Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 expression and its relationship with T cell activation in human immunodeficiency virus/acquired immune deficiency syndrome patients with lung-spleen Qi deficiency syndrome pattern

LIU Zhen,ZHANG Qingyan,DENG Bowen,SANG Feng,WANG Danni,KANG Ning,LI Jie,ZHANG Min,LIANG Shoupei,DUAN Chenchen,LIU Zhibin,XU Qianlei,LI Qiang

LIU Zhen,ZHANG Qingyan,DENG Bowen,SANG Feng,LI Jie,DUAN Chenchen,LI Qiang,Henan Key Laboratory of Viral Diseases Prevention and Treatment of Traditional Chinese Medicine,the First Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450000,China

WANG Danni,ZHANG Min,LIU Zhibin,XU Qianlei,Department of Acquired Immune Deficiency Syndrome Treatment and Research Center,The First Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450000,China

KANG Ning,Department of Obstetrics and Gynecology,the First Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450000,China

LIANG Shoupei,Medical Department,Medical Department of Zhengzhou People’s Hospital,Zhengzhou 450000,China

Abstract OBJECTIVE:To investigate the relationship between antiviral restriction factor Sterile Alpha Motif and Histidine-Aspartic acid domain-containing protein 1(SAMHD1) expression and T cell activation,furthermore,identifying objective indexes of lung-spleen Qi deficiency symptom pattern.METHODS:We assessed the profile of T lymphocyte subsets,characteristics of SAMHD1 and human leukocyte antigen DR (HLA-DR) expression in lungspleen Qi deficiency patients.At the same time,people living with human immunodeficiency virus/ acquired immune deficiency syndrome (HIV/AIDS) (PLWHA)without obvious clinical symptoms and healthy donors in this area were used as controls.RESULTS:Immunohematologic indexes lower CD4 count,lower CD4/CD8 ratio and higher SAMHD1 level were found in lung-spleen Qi deficiency patients.Furthermore,we demonstrated a positive relationship between SAMHD1 and HLA-DR level as well as with interferon factor in lung-spleen Qi deficiency syndrome and patients without obvious clinical signs and symptoms groups.CONCLUSIONS:These data indicated the positive relationship between SAMHD1 and T cell activation which further elucidated the role of SAMHD1 in cellular immune response.Furthermore,combination of T lymphocyte subsets counts and SAMHD1 level may be used as clinical and biological reference basis for the differentiation and diagnosis of HIV/ AIDS traditional Chinese medicine syndromes.

Keywords:HIV;acquired immunodeficiency syndrome;symptoms complex;SAM domain and HD domain-containing protein 1;T-lymphocytes

1.INTRODUCTION

Acquired immune deficiency syndrome (AIDS),which was first recognized in the early 1980s,can seriously evoke deficiency of Immune function.1Human leukocyte antigen (HLA) mediated virus-specific cytotoxic T lymphocyte reaction (CTL) plays an important role in controlling viral replication and disease progression.2,3Cytotoxic T lymphocytes,the main effector cells of specific cytotoxicity are functional subsets of CD8+T cells.Activated CD8+T lymphocytes can produce perforin and granzyme leading to cell lysis and death,as well as secreting immune effector molecules such as IFN,TNF and MIP-1α/β to inhibit virus replication.4Correspondingly,CD4+T lymphocytes are the main target of HIV,and also the helper cells of specific immune response.5Specific cellular immune effects depend on T cell activation,and in this process interferon is one of the antiviral effectors that need to be emphasized.Sterile Alpha Motif and Histidine-Aspartic acid domain-containing protein 1 (SAMHD1),a newly discovered antiviral factor,can be stimulated by immune effector molecule interferon.

