TAO Zhuang,WANG Jian，CHEN Xin,LI Yonghong,YAN Yuguang,ZHANG Ao,ZOU Wen,LIU Ying

TAO Zhuang,Research Center of AIDS Treatment with Traditional Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China;Institute of Basic Theory for Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China

WANG Jian,ZOU Wen,LIU Ying,Research Center of AIDS Treatment with Traditional Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China

CHEN Xin,Department of Integrated Traditional Chinese and Wetern Medicine,Kunming Hospital of Traditional Chinese Medicine,Kunming 650000,China

LI Yonghong,YAN Yuguang,Department of Infection,No.6 People's Hospital of Shenyang City,Shenyang 110006,China

ZHANG Ao,University of Chinese Academy of SoCIal SCIences,Beijing,100102,China

Abstract OBJECTIVE: To evaluate the effects of the Wenshen Jianpi recipe (溫腎健脾方,WJR) on immune reconstruction and natural killer (NK) cells in immunological non-responders (INRs) of people living with human immunodeficiency virus (HIV) (PLWH) and propose new therapeutic strategies for HIV.METHODS: Based on Traditional Chinese Medicine treatment principle “invigorating Qi and warming Yang in the spleen and kidneys”,WJR combined with antiretroviral therapy (ART) therapy was performed in a randomized,double-blind,placebo-controlled study of 60 patients with non-responders.The randomized process was executed by the Clinical Evaluation Center of China Academy of Chinese Medical Sciences.Sixty patients who met the inclusion criteria obtained random numbers(that is the drug number) was randomly divided into a treatment group and a placebo control group according to a 1∶1 ratio.CD4+T cell counts and natural killer (NK)cells counts were evaluated at baseline and 12-week,24-week follow-ups.RESULTS: Four participants received random numbers and did not enter the group due to the patient's own reasons.A total of 56 patients were enrolled,including 28 in the treatment group and 28 in the control group.CD4+T cell counts in the treatment group were significantly increased at week 24 (P =0.01 ＜ 0.05),which were significantly higher than those in the control group (P =0.01 ＜ 0.05).Although no significant differences were observed between two groups,the CD56briCD16-NK cell counts in the treatment group were significantly increased after duration.and CD56dimCD16+NK cell counts in the treatment group were significantly higher than those in the control group after 24 weeks of treatment (P =0.025＜ 0.05).As compared with the control group,the treatment group had significantly lower CD56negCD16+NK cell counts after 24 weeks of treatment (P =0.023 ＜0.05).CONCLUSIONS: WJR promotes the immune reconstruction of INRs and redistribution of NK cell subsets,notably decreasing CD56negCD16+NK cell counts in INRs.However,the redistribution of NK cell subsets is not beneficial for immune reconstruction in INRs.Further large-scale RCTs are required to evaluate the effect of WJR on immune recovery in INRs and decipher the underlying mechanism.

Keywords: HIV;acquired immunodeficiency syndrome;killer cells,natural;immunological non-responders;antiretroviral therapy;Wenshen Jianpi recipe;immune reconstruction;randomized controlled trial

1.INTRODUCTION

Derived from hematopoietic stem cells,natural killer(NK) cells are widely distributing in bone marrow,liver,lymph nodes,spleen,lungs,and mucosa.The number of NK cells in the liver and lungs accounts for 10%-30% of the total number of lymphocytes and 10%-15% of the lymphocytes in peripheral blood.1NK cells play a vital role in the immune defense mechanism against viruses and tumors.Acquired immunodeficiency syndrome(AIDS) is an infectious disease caused by the human immunodeficiency virus (HIV),which results in the progressive damage of immune function,eventually resulting in various opportunistic infections and cancer.2The role of NK cells in HIV infection involved the elimination of target cells through the release of perforin or granzymes,3modulating the adaptive response by crosstalk with dendritic cells (DCs).4These manifestations enhance the antiviral response and limit the viral spread by inducing several cytokines and chemokines.5,6

Traditional Chinese Medicine (TCM) has demonstrated substantial efficacy for HIV/AIDS treatment through the induction of immune reconstitution,reduction of antiretroviral therapy (ART) induced side effects,and delaying the disease progression.7,8Many Chinese herbal Medicines (CHM) play beneficial roles in HIV/AIDS treatment by showing immunomodulatory effects through their influence on NK cells.9-12Although the Wenshen Jianpi recipe (溫腎健脾方,WJR) is reported to increase the CD4+T cell counts in HIV-infected immunological non-responders (INRs),13the underlying mechanism is still unclear.To the best of our knowledge,this study reports the result of the randomized controlled trial (RCT) exploring the effect of WJR on NK cells,explore its potential mechanism,and systematically evaluates the efficacy of WJR in INRs.

