CD14+CD16-Low Monocyte Subset Predicts Non-Progressive HIV Disease: Evidence of A New Prognostic and Diagnostic Biomarker

1.    Introduction

The phenotypic pliability and the differentiation ability of monocytes empower this cell type to play a crucial role in HIV pathogenesis through cellular differentiation, phagocytosis, and antigen presentation. Compared to T cells and macrophages, monocytes are much less permissive to HIV infection [1,2], although all these cells express HIV receptor CD4 and co-receptors CCR5 and/or CXCR4. In spite of less than 1% of circulating monocytes directly infected in vivo, infectious virus can be isolated from circulating monocytes in untreated patients and HAART responders [2,3], which could become a dominant source of plasma virus in HAART responders in whom HIV replication in activated T cells is blocked [4]. In addition, monocytes represent an important cellular reservoir by harboring and trafficking HIV into various tissue compartments through differentiating into tissue macrophages or dendritic cells, which enable productive HIV replication [4]. Furthermore, undifferentiated monocytic precursor cells, such as CD34+ progenitor cells, may be infected with HIV and pass on the virus to progeny monocytes and keep on renewing the viral pool in peripheral blood monocytes [5,6]. Monocyte subpopulations exist with differing levels of maturation and functions. Monocytes that express CD14, the LPS receptor, and CD16, the Fcylll receptor, are a mature population of cells that are highly susceptible to HIV.

Based on these sub-populations, monocytes can be subcategorized into subsets based on the surface expression of CD14 and CD16 antigens. CD14+CD16+ monocytes are present in significantly greater numbers in HIV-infected people, despite viral suppression, in contrast to individuals without HIV, but the modulation of such populations, as we have visualized by sub-categorizing, has never been looked into at various stages of HIV disease. Although the CD4+ T cell counts have been shown to predict HIV viremia, the actual predictive value of surface antigen changes on monocytes, particularly CD16 at different stages of plasma viremia has also not been evaluated. In this study, we have derived ex vivo monocytes from HIV positive patients with detectable (84-231,000 copies of HIV RNA/ml plasma) and Below Detectable Levels (BDL) of plasma viremia (<40 copies of HIV RNA/ml plasma), HIV negative individuals and therapy naïve HIV+ Long-Term Non-Progressors (LTNP) who have been infected with HIV for >25 years, remained therapy naïve and have maintained below detectable levels of plasma viremia throughout the course of infection. Since CD14+CD16+ monocytes represent an important heterogeneous cell population that is often targeted, particularly for HIV-1 entry, we evaluated the effects of HIV infection and distinct subsets of ex-vivo-derived CD16+ monocytes. We subdivided monocytes into CD14+CD16-high, CD14+CD16-medium and CD14+CD16-low populations and visualized the phenotypic changes in expression of CD16 antigen in CD14+ monocytes in HIV+ patients during different stages of plasma viremia.

2.  Methods and Materials

2.1.  Derivation and Processing of Ex-Vivo Monocytes from HIV Patients

The human Peripheral Blood Mononuclear Cells (PBMCs) were obtained from the EDTA blood of 50 HIV positive individuals [14 viremic patients; 29 BDL on Highly Active Antiretroviral Therapy (HAART) and 7 therapies naïve, aviremic LTNPs] and 4 HIV negative donors (See Table 1 and supplementary File 1 for Patient clinical details and raw Excel data). The work was cleared by the human Ethics Committee of the Sydney West Western Area Health Research Committee, Westmead Hospital, Sydney, NSW. Australia. All blood samples were collected strictly after individual informed written consent.

2.2.  Immuno-Staining and Flow Cytometry Analysis on Human Peripheral Blood Mononuclear Cells Samples

A two-color antibody panel was used to identify the CD14 and CD16 antigen expression on monocytes. Cells were ficolled and stained with CD14-PE (BD Biosciences, Australia) and CD16-Alex Fluor 647 (BD Biosciences, Australia) for 20 min at 40C. Following washing with PBS, cells were fixed with (2% Paraformaldehyde) for 10 Min at room temperature, washed and then re suspended in PBS before flow cytometry. Flow cytometry was performed on a Canto A cytometer (BD Biosciences, Australia) using DIVA 6.1.2 software (BD Biosciences). Monocyte population was first identified by FSC and SSC dot plot. Following gating on the CD14 positive population, based on the fluorescent intensity, CD16 expression on CD14 positive monocytes was divided into three groups, CD16-low, CD16-med and CD16-high. The same gating strategy was used across all samples. Percentages of three CD16 populations were analyzed by FlowJo software (v10.0.7; Treestar, USA).

