A pilot study on the effects of probiotic supplementation on neuropsychological performance and microRNA‐29a‐c levels in antiretroviral‐treated HIV‐1‐infected patients

Abstract Introduction The gut microbiota is involved in the regulation of cognition, mood, anxiety, and pain, and can impact cognitive functions by producing neuroactive substances or releasing bacterial by‐products and metabolites. No information is available on the effects of a probiotic supplementation on brain function of HIV+ subjects. In light of the above considerations, we performed a pilot study in cART‐treated HIV‐1‐positive patients with long‐term virologic suppression. The aims were to analyze the effect of high‐concentration multistrain probiotic supplementation (Vivomixx®; Visbiome®) on several neurocognitive abilities and to evaluate the safety of this supplementation. Methods To address those issues, neurocognitive performances were explored by administering neuropsychological tests; moreover, miRNA‐29a‐c levels were measured in cerebrospinal fluid (CSF) to confirm the persistent undetectable levels of HIV‐RNA in the central nervous system after probiotic supplementation. Results Our results show that the Rey auditory verbal learning test (RAVLT) (immediate and delayed recall), Rey‐Osterrieth complex figure test (ROCF) (copy immediate and delayed recall), phonological verbal fluency (PVF) test, Toronto alexithymia scale‐20 (Tas‐20), State‐trait anxiety inventory Y‐2 (STAY Y‐2), and time and weight estimation test (STEP) scores improved significantly during the study. Moreover, we found unchanged levels, associated to high degree of individual variability, in miRNA‐29 levels in CSF collected before and after probiotic supplementation. Conclusions In conclusion, we observed that HIV patients treated with 6 months of this probiotic supplementation appear to have an improvement in some neurocognitive functions; moreover, this approach is safe and did not modify significantly the levels of miRNA in CSF. Further studies are needed to better understand the contribution of the probiotics in modulating gut–brain‐axis in HIV patients.

20 (Tas-20), State-trait anxiety inventory Y-2 (STAY Y-2), and time and weight estimation test (STEP) scores improved significantly during the study. Moreover, we found unchanged levels, associated to high degree of individual variability, in miRNA-29 levels in CSF collected before and after probiotic supplementation.

Conclusions:
In conclusion, we observed that HIV patients treated with 6 months of this probiotic supplementation appear to have an improvement in some neurocognitive functions; moreover, this approach is safe and did not modify significantly the levels of miRNA in CSF. Further studies are needed to better understand the contribution of the probiotics in modulating gut-brain-axis in HIV patients.

| INTRODUCTION
Probiotic supplementation has become a common practice to control the dysbiosis associated to many chronic inflammatory conditions, such as inflammatory bowel diseases (IBD) with positive effects on the clinical outcome (Shen et al., 2014).
HIV is a chronic inflammatory disease in which a proinflammatory change in the gut microflora associated to a number of structural, immunological, and functional changes are present (Ziberman-Schapira et al., 2016).
Consequently, the administration of probiotics to HIV patients has a rationale, even though the administration of live bacteria in the presence of a viral infection and associated immunosuppression could be a risk for sepsis and abscesses (Haghighat & Crum-Cianflone, 2015).
Few studies have been published on the role of a probiotic supplementation on SIV+ monkeys and on HIV+ humans, with promising results regarding the immune reconstitution of the gut barrier and immune response (Cunningham-Rundles et al., 2011;d'Ettorre, Ceccarelli, Andreotti, et al., 2015;d'Ettorre, Ceccarelli, Giustini, et al., 2015;Ortiz et al., 2016;Villar-García et al., 1999). No information is available on the effects of a probiotic supplementation on brain function and behavior of HIV+ subjects. The gut microbiota is involved in the regulation of cognition, mood, anxiety and pain as previously shown by studies performed in germ-free animal models or in animals exposed to pathogenic bacterial infections treated by antibiotic drugs or probiotic supplementations (Cryan & Dinan, 2012). The gut microbiota can impact mood and cognitive functions (Desbonnet, Garrett, Clarke, Bienenstock, & Dinan, 2008;Desbonnet et al., 2010) in different ways, i.e., by producing neuroactive substances or releasing bacterial byproducts and metabolites.
In light of the above considerations and the fact that neurocognitive impairment affects more than 50% of HIV-1-infected individuals (Nightingale et al., 2014), we performed a pilot study in cART-treated HIV-1-positive patients with long-term virologic suppression. The aims were (1) to analyze the effect of high-concentration multistrain probiotic (Vivomixx ® Visbiome ® ) supplementation on neuropsychological tests exploring several neurocognitive abilities and (2) to evaluate the safety of this supplementation.
The primary endpoint aimed at evaluating the effect of this specific kind of probiotics supplementation on the following neuropsychological tests: episodic declarative memory (Rey Auditory Verbal Learning Test RAVLT), visual perception and long-term visual memory function (Rey-Osterrieth Complex Figure, ROCF) To evaluate the safety of the product at the neurological level, a lumbar puncture before and after the probiotic supplementation was performed. Due to the invasive procedure of the lumbar puncture and associated risks, a control group was excluded. CSF miRNA-29a-c levels were evaluated. miRNAs are short single-stranded noncoding RNAs that have been shown to play a role in modulating HIV-1 infection, either directly or indirectly (Monteleone et al., 2015;Müller et al., 2016;Su, Aloi, & Garden, 2016;Swaminathan, Navas-Martín, & Martín-García, 2014).