2.METHODS

2.1.Study design

This study is a randomized,double-blinded,and placecontrolled trial conducted based on the CONSORT 2010 statement14in Kunming Hospital of Traditional Chinese Medicine (KHTCM) and No.6 People's Hospital of Shenyang City (PHS) in China between July 2019 and November 2019.We registered the clinical trial protocol with the Chinese Clinical Trial Registry (DOL No.ChiCTR-INR-16009369).The institutional review board of PHS reviewed and approved the protocol and consent forms before the start of the study (DOL No.2016-05-001-02).All participants signed written informed consent forms before enrollment.

2.2.Ethics statement

Informed consent was obtained from every participant,and the study was approved by the review board of No.6 PHS.The study was in compliance with local regulations and the Declaration of Helsinki.

2.3.Patient enrollment

The diagnostic criteria were accorded with Chinese guidelines for diagnosis and treatment of HIV/AIDS(2018) and Consensus of integrative Medicine treatment experts on poor reconstruction of HIV immune function(2019).

The inclusion criteria are as follows.(a) Male or female patients aged from 18 to 55 years old;(b) positive test for HIV antibody confirmed by Western blot assay;(c) less than 12 months treatment of ART,and plasma HIV viral load less than 40 copies/mL for more than 12 months;(d)CD4+T cell counts less than 350 cells/μL;(e) Consent to participate in this study with signed informed consent.The exclusion criteria are as follows.(a) uncontrolled severe opportunistic infection prior to enrollment;(b)participating in other clinical trial within a month prior to enrollment;(c) receiving immunomodulator within a month;(d) WBC ＜ 2×109/L,N ＜ 1.0 × 109/L,Hb ＜ 90 g/L,PLT ＜ 75 × 109/L,or with liver dysfunction (AST/ALT/T-BIL ≥ 2 times of the upper limit of reference value) or kidney dysfunction (lower creatinine clearance rate);(e) pregnant or lactating women,or women preparing for pregnancy;(f) combined with other serious diseases (such as tumor,liver cirrhosis,cardiovascular and cerebrovascular diseases,etc.;(g) with mental or language impairment and difficulty in fully understanding the trial or failing to cooperate.

2.4.Intervention

WJR (5.5 g/d,po,bid) combined with ART (AZT/TDF+3TC+NVP/EFV,or TDF+3TC+Lpv/r) were administered to the participants of the treatment group,including Renshen (Radix Ginseng) 15 g,Lingzhi(Ganoderma Lucidum) 15 g,Baizhu (Rhizoma Atractylodis Macrocephalae) 15 g,Fuling (Poria) 15 g,Tusizi(Semen Cuscutae) 15 g,Bajitian (Radix Morindae Officinalis) 15 g,and Gouqizi (Fructus Lycii) 15 g,which explored with the functions of invigoratingQiand warmingYangin the spleen and kidneys.The control group was treated with ART and simulant WJR (SWJR),WJR and SWJR were procured from Tianjiang Pharma Group Co.Ltd.,batch No.AS1603301 (Jiangyin,China).The total treatment duration was 24 weeks for the two groups.

2.5.Outcome measures

Immunological parameters and related NK cell indicators were evaluated in our study.CD4+T cell counts,different NK cell subsets including CD56briCD16-,CD56dimCD16+,and CD56negCD16+and safety safety indexes were evaluated at baseline,week 12,and week 24.

2.6.Statistical analysis

SPSS (vers 26.0,IBM Corp.,Armonk,NY,USA)software were used for statistical analysis.For continuous variables,mean ± standard deviation (±s)or interquartile range (IQR) were used for descriptive statistics;Shapiro-Wilk (SW) test was used for testing normality,based on which either Two-Independent-Samplest-test or Mann-WhitneyU-test was used for evaluating statistical differences between the two groups.The comparison of binary variables was made using χ2test or Fisher’s exact probability method.Correlation analysis among the levels of NK cell subsets and CD4+T cell counts was done using Pearson Correlation Analysis method.P＜ 0.05 was considered statistically significant.

3.RESULTS

3.1.Participant characteristics

A total of 60 participants were included in our study,four participants received random numbers and did not enter the group due to the patient's own reasons.A total of 56 patients were enrolled,including 28 in the treatment group and 28 in the control group.There were no differences in the demographic and clinical characteristics between the two groups.The flow diagram of the selection process and characteristics of the included participants are shown in Figure 1,Table 1.