2.3.  Statistical Analysis

Results are expressed as mean ± standard error. Differences among groups were measured using Student’s t test with one-tailed distribution and two sample equal variance test, P<0.05 was considered significant. The relationship of CD16 with CD4 count and viral load was determined by the correlation coefficient with the formula shown below.

3. Results

By categorizing monocyte population into CD14+CD16-high, CD14+CD16-medium and CD14+CD16-low, we could discriminate between viremic and aviremic HIV patients (Figure 1). There was considerable elevation of CD14+CD16-low population (80%) in HIV-negative individuals and 57% in LTNPS (p<0.0114), as opposed to 8% in HAART-treated viremic and aviremic (BDL) groups (BDL vs neg P<0.0000374 and Viremic vs neg P<0.0000056) (Figure 2, Table 3). Notable was that the CD14+CD16-low population never recovered despite complete viral control during HAART in the BDL group, suggesting their low levels as definite indicators of quality of monocyte in BDL and viremic groups when compared against their elevated levels in the LTNPs, who control the plasma virus naturally (LTNP vs BDL p<0.0075102 and LTNP vs Viremic p<0.0015809). Moreover, the HAART-treated groups (viremic and BDL) were also characterized by the elevated levels of CD14+CD16-high population (33 and 45%, respectively) (Figure 2, Table 2,3), when compared against LTNPs (BDL vs LTNP p<0.0000180 and Viremic vs LTNP p<0.0009354) and negative controls, which showed low levels of CD14+CD16-high populations. Although LTNPs were comparable in the expression of CD14+CD16-high populations with HIV negative donors (p<0.6796), they could be segregated from LTNPs based on CD14+CD16-medium and CD14+CD16-low populations (p<0.0048514 and p<0.0114045, respectively) (Table 3).

From these data two significant aspects of phenotypic regulation of monocytes are clear that the high levels of CD14+CD16- low and low levels CD14+CD16-high of populations characterize LTNPs and HIV-negative individuals implying that although the LTNPS are closer to HIV negative individuals when compared against the viremic and BDL groups, they could be segregated from each other by both CD14+CD16-medium and -low populations. Secondly, that the CD14+CD16-low and - high populations of monocytes were strong and reliable indicators of plasma viremia, immune deterioration and the quality of monocytes during viremia. It also raises the possibility that this phenotypic modulation in monocyte may also be linked to the deterioration in quality of monocytes, which even fails to recover despite HAART as apparent in BDL HIV patients on HAART.

We also evaluated, if this modulation in monocytic subset was linked to CD4+T cell counts, but we obtained inverse relationship between monocytic deterioration and CD4+T cells counts. The correlation between CD16-med vs CD4 count was 0.31176704 for the BDL group, while in Viremic group the CD16-high vs CD4 count showed p value of 0.42996958, with R2 values for both groups at 0.0972; 0.18487, respectively, suggesting that monocytic deterioration has less bearing on overall CD4+ T cell counts (supplementary file 1).

4. Discussion

Quality of monocyte subsets and their modulation in vivo plays a vital role in guiding immune responses during HIV infection [7]. The data shown in our study not only highlights the significance of CD16+ monocytes in HIV infection but also demonstrates the vital dynamics of these monocyte subsets exhibited in viral suppression and disease progression. This novel way of visualizing the trichotomy between low, medium and high subsets of CD14+CD16+ monocytes from HIV- and HIV+ groups has allowed us for the first time in elucidating not only the immunologic relationship they hold with different stages of HIV disease, but also their association with the natural control of HIV disease in therapy naïve Elite Controllers (EC)- a phenomenon not shown previously. We believe that this could offer new insights into the roles of innate immunity in HIV pathogenesis, underpinning their role as new biomarkers in HIV disease diagnosis and prognosis.