| Study design, recruitment, and study eligibility criteria
This is a longitudinal, nonrandomized designed, single-arm, pilot study ( Figure 1) registered on the ClinicalTrials.gov registry with identifier number NCT02276326. The inclusion criteria allowed to enroll HIVpositive patients (1) who had signed the informed consent, (2) women or men at least 18 years of age, (3) in HAART with HIV-RNA <50 copies/ml, and CD4 counts >400 cells/mm 3 . Exclusion criteria were: (1) patients with known allergy or intolerance to the product, (2) diarrhea, (3) history of or current inflammatory diseases of the small or large intestine, (4) any past or current systemic malignancy, (5) previous or actual drug addiction, (6) use of antibiotics or probiotics during the 3 weeks prior the enrollment, (7) previous or actual psychiatric disorders, (8) previous or actual neurological disorders, and (9) pregnancy. All HIV-1-infected patients underwent neuropsychological testing and lumbar puncture before (T0) and after (T6) probiotic supplementation of cART, in this way, each patient was the control of oneself. (Figure 1). Because lumbar puncture is considered a procedure with several risks and in literature there is no reported study where lumbar puncture was performed in the control arm, our Institution considered it unethical to have a control group undergoing such a procedure.
Patients received a high-concentration lyophilized multistrain probiotic supplement twice a day for 6 months (

| Ethics statement
The researchers declare compliance with ethical practices upon submission of a manuscript and that clinical investigations were con-

| Cerebrospinal fluid measurements
CSF was collected by lumbar puncture, centrifuged, and cell-free supernatant samples stored in aliquots at −70°C until analysis of miRNA-29a/b/c species.

| Statistical analysis
Statistical analyses were done using SPSS software, version 20.00 (IBM, Somers, NY, USA). Results obtained from neuropsychological assessment and levels of miRNA-29 before (T0) and after 6 months of probiotic (T6) supplementation were evaluated using the Wilcoxon test.
Differences in MiRNA-29a-c expression at T0 and T6 were assessed by the Kruskal-Wallis test. The correlations between MiRNA-29a-c levels and neurological test scores were evaluated using the Pearson's test.
Differences were considered statistically significant when p < .05. As reported in the exclusion criteria, no patients enrolled presented previous or actual drug addiction, psychiatric disorders or neurological diseases. Average education level was 12 years. By contrast, no significant differences were observed for the other neuropsychological test scores examined before and after probiotic intake.

| Expression of microRNA-29a-c
The analysis of miRNA-29a-c levels in CSF collected from HIV-1infected patients before (T0) and after 6 months of daily (T6)

| Bacterial DNA isolation from fecal samples
We found that fecal Bifidobacteria spp. increased significantly in all patients compared to their basal level, after 2 months of supplementation (p < .05) and remained stable through the study (data not shown) and this was considered as a proof of compliance to the treatment.

| Safety of probiotic supplementation
All patients received a high-concentration lyophilized multistrain probiotic powder supplement twice a day for 6 months (1.8 × 10 9 ). This dosage was well-tolerated throughout the course of the study and side effects were not reported.