Figure 1 Participants flowchart

3.2.Differences between the recoveries of CD4+T cell counts of the groups

No significant differences in CD4+T cell counts in the treatment and control groups were observed (P=0.61 ＞ 0.05) after 12 weeks.After 24 weeks of WJR treatment,CD4+T cell counts increased to a mean of 275.00 cells/μL (95%CI: 240.76,309.23 cells/μL),which was higher than that the counts in single ART treatment group 214.95 cells/μ L (95%CI: 182.82,247.08 cells/μL).A significant increase on CD4+T cell counts was observed in the WJR treatment group after 24 weeks (P=0.01＜ 0.05).The complete CD4+T cell counts before and after WJR treatment are summarized in Table 2.

3.3.Comparison of levels of NK cell subsets (CD56bri CD16-,CD56dimCD16+and CD56negCD16+) before and after WJR treatment

The levels of NK cell subsets before and after WJR treatment were analyzed in this study.Although the CD56briCD16-NK cell counts were significantly increased in the WJR treatment group after 24 weeks of treatment,there were no significant differences in the CD56briCD16-NK cell counts between the two groups after 12 and 24 weeks of treatment (P＞ 0.05).Compared to single ART treatment,24-week treatment with WJR increased CD56dimCD16+NK cell counts [mean,342.00 cells/μL (95%CI: 252.31,432.47cells/μL];however,no differences in the two groups after 12 and 24 weeks treatment were observed in the CD56dim CD16+NK cell counts (P＞ 0.05).We observed significant differences in the CD56negCD16+NK cell counts in the WJR treatment group after 24 weeks of treatment (P＜ 0.05).As compared with the CD56negCD16+NK cell counts in the single ART treatment group,the counts in the WJR groups were lower with a mean of 172.50 cells/μL (95%CI: 174.11,245.39 cells/μl,12-week group).The counts of the NK cell subsets before and after WJR treatment are summarized in Table 3.

Table 1 Participants’ characteristics

Table 2 CD4+T cell counts recovery before and after WJR treatment (cells/μL)

Table 3 Counts of NK cell subsets before and after WJR treatment (cells/mm3)

At week 12,we observed no significant differences in the CD56briCD16-NK cell counts,CD56dimCD16+NK cell counts and CD56negCD16+NK cell counts between the two groups (P＞ 0.05).At week 24,there were no significant differences in the CD56briCD16-NK cell counts between the two groups (P=0.571 ＞ 0.05).CD56dimCD16+NK cell counts in the treatment group were much higher than that in the control group (P=0.025 ＜ 0.05),and CD56negCD16+NK cell counts in the treatment group were significantly lower than that in the control group (P=0.023 ＜ 0.05).At week 12,there were no significant differences in the CD56briCD16-NK cell counts,CD56dimCD16+NK cell counts,and CD56negCD16+NK cell counts in two groups (P＞ 0.05)before and after treatment.While significant increases were observed in CD56briCD16-NK cell counts afterfull duration for treatment group (P=0.008 ＜ 0.05),CD56negCD16+NK cell counts in the WJP treatment group were significantly decreased after 24 weeks treatment (P=0.003 ＜ 0.05).

3.4.Correlation between the levels of NK cell subsets and CD4+T cell counts

We found no correlation between NK cell subsets' levels and CD4+T cell counts (r2＜ 0.001,P=0.47) (Figure 2).

3.5.Correlation between the levels of different NK cell subsets

We found a low correlation between the levels of different NK cell subsets (Figure 3).

Figure 2 Correlation between the levels of NK cell subsets and CD4+T cell counts using Pearson correlation analysis

Figure 3 Correlation between the levels of different NK cell subsets using Pearson correlation analysis

A very low correlation was observed among the levels of different subsets of NK cells: between CD56dimCD16+NK cell counts and CD56negCD16+NK cell counts (r2=0.18,P＜ 0.001;95%CI: 0.19,0.38);between CD56 briCD16-NK cell counts and CD56negCD16+NK cell counts (r2=0.04,P=0.01;95%CI: 1.13,8.45);between CD56briCD16-NK cell counts and CD56dimCD16+NK cell counts (r2=0.13,P＜ 0.0001;95%CI: 7.74,18.22).

3.6.Safety evaluation

During our treatment duration,there were no adverse reactions in the two groups and the blood routine,urine routine,liver and renal function,and ECG of the two groups were also normal,indicating WJR with ART was well-tolerated.