SAMHD1,first discovered by Professor Cao Xuetao in human dendritic cell cDNA library in 2000,6is considered to be the fourth restriction factor against HIV-1 in primates after APOBEC3G,TRIM5α and BST-2.7HD domain,main functional structure of SAMHD1 protein,not only participates in nucleotide metabolism,but also has dNTPase and RNase activity,adjusting the level of deoxynucleoside triphosphates pool which is essential for HIV virus replication.8The discovery of SAMHD1,an type I interferon inducible protein in dendritic cells,hinted at the importance of SAMHD1 as an effector of innate immunity.Subsequently,SAMHD1 was found to play an important role in macrophage and T cell immune respond as well as for viral latency.9-11Thus it can be seen that SAMHD1 protein involves in the immune regulation basing on its function of monitoring and controlling nucleotide pool.However,relationship between SAMHD1 expression and T cell activation has not been conclusively reported.

At present,cART is the most effective treatment strategy for AIDS,and has the advantage of immunohematologic and virologic outcomes.However,the lifelong medication involved in cART is often limited by drug toxicity,poor adherence to therapy,and drug resistance.12Traditional Chinese medicine (TCM) has become an effective complementary and alternative therapy for AIDS and its complications.13Effective indications of TCM on AIDS include the alleviation of HIV related signs and symptoms,14improvements in quality of life,15improvements in long-term survival,16counteraction of the adverse side effects of antiviral drugs,17and promotion of immune reconstitution.18Compared with western medicine,TCM treatment emphasizes holistic nature of the body and is based on syndrome differentiation.19Our research group found that lung-spleenQideficiency syndrome was one of the common basic TCM syndromes in AIDS patients.20According to the epidemiological investigation of human immunodeficiency virus/ acquired immune deficiency syndrome (HIV/AIDS) by Wanget al,21spleenQideficiency was the main symptom in the early stage of AIDS,and with the progress of the disease,the syndromes showed regular changes ofQideficiency,Qideficiency with dampness,deficiency of bothQiandYin,andYangdeficiency.Identifying objective and quantitative diagnostic criteria of TCM syndromes are indispensable prerequisite for effective TCM treatment,whereas researches based on TCM syndromes of AIDS are still limited.

In this study,Relationship between SAMHD1 expression and T cell activation was analyzed to reveal the role of SAMHD1 in cellular immune response.Moreover,T lymphocyte subsets level,expression of SAMHD1 and cell activation level in peripheral T lymphocytes of patients with different groups were analyzed in order to provide the reference basis for the objectivity and quantification of TCM dialectics of AIDS.

2.MATERIALS AND METHODS

2.1.General information

A total of 123 HIV/ AIDS patients (63 males and 60 females,aged 16 to 45 years old) in Zhengzhou,Nanyang and Zhumadian of Henan province were selected in 2020,including 51 lung-spleenQideficiency syndrome(LSQD) patients and 72 individuals without obvious clinical signs and symptoms (WCSS).Twenty-four healthy donors (HD) were obtained from the same area,comparable in gender and age.Sample collection was approved by the Ethics Committee of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine (2018HL-050-01),and all subjects collected anticoagulant whole blood with informed consent.

Both western and Chinese medicine criteria were used to divide HIV/AIDS patients into two groups.First,participants were satisfied with diagnostic criteria of western medicine derived from ‘AIDS Treatment Guidelines (2018 version)’.22Then,diagnostic criteria of lung-spleenQideficiency syndrome referred to the summary items drawn up by our previous researches with main symptoms and signs standards including susceptible to cold,asthma,diarrhea,spontaneous sweating,cough,thin sputum,pale tongue,thin white coating,weak pulse and so on.23Patients were filtered when they satisfied one of the following criteria:pregnant women or nursing mothers;with severe opportunistic infection that were not caused by HIV/AIDS;with obnubilation,dementia,or other psychiatric/psychological disorders;unwilling to cooperate or failing to provide informed consent for whatever reason.

