained 3 months after treatment discontinuation. In addition, β2-microglobulin levels showed a significant decrease at week 24 (P = 0.05). However, given the significant differences between the 2 randomized groups in baseline β2-microglobulin values, this result must be interpreted with caution (see Figures S1–S3, Supplemental Digital Content, http://links.lww.com/QAI/A612).
In OT qualitative analysis, a significant decrease in LBP was again observed at 12 weeks after treatment discontinuation (decrease in 57.9% and 6.2% in the probiotic and placebo group, respectively; P = 0.002). Differences between groups are shown in Table 3.
The immune markers IgE, IgG, interferon gamma, and IL-10 did not show a significant impact of probiotic supplementation. T-CD4+ and T-CD8+ cell counts (absolute and percentage values) did not change between baseline and week 24. No statistically significant differences were found in inflammation and immunity parameters after intervention in patients in the discordant group compared with patients with good immune restoration.
We report a significant decline in LPB and IL-6 levels in a small cohort of HIV-infected, antiretroviral therapy (ART)-treated patients supplemented with short-term treatment of S. boulardii. However, parameters such as sCD14, TNF-α, Hs-CRP, and other immune parameters did not vary in this trial. Recently, it has been suggested that specific intestinal microbiota may be beneficial to control microbial translocation and, therefore, improve immunity in HIV patients.34 It seems clear that the study of the microbiome will open new doors to concepts related to the pathogenesis of common conditions.35 Therefore, treatments that modify the intestinal ecosystem toward a more beneficial one may give rise to a new adjuvant therapy that can be used to treat chronic HIV infection.
Previous trials performed in developing countries reported that using Bifidobacterium bifidum and Streptococcus thermophilus in HIV-infected children without ART in Brazil,36Lactobacillus rhamnosus GR-1 in HIV-infected women naive to ART in Nigeria,37 and L. rhamnosus R-1 and L. reuteri RC-14 in Tanzania22 could maintain immune function and reduce liquid stool episodes. Recently, a study by Klatt et al38 with simian immunodeficiency virus-infected, ART-treated pigtail macaques found that symbiotic (probiotic/prebiotic) supplementation in the GI tract had the following effects: increased frequency and functionality of antigen-presenting cells in the gut; enhanced reconstitution and functionality of colonic, but not peripheral, T-CD4+ cells; and reduced fibrosis of lymphoid follicles in the colon. But not all probiotics have shown the same effectiveness: previous studies using several diarrheal disease models with a predominant inflammatory component showed that the mechanisms underlying the anti-inflammatory effect of S. boulardii involved modifying the signaling pathways in intestinal host cells participating in the pathogenesis of inflammation.31 Thus, S. boulardii plays a potential anti-inflammatory role in the host because of its ability to modulate dendritic cell phenotype, function, and migration by inhibiting dendritic cell responses to bacterial antigens, such as LPS.29,30
LBP is closely related to the endotoxin-binding protein, and they function in a coordinated manner to facilitate an integrated host response to invading Gram-negative bacteria. LBP also permits highly sensitive proinflammatory responses to small numbers of bacteria at the onset of bacterial invasion and, later, efficient elimination of viable bacteria and their remnants as well as of endotoxin-driven inflammation.4,39 Previous studies in HIV patients and in patients with other diseases commonly associated with bacterial translocation, such as cirrhosis, have shown a correlation among elevated levels of LBP, sCD14, and proinflammatory cytokines.40,41 The connection between serum LBP and LPS levels confirms that LBP is a useful marker for bacterial translocation5,42 and disease activity. Further studies may confirm the usefulness of detecting this marker to determine microbial translocation in HIV patients.
This is the first randomized, placebo-controlled pilot study to assess the impact of S. boulardii use on microbial translocation and inflammation among virologically suppressed HIV patients effectively treated with HAART. LBP correlates with parameters of immune activation (sCD14) and inflammation (IL-6) in peripheral blood, whereas previous studies have demonstrated the greatest effect of probiotics in GI tissues.38 These results suggest that serum LBP levels could be used as a marker of bacterial translocation, although the clinical significance of this finding remains to be determined. It seems that treatment with S. boulardii could decrease microbial translocation (LBP) and inflammation parameters (IL-6 and β2 microglobulin) in HIV patients with virologic suppression. Both ITT and OT analyses were performed, excluding patients with severe clinical events or poor adherence to avoid confounding factors that could potentially affect the results, like antibiotic therapy. Because this is a study of inflammation and bacterial translocation, patients with good adherence and no serious clinical events (e.g., infection) were analyzed separately during the study. This group of probiotic-treated patients with no possible confounding factor not only showed a decrease in bacterial translocation but also a decrease in inflammation, as measured by IL-6. Moreover, in this group, treatment-induced changes persisted throughout the study (24 weeks). These observations, if confirmed in larger studies, have significant clinical relevance because of the clearly established association between persistent elevation of IL-6 and increased morbidity and mortality in HIV-infected patients.43–45
This study did not show a significant impact of S. boulardii on immune function as measured by T-CD4+ and T-CD8+ cell counts or percentage. No adverse events were associated with the use of probiotics.
The study has some limitations. Adherence to HAART and to placebo or probiotic treatment was assessed by interview, and no objective measurement of adherence was used; however, selected patients showed high adherence in the past, and all had long-term viral suppression. The daily probiotic dose administered to patients and the 12-week duration of treatment followed the recommendations outlined in previous studies using S. boulardii to treat diseases characterized by an increase in gut permeability and bacterial translocation.27
The major limitation of the study is the small sample size. In addition, a significant proportion of patients had a potentially confounding factor that made it necessary to perform 2 types of analyses. The ITT analysis showed a decrease in LBP while keeping the probiotic treatment. The OT analysis in patients without confounding factors showed a decrease in LBP; IL-6 also appeared to decrease when probiotics were withdrawn. The optimal values of LBP or IL-6 are unknown; therefore, the clinical significance of these findings remains unclear.
This randomized, double-blind study suggests that the use of some probiotics (S. boulardii) reduces LBP and thus bacterial translocation and systemic inflammation in HIV-treated patients with virologic suppression. Nevertheless, this short course of probiotics did not demonstrate any effectiveness in significantly altering the immune repertoire in HIV-infected patients. Probiotic treatment is well tolerated, inexpensive, and offers an exciting new approach to adjunctive treatment of HIV infection.
The present data suggest that, as in other diseases, the use of mucosal adjuvants may be useful to supplement antiretroviral therapy in HIV-infected individuals, mitigating residual GI inflammation and decreasing intestinal permeability. We also report a significant decrease in IL-6, a factor directly related to morbidity and mortality. Hence, future studies incorporating a longer duration of treatment and greater numbers of study participants will be essential to define the optimal dose and duration of probiotic treatment, the effect of probiotics on microbiota composition, and which patients may benefit the most from probiotic treatment.
The authors thank all the patients who participated in the study. The authors thank Elaine M. Lilly, PhD, for helpful advice and critical reading of the article.
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