4.DISCUSSION

This study is the first study that explores WJR,a welltolerated TCM prescription,with immune recovery and the NK cell subsets redistribution properties in INRs.NK cells play multiple roles in the immune defense mechanisms of protecting against HIV;NK cells kill HIV through cytotoxic elimination of target cells3and limit viral spread through secretion of cytokines and modulate adaptive response through crosstalk with DCs.15,16

A study on the distribution of TCM syndromes showed that PLWH have deficiency ofYang,Yin,andQi,multiple deficiency syndromes ofYangandYin,weakness of spleen and kidney,and metabolic disorder ofQideficiency and blood stasis.17A cross-sectional study18that included 486 PLWH showed that 12.96% of PLWH met the diagnostic criteria of INRs characterized by deficiency syndrome mostly involving spleen and kidney.Compared with healthy controls,PLWH in coldsyndrome and heat-syndrome groups had a primordialyangdeficiency.19,20We hypothesize that INRs mostly haveYangandQideficiency of spleen and kidney;therefore,we should start from TCM treatment of invigoratingQiand warmingYangin the spleen and kidneys.WJR consists ofRenshen (Radix Ginseng),Lingzhi (Ganoderma Lucidum),Baizhu (Rhizoma Atractylodis Macrocephalae),Fuling (Poria),Tusizi(Semen Cuscutae),Bajitian (Radix Morindae Officinalis),and Gouqizi (Fructus Lycii),which have functions of invigoratingQiand warmingYangin the spleen and kidneys.Our previous study demonstrated that prescriptions invigoratingQiand warmingYangin the spleen and kidneys significantly promoted recovery of CD4+T cell counts in PLWH and INRs13by increasing the expression of Toll-like receptors (TLRs).These observations highlighted its mechanism of promoting immune regulation.21,22The present study demonstrated that an optimization prescription -WJR could promote the immune reconstruction of INRs by increasing CD4+T cell counts.

NK cells are generally divided into two subsets according to the expression of CD56 on their surface:CD56briCD16-and CD56dimCD16+.These subsets have different cell functions.For instance,CD56 briCD16-NK cells mainly produce a high amount ofcytokines,including interferon-gamma,tumor necrosis factor-alpha and granulocyte-macrophage colonystimulating factor and a small number of lytic granules;CD56dimCD16+NK cells,the mature stages of NK cells,mainly produce lytic granules that play an essential role in the cytotoxic activity of NK cells.CD56negCD16+,a subset of NK cells,has low cytotoxic activity and cytokine production.

Infection with HIV can change the distribution of NK cell subsets.In one study,CD56dimCD16-NK cell counts decreased significantly,while CD56negCD16+NK cell counts increased during HIV/AIDS infection.23Although NK cell subsets' counts can be recovered up to a certain extent after long-term ART treatment,the imbalance in NK cell subsets' distribution persists in PLWH.24,25Compared with immunological responders and healthy controls,INRs have a lower absolute NK cell count and a lower percentage of NK cells among peripheral blood mononuclear cells;however,no significant differences in the percentages of CD56dim-CD16-NK cell counts and CD56briCD16-NK cell counts among the three groups were observed.26In our study,we found a significant correlation between the counts of different NK cell subsets.WJR induced a balance in NK cell subsets' distribution by significantly decreasing the CD56negCD16+NK cell counts,increasing the CD56briCD16-NK cell counts and CD56dimCD16+NK cell counts,although no significant differences in the counts of the CD56briCD16-NK cell counts were observed when compared with the control group.And the CD56dimCD16+NK cell counts were higher,and CD56negCD16+NK cell counts were lower in the WJR group.These findings suggest that WJR treatment effectively promotes the redistribution of NK cell subsets,mainly balancing the CD56negCD16+NK cell counts and the counts of different NK cell subsets that play a role in HIV pathogenesis.

We observed no correlation between the counts of NK cell subsets and CD4+T cells in both groups.These results suggested that redistribution of NK cell subsets was not beneficial for immune reconstruction in INRs.Other studies showed activation of NK cells and NK cell subsets significantly correlated with CD4+T cell counts.25,26The levels of activation indicators increased in CD56negCD16+NK cells counts were adverse to CD4+T cell counts,26,28,29indicating that the mechanism of WJR-induced immune reconstruction in INRs may involve balancing the levels of activation indicators on NK cells and NK cell subsets.Further studies investigating the activation and functional indicators on NK cells and NK cell subsets are required.

Our study has some limitations.First,the limited sample size in our study may have influenced the study outcomes.Second,relevant indicators such as activation and functional markers on NK cells and NK cells'cytotoxicity should have been considered.Based on our study's findings,large-scale RCTs are required further to strengthen the applicability of WJR in clinical practice.In conclusion,WJR can promote the immune reconstruction of INRs and redistribution of NK cell subsets,mainly decreasing the CD56negCD16+NK cell counts in INRs.However,the findings suggest that NK cell subsets' redistribution is not beneficial for immune reconstruction in INRs.Further large-scale RCTs are required to evaluate the effect of WJR on immune recovery and decipher the underlying mechanism.30-32