| DISCUSSION
Neurocognitive disorder affects more than 50% of antiretroviraltreated HIV subjects (Heaton et al., 2010). An improvement, but not complete recovery, of neurocognitive impairment has been reported in the literature, but only after the cART was intensified which often is not a feasible avenue due to the untoward effects and chronic nature of the disease (Stilling, Dinan, & Cryan, 2014).
We hypothesized that a new and safe approach for the improvement of the neuropsychological performance of these subjects could be the administration of probiotics. In the last years, there has been an increase in experimental research, conducted mainly on animals, aimed to explore the contribution of the microbiota in modulating gut-brain-axis (GBA). The discovery that differential microbial composition is associated with alterations in behavior and cognition has significantly contributed to establish the "microbiota-gut-brain-axis" as an extension of the well-accepted "gut-brain-axis" concept. This concept is used to describe the bidirectional communication between the CNS and intestinal organs (Gates et al., 2016).
Recent studies exploring the effects of different gut microbiota composition on impairment of cognition and behavior have shown that alterations of microbioma composition are connected to stress, modification of mood and behavior, worsening of cognition and that probiotics can play a protective role (Carabotti, Scirocco, Maselli, & Severi, 2015;Jiang et al., 2015;Steenbergen, Sellaro, van Hemert, Bosch, & Colzato, 2015).
Since no data were available, our primary study end point was to verify the safety of a high-concentration multistrain probiotic supplementation at the neurological level. Each patient underwent a lumbar puncture before and after probiotic supplementation to verify any changes in CSF.
All HIV-1-positive patients received a high-concentration lyophilized multistrain probiotic powder supplement twice a day for 6 months (1.8 × 10^9). This dosage was well-tolerated throughout the course of the study and the potential side effects which had been previously reported for other and different formulations were not reported here (Anukam, Osazuwa, Osadolor, Bruce, & Reid, 2008;Haghighat & Crum-Cianflone, 2015;Hummelen et al., 2011). We found that fecal Bifidobacteria spp increased in all patients compared T A B L E 1 Results of neuropsychological test administered before and after 6 months of probiotic supplementation AAT (Aachen Aphasia Test) 9.00 (9.00-9.00) 9.00 (9.00-9.00) 1.0000 CBTT-Forward (Corsi block-tapping task) 4.37 (4.00-5.00) 5.25 ( to their basal level, at 2 months of supplementation and remained stable throughout the study confirming adherence of the patients to the regimen.
Our results showed that probiotics do not affect miRNA-29a-c levels or the signature of miRNA-29-a-c in CSF collected from cART-fully suppressed HIV-1-positive patients confirming the neurological safety of the probiotic formulation, a finding never described before in the literature.
Moreover, our patients presented a relevant improvement of performance in the neuropsychological and behavioral tests after 6 months of probiotic intake. MMSE was not modified by 6 months of probiotic supplementation while RAVLT (immediate and delayed recall), ROCF (immediate and delayed recall), PVF, STAY Y-2, and STEP (total, time and weight) appeared significantly improved.
Our data seem to demonstrate that the cognitive domains more involved are the short and long memory and the abstract reasoning (one of the aspects involved in the executive functions). Such results, seem to be aligned with what has emerged from a previously study, in which it has been highlighted that a crucial role in the bidirectional communication of the gut-brain-axis, is covered by the hypothalamic-pituitary adrenal (HPA) axis. As well known, the HPA axis is considered the core stress efferent axis that coordinates the adaptive responses of the organism to stressors of any kind, and also it is a part of the limbic system, a crucial zone of the brain predominantly involved in memory and emotional responses (Carabotti et al., 2015;Gates et al., 2016).
Pending the confirmation by larger study, probiotics supplementation seems to be a safe dietary approach to improve neurocognitive performance in HIV-1-positive patients, in particular memory functions. The same multistrain high-concentration probiotic has been shown to increase the brain-derived neurotrophic factor (BDNF) expression and to attenuate age-related alterations in the hippocampus of rats (Reitan, 1992).
On a pure speculative basis, according to what has been observed in animal models, probiotics may act on the hippocampal regions controlling memory functions in HIV-positive subjects undergoing antiretroviral treatment (Mayer et al., 2015), so the manipulation of microbial taxa trough probiotics may provide a means by which cognition may be altered in a reproducible and consistent manner in order to achieve a beneficial outcome for the patients in all daily activities in which, are required cognitive functions as memory (Lyte et al., 2016). This study suffers for the small sample size and the related statistical concerns and lacks proper control for multiple correlations.
However, the small sample size is justified by the need to verify the safety of this high-concentration multistrain probiotic supplementation. Usually, few thousand million probiotic bacteria have been administered to AIDS patients and a prudent approach (pilot study) consequently was mandatory. It is evident that a small sample size lim- Furthermore, although our data suggest the improvement of some cognitive domains, we cannot totally exclude the possible influence of the practical effect on neuropsychological performances. The use of parallel/alternate forms is one possible approach to minimize practice effect, but it does not fully eliminate concerns about this topic.
Our findings seem to be encouraging, but future studies, carried out with specific methodological procedures, are needed to support their validity.
In conclusion, despite the above limitations justified by the complexity of the procedures and the ethical boundaries, the findings of this pilot study indicate that HIV-1-positive patients receiving probiotic formulation twice a day for 6 months had significantly improved some neurological cognitive functions (short and long memory and abstract reasoning), thus supporting the concept that modifications of the gut flora can provide specific neurological benefits in HIV-1 patients in the absence of significant side effects.

ACKNOWLEDGMENT
We thank all the patients who agreed to participate.