Normocaloric Low Cholesterol Diet Modulates Th17/Treg Balance in Patients with Chronic Hepatitis C Virus Infection

Hepatitis C virus (HCV) infection is associated with hepatic and extrahepatic manifestations, including immunological disorders. Chronic Hepatitis C (CHC) is often characterized by cholesterol and lipid metabolism alterations, leading to hepatic steatosis. Cholesterol metabolism, in fact, is crucial for the viral life cycle. Recent works described that a higher dietary cholesterol intake is associated with the progression of HCV-related liver disease. CHC patients have increased levels of T helper 17 (Th17)-cells, a lymphocytic population involved in the pathogenesis of liver inflammation and autoimmune hepatitis. The balance between Th17 and regulatory T (Treg) cells is crucial for chronic inflammation and autoimmunity. Th17-cell differentiation is deeply influenced by the activation LXRs, nuclear receptors modulating cholesterol homeostasis. Moreover, HCV may affect these nuclear receptors, and cholesterol metabolism, through both direct and indirect mechanisms. On these bases, we hypothesized that modulation of cholesterol levels through Normocaloric Low Cholesterol Diet (NLCD) may represent an innovative strategy to reduce the progression of HCV infection, through the modulation of peripheral Th17/Treg balance. To this end, we performed a pilot study to investigate whether a Normocaloric Low Cholesterol Diet may be able to modulate Th17/Treg balance in patients affected by chronic HCV infection. After 30 days of NLCD CHC patients showed a significant reduction in Th17 cells frequency, which correlated with strong reduction of IL-17 and IL-22 serum levels. At the same time, we appreciated an increase in the percentage of Treg cells, thus improving Treg/Th17balance. Moreover, we observed an increased expression of LXRs and their target genes: SREBP-1c and ABCA-1. In conclusion, NLCD finely regulates Th17/Treg balance, improving immune system response in CHC patients. This study could pave the way for new treatments of CHC patients, suggesting that change in lifestyle could support the management of these patients, promoting well-being and possibly hindering disease progression. Trial Registration ClinicalTrials.gov NCT02038387


Introduction
Background 2 Scientific background and explanation of rationale Theories used in designing behavioral interventions

Methods
Participants 3 Eligibility criteria for participants, including criteria at different levels in recruitment/sampling plan (e.g., cities, clinics, subjects) Method of recruitment (e.g., referral, self-selection), including the sampling method if a systematic sampling plan was implemented Recruitment setting Settings and locations where the data were collected Interventions 4 Details of the interventions intended for each study condition and how and when they were actually administered, specifically including: Unit of assignment (the unit being assigned to study condition, e.g., individual, group, community) Method used to assign units to study conditions, including details of any restriction (e.g., blocking, stratification, minimization) Inclusion of aspects employed to help minimize potential bias induced due to non-randomization (e.g., matching) Whether or not participants, those administering the interventions, and those assessing the outcomes were blinded to study condition assignment; if so, statement regarding how the blinding was accomplished and how it was assessed.
Unit of Analysis 10 Description of the smallest unit that is being analyzed to assess intervention effects (e.g., individual, group, or community) If the unit of analysis differs from the unit of assignment, the analytical method used to account for this (e.g., adjusting the standard error estimates by the design effect or using multilevel analysis) Statistical Methods

11
Statistical methods used to compare study groups for primary methods outcome(s), including complex methods of correlated data Statistical methods used for additional analyses, such as a subgroup analyses and adjusted analysis Methods for imputing missing data, if used Statistical software or programs used

Participant flow 12
Flow of participants through each stage of the study: enrollment, assignment, allocation, and intervention exposure, follow-up, analysis (a diagram is strongly recommended) o Enrollment: the numbers of participants screened for eligibility, found to be eligible or not eligible, declined to be enrolled, and enrolled in the study o Assignment: the numbers of participants assigned to a study condition o Allocation and intervention exposure: the number of participants assigned to each study condition and the number of participants who received each intervention o Follow-up: the number of participants who completed the followup or did not complete the follow-up (i.e., lost to follow-up), by study condition Number of participants (denominator) included in each analysis for each study condition, particularly when the denominators change for different outcomes; statement of the results in absolute numbers when feasible Indication of whether the analysis strategy was "intention to treat" or, if not, description of how non-compliers were treated in the analyses Outcomes and estimation 17 For each primary and secondary outcome, a summary of results for each estimation study condition, and the estimated effect size and a confidence interval to indicate the precision Inclusion of null and negative findings Inclusion of results from testing pre-specified causal pathways through which the intervention was intended to operate, if any Ancillary analyses 18 Summary of other analyses performed, including subgroup or restricted analyses, indicating which are pre-specified or exploratory Adverse events 19 Summary of all important adverse events or unintended effects in each study condition (including summary measures, effect size estimates, and confidence intervals)

Interpretation 20
Interpretation of the results, taking into account study hypotheses, sources of potential bias, imprecision of measures, multiplicative analyses, and other limitations or weaknesses of the study Discussion of results taking into account the mechanism by which the intervention was intended to work (causal pathways) or alternative mechanisms or explanations Discussion of the success of and barriers to implementing the intervention, fidelity of implementation Discussion of research, programmatic, or policy implications Generalizability 21 Generalizability (external validity) of the trial findings, taking into account the study population, the characteristics of the intervention, length of follow-up, incentives, compliance rates, specific sites/settings involved in the study, and other contextual issues Overall Evidence

22
General interpretation of the results in the context of current evidence and current theory

Results
Clinical parameters of CHC and NAFLD/NASH patients after NLCD

Discussion
Discussion