2.2.T lymphocyte subsets count testing

T lymphocyte subsets count from EDTA anticoagulated whole blood was performed by using TruCounts (FITCCD3Ab,PE-CD4Ab,PerCP-CD45Ab and APC-CD8Ab)(BD,New Jersey,USA) and FACS Canto∥ (BD,New Jersey,USA).Canto software automatically acquired and analyzed data.

2.3.SAMHD1 protein detecting

Flow cytometric intracellular staining was carried out as described previously.24Briefly,100 uL whole blood was stained with surface antibodies CD4 and CD8 (BD,New Jersey,USA),then the red blood cells were lysed with lysing solution.After rupturing cell nuclear envelopes with eBioscience Foxp3/Transcription Factor Staining Buffer Set,cells were washed with PBS and then stained with SAMHD1 antibody (Abcom) and fluorescent secondary antibody (Proteintech),ultimately washed or resuspended with PBS plus 1% BSA.Data were acquired using the BD Canto∥ flow cytometer (BD,New Jersey,USA) Diva software,and the mean fluorescence intensity (MFI) of SAMHD1 in T lymphocyte subsets was analyzed by Flowjo software.

2.4.T cell activation assays

The expression of HLA-DR,a marker of cell activation,was detected by flow cytometry as usual.25HLA-DR expression level in CD4+/ CD8+T cells was performed by using surface staining antibody (BD,New Jersey,USA).Data were acquired using the BD Canto∥ flow cytometer (BD,New Jersey,USA) Diva software,and the expression percentage of HLA-DR in T lymphocyte subsets was analyzed by Flowjo software.

2.5.mRNA Detecting of SAMHD1 and interferon-γ(IFN-γ)

Peripheral blood mononuclear cell (PBMC) isolated by Ficoll-paqueTM Plus (GE,Boston,USA) density gradient centrifugation were used to extract RNA(Solarbio,China),subsequently cDNA was synthesized(Thermo,Massachusetts,USA).The mRNA expression level of SAMHD1 and IFN-γ was detected by specific primers and SYBR Green Fluorescence quantitative detection kit (QIAGEN,NY,USA),meanwhile glyceraldehyde-3-phosphate dehydrogenase was used as control.

2.6.Statistical analysis

IBM SPSS Statistics for Windows,Version 18.0 and GraphPad Prism software (San Diego,CA,USA) were used for statistical analysis and plotting.Differences of T lymphocyte subsets,SAMHD1 and HLA-DR levels among LSQD,WCSS and HD groups were analyzed by analysis of variance (ANOVA),and if there was a statistical difference among the three groups,differences between each two groups were compared using LDS method in pairs.Pearson or Spearman rank correlation was used to analyze the correlation between SAMHD1 expression and T cell activation.All statistical tests were two-sided and the hypothesis test level α was equal to 0.05.

3.RESULTS

3.1.T lymphocyte subsets profiling

To gain immunohematologic insights into the differences of TCM syndromes,147 participants were obtained,including 51 LSQD patients,72 WCSS patients and 24 HD as the control.As showed in Table 1,absolute CD4 count,CD8 count and CD4/ CD8 ratio were recognized,respectively.Statistical analysis (ANOVA) showed that there were significant differences in CD4 count and CD4/CD8 ratio among the three groups (CD4 count:F=7.955,P=0.01;CD4/CD8 ratio:F=30.99,P＜ 0.001),further analysis of pairwise comparison showed that WCSS and LSQD were significantly lower than HD based on absolute count of CD4 (WCSSvsHD:P=0.004;LSQDvsHD:P＜ 0.001),meanwhile,CD4/ CD8 ratio of WCSS and LSQD groups were significantly lower than HD (WCSSvsHD:P＜ 0.001;LSQDvsHD:P＜ 0.001).On the contrary,there was no statistic difference in index of absolute CD8 count (F=2.365,P=0.098).