There is a significant rise in the proportions of non-classical monocytes in HIV-1 disease [8,9]. This heterogeneous subset which represents a minor sub-population of monocytes in healthy individuals, increases in peripheral blood and may represent up to 40% of total circulating monocytes during HIV infection and in patients with AIDS [9]. An important goal in clinical manifestation and diagnosis of HIV infection is to find laboratory parameters to monitor the disease progression. By further diversifying the established classification of non-classical and intermediate monocytes population into three subsets based of on CD16+ antigen, we sub categorized them into low, medium and high clusters. Through this way of visualizing, we could clearly discriminate between viremic and aviremic HIV patients (Figure 1 and Table 3). There was considerable and statistically significant elevation of CD14+CD16-low population (80%) in HIV-negative individuals in comparison to 57% in the LTNPs (p<0.0114). In contrast, it was only 8% in HAART-treated viremic and aviremic groups (BDL vs neg P<0.0000374 and Viremic vs Negatives P<0.0000056) (Figure 2 and Table 2,3).

Notably, the CD14+CD16-low population never recovered despite complete viral control during HAART in the BDL group, suggesting their low levels as definite indicators of quality of monocyte in the BDL and viremic groups when compared against their high levels in the LTNPs, who control the plasma virus naturally (LTNP vs BDL p<0.0075102 and LTNP vs Viremic p<0.0015809). And secondly, this also highlights the fact that even in the face of complete control of viremia during HAART, the CD14+CD16-low population failed to recover, suggesting HAART has no bearing on the recovery of this population, which may be one of the underlying reasons for partial immune restoration during HAART.

The robust maintenance and elevation of CD16-low populations and substantially low levels of CD16-high populations distinctively in HIV-negative and non-progressing HIV+ individuals correlated with the natural control of HIV in the LTNPs, thereby demonstrating the ability of this subset in predicting the strength of the immune system at different stages of HIV disease and their possible role in innate immunity. Further comparing CD16-low population at complete control of plasma viremia under HAART as opposed to its natural control in LTNPs, it appears that the quality of immune cells and the overall strength of the immune system is vital for this cell subset, and even a little virus compromises their quality. This fact emerges from the comparison of LTNPs with HIV-negative individuals, where the difference becomes apparent despite the two groups sharing closeness. This further suggests that LTNPs may maintain therapy naïve and virus-free status, the overall numbers of CD16-low population in the blood can serve not only as excellent indicators of even very low and below detectable levels viremic states as seen in case of LTNPs, but also in stratifying individuals based on the strength of their immune system.

Further to this, the HAART-treated groups (viremic and BDL) were also characterized by elevated levels of CD14+CD16-high populations (45% and 33% respectively) (Figure 2 and Table 2), when compared against LTNPs (BDL vs LTNP p<0.0000180 and Viremic vs LTNP p<0.0009354) and negative controls, who displayed low levels of CD14+CD16-high populations. Although LTNPs were comparable in the expression of CD14+CD16-high populations with HIV negative donors (p<0.6796), the HIV- donors could only be segregated from the LTNPs based on CD14+CD16-medium and CD14+CD16-low populations (p<0.0048514 and p<0.0114045, respectively) (Figure 2 and Table 3), suggesting a clear demarcation between HIV- and HIV+ individuals, raising a possibility of subliminal infection in LTNPs which is under a tight natural control.

Furthermore, HIV infectivity correlated with elevated CD16-mediium monocytes population, underscoring the distinction between HIV positive patients and negative individuals, which was highlighted to a measurable extent by the expression of CD16-medium population relatively pronounced in the HIV+ group 47% (BDL) and 58% (Viremic), as opposed to 39% (LNTP) and 15% (healthy donors)- the HIV negative group, implying the functional relevance of CD16-low and CD16-med monocytic populations in discriminating LTNPs from the negative donors. Thus, for the maintenance of the LTNP status, it was the high levels of CD16-low and low-levels of CD14_CD16-high appeared essential, which essentially coincides with the levels HIV- healthy individuals.

Theiblemont et al., (1995) [9] suggested that IN HIV Infection the expansion of CD14 low CD16 high monocyte subset, which produce high amount of TNF-alpha and IL-1 alpha may participate in the immune dysfunction observed during HIV infection. Thus their elevated levels in patients with viremia, is consistent with our data. Also consistent is their low levels in healthy individuals, in addition to LTNPs-which Thieblemont et al., did not show. The CD14lowCD16high circulating monocytes co-express MAX.1, p150, 95 and HLA- DR, which are typical of tissue macrophage markers. These cells also express higher levels of Intracellular Interleukin (IL)-1 alpha and Tumor Necrosis Factor (TNF)-alpha than the CD14highCD16low monocyte population from the same patients, which could form the biological basis of natural viremia control in LTNPs as seen our study.