Table 1 Comparison of T lymphocyte subsets in LSQD,WCSS and HD groups

3.2.Characteristics of SAMHD1 and HLA-DR expression

To further discover intracellular protein biomarkers in TCM Syndromes of AIDS,expression levels of SAMHD1 and HLA-DR in T lymphocyte subsets were detected by flow cytometry based on intracellular factor or surface staining respectively.SAMHD1 MFI in CD4+T cells (SAMHD1 MFI/ CD4) were 28033,41296 and 47775 in HD,WCSS and LSQD groups,respectively.SAMHD1 MFI in CD8+T cells (SAMHD1 MFI/ CD8)were 32395,47808 and 55036 in HD,WCSS and LSQD groups,respectively.Percentage of HLA-DR expression in CD4 surface (HLA-DR/ CD4) were 26.85,23.30 and 30.28 in HD,WCSS and LSQD groups,respectively.Percentage of HLA-DR expression in CD8 surface(HLA-DR/ CD8) were 49.99,54.92 and 60.24 in HD,WCSS and LSQD groups,respectively (Figure 1).Statistical analysis showed that there were significant differences in SAMHD1 MFI among the three groups(SAMHD1 MFI/ CD4:F=10.514,P＜ 0.001;SAMHD1 MFI/ CD8:F=8.977,P＜ 0.001).Compared with WCSS and HD groups,SAMHD1 MFI in CD4 cell was higher in LSQD group (LSQDvsWCSS:P=0.044,LSQDvsHD:P＜ 0.001,WCSSvsHD:P=0.002),meanwhile,pairwise comparison showed that WCSS and LSQD were significantly higher than HD in SAMHD1 MFI/ CD8 (LSQDvsHD:P＜ 0.001,WCSSvsHD:P=0.003).Whereas,there was no significant difference in HLA-DR expression among the three groups (HLA-DR/ CD4:F=2.014,P=0.137;HLA-DR/ CD8:F=2.537,P=0.083).

3.3.Positive relationship between SAMHD1 and T cell activation

To explore the correlation between SAMHD1 expression and the level of cell activation during HIV-1 infection,we analyzed the relationship between the intensity of SAMHD1 and the percentage of HLA-DR in corresponding subpopulation,a strong positive association was found in CD8+T lymphocytes (LSQD:r=0.363,P=0.008;WSCC:r=0.246,P=0.037),but in CD4+T lymphocytes,the relationship between them is weak (LSQD:r=0.114,P=0.428;WSCC:r=0.099,P=0.408).Furthermore,mRNA levels of SAMHD1 and IFN-γ in PBMC were detected and the relationship between them were analyzed,positive relationship(LSQD:r=0.4776,P=0.0004;WSCC:r=0.395,P=0.0006) using Spearman rank correlation analysis further confirmed association between SAMHD1 and cell activation (Figure 2).

Figure 1 Comparation of SAMHD1 MFI and HLA-DR percentage in HD,WCSS and LSQD groups

Figure 2 Correlation of SAMHD1 expression and T cell activation in HIV/AIDS patients

4.DISCUSSION

Although TCM has been long practiced effectively for HIV/AIDS treatment,objective and biological evidences for classification and diagnosis of the TCM syndromes are still limiting to date.Deferentially expressed proteins or molecules inQiandYindeficiency,dampness-heat retention,accumulation of heat-toxicity,Yangdeficiency of spleen and kidney syndromes were previously researched.26,27In this study,we focused on lung-spleenQideficiency syndrome,one of the top popular TCM syndromes of HIV/ AIDS to characterize the levels of T lymphocyte subsets and SAMHD1 protein expression.In line with previous studies,28immun-ohematologic indexes CD4+T lymphocyte count and CD4/ CD8 ratio in Lung-spleenQideficiency syndrome group were lower compared to healthy donors,besides,we incorporated PLWHA without obvious clinical symptoms as pure control population so as to find out the specificity of the index changes in lung-spleenQideficiency individuals.The same trends were found when compared with WCSS group.These data suggested that worse impairment of immune function occured in LSQD patients.

We observed higher expression of SAMHD1 and HLADR in PLWHA compared with healthy controllers,in agreement with those in other reports.Previously research showed that a subset of HIV+elite controllers with lower T cell proliferation levels expressed higher SAMHD1 compared with healthy donors or viraemic progressors.29Additionally,study on changes in the level of cellular activation during disease progression suggested that CD38 and HLA-DR expression intensity in HIV-infected patients were higher than those in healthy volunteers.30Herein,Flow cytometry was used to detect the expression of SAMHD1 and HLA-DR in T cell subsets,in view of the strong and continuous expression of SAMHD1,mean fluorescence intensity was used to analyze the expression level of SAMHD1 in both CD4+and CD8+T lymphocytes.Statistical results indicated that expression of SAMHD1 in T cell subsets was significantly different among LSQD,WCSS and HD groups,pairwise comparison showed that the expression of SAMHD1 in LSQD and WCSS groups was higher than that in healthy subjects,especially in LSQD group.Meanwhile,HLA-DR expression in T cell subsets did not show significantly different among the three groups.These findings extend those syndrome studies confirming a more intensive objectification index in syndrome classification,so that TCM treatment of AIDS is no longer limited to the summary of experience,but based on clinical practice with scientific basis.Previous studies on the relationship between SAMHD1 expression and cell activation have been controversial.Using purified circulating CD4+T cells from uninfected donors,blocking B cell lymphoma 6 resulted in downregulation pSAMHD1 expression and T cell activation which indicated the positive relationship between them.31On the contrary,elevated SAMHD1 expression was found in the peripheral blood mononuclear cells of all patients with HIV-1,instead,immune activation decreased in HIV-1 elite controllers.32To further address the relationship between SAMHD1 expression and T cell immune activation,we analyzed the relationship between percentage of HLA-DR expression and SAMHD1 mean fluorescence intensity in corresponding cells based on a relatively large number of clinical samples.Interestingly,expression of SAMHD1 and HLA-DR was positively correlated in CD8+T lymphocytes,the relationship was weak in CD4+T lymphocytes.Similar to previous studies,33,34the positive correlation between SAMHD1 and IFN-γ in PBMC further confirmed the relationship between SAMHD1 protein and cell activation.The possible reason for this phenomenon is that HIV infection causes stress response,then activates immune cells,subsequently works up interferon production,and eventually highly expressing SAMHD1 to play an immune effect.

Together,these data indicated that SAMHD1 expression level positively associated with the level of T cell activation,which partly revealed complex interconnection between virological and immunological functions of SAMHD1.SAMHD1 acted as a sensor of foreign virus in the immune system,and then exerted immune response.However,the mechanism of SAMHD1 in regulating immune response needs to be further researched.Combination of T lymphocyte count and SAMHD1 level may be used as objective and quantitative reference index for the diagnosis of lungspleenQideficiency syndrome,extending existed research results of TCM syndromes.

In conclusion,this analysis provided results from three different groups including LSQD and WCSS HIV/AIDS patients in Henan province of China where HIV epidemic was still prevalent and healthy donor group in the same area.We demonstrated lower CD4+T lymphocyte count,lower CD4/ CD8 ratio and higher SAMHD1 in lung-spleenQideficiency syndrome patients,expression of SAMHD1 correlated positively with HLA-DR as well as interferon.These findings provided a clear indication about the relationship between SAMHD1 and cell activation which further revealed the complex antiviral and immunomodulatory functions of SAMHD1.Meanwhile,the data suggested that combination of T lymphocyte subsets count and SAMHD1 level may be used as objective and quantitative reference index for the diagnosis of lungspleenQideficiency syndrome.

5.ACKNOWLEDGMENTS

The author(s) are grateful to the clinicians who administer AIDS patients and establish medical record registers database.The authors also would like to thank the experiment participants and leaders of National Pilot Project for AIDS Treatment with TCM.Their efforts made this program possible.

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