5. Conclusions

The robust maintenance and elevation of CD14+CD16-low populations and low levels of CD14+CD16-high populations uniquely in HIV-negative and non-progressing HIV+ individuals correlate with natural control of HIV in LTNPs and is able to predict viremia, strength of the immune system and quality of both monocytes and T cells. LTNPs and HIV-negative individuals could be segregated based on CD14+CD16-medium populations, and despite the elevation of this population in the BDL and viremic groups, they significantly differed from LTNPs (p<0.0000066), suggesting possible differences in the quality of these monocytes in the LTNP group. These data may allow the development of new diagnostic and prognostic tools for the prediction of HIV disease staging in HIV patients.

6. Financial and Competing Interest Disclosure

NKS is thankful to the NHMRC Development grant for funding the project. Viviane Conceicao is thankful to the University of Sydney for A University of Sydney Postgraduate Award (IUPA) scholarship and Westmead Medical Research Foundation for a top-up grant. SP is thankful to the Arin Apcarin WMI Scholarship for his work. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

SUPPLEMENTARY DATA

        The spread sheet includes the viral load number as a Logrithmic scale (log10) for each patient. The CD4/CD8 count was also included for each patient. N:B... those highlighted in blue are those that did not have the CD4/CD8 stats available hence (n/a) highlighted in red is that of subjects with high CD16 low percentage. normal range for CD4count (380-1390) normal range for CD8+ T cell count (200-690).

	CD16high		CD16med	CD16low		Viral load (Log10)		CD4 count		CD8 count	Group		Viral Load (new)
3: Bn_CD14-PE+CD16AF647.fcs	1.99		66	31.6		BDL		476		571	BDL		40
9: BrAl_CD14-PE+CD16-AF647.fcs	66.6		30.8	2.53		BDL<40		682		946	BDL		40
11: BRJU_CD14-PE+CD16-AF647.fcs	73		24.2	2.84		BDL		706		706	BDL		40
13: CRBR_Cd14-PE+CD16-AF647.fcs	7.06		66.7	26.2		BDL		363		1597	BDL		40
15: DEBR_CD14-PE+CD16AF647.fcs	75.3		19.8	4.81		BDL<40		45		566	BDL		40
19: DMR_CD14-PE+CD16AF647.fcs	2.32		73.9	23.7		BDL		515		1576	BDL		40
23: Ga Wa_CD14-PE+CD16-AF647.fcs	49.6		48.3	2.04		BDL		435		435	BDL		40
27: GA_CD14-PE+CD16-AF647.fcs	8.72		61.9	29.4		BDL		437		494	BDL		40
29: GS_CD14-PE+CD16-AF647.fcs	61.6		36.2	2.19		BDL<40		338		520	BDL		40
31: G G_CD14-PE+CD16-AF647.fcs	30.7		57.6	11.7		3.2:1690		368		1472	BDL		40
35: HA_CD14-PE+CD16-AF647.fcs	59.6		36.1	4.28		BDL		615		315	BDL		40
37: Harris_CD14-PE+CD16AF647.fcs	5.23		77.1	17.5		BDL		437		760	BDL		40
39: HK_CD14-PE+CD16-AF647.fcs	43.3		53.8	2.83		BDL		443		586	BDL		40
41: iAK_CD14-PE+CD16-AF647.fcs	69.3		29.3	1.34		BDL		n/a		n/a	BDL		40
47: KA_CD14-PE+CD16AF647.fcs	84.8		14.4	0.82		BDL		377		406	BDL		40
49: KJ_CD14-PE+CD16AF647.fcs	9.96		76.8	13.2		BDL		922		1920	BDL		40
51: lu 700108_CD14-PE+CD16AF647.fcs	79.1		19.8	1.04		BDL		592		444	BDL		40
59: m P_CD14-PE+CD16-AF647.fcs	63.8		35.5	0.693		BDL		n/a		n/a	BDL		40
61: mJ_CD14-PE+CD16-AF647.fcs	55.9		40.9	3.13		BDL		608		496	BDL		40
63: MI_CD14-PE+CD16AF647.fcs	16.7		76.6	6.64		BDL<40		1078		868	BDL		40
71: pO T_CD14-PE+CD16-AF647.fcs	83.6		15.8	0.547		BDL		n/a		n/a	BDL		40
75: SAR_CD14-PE+CD16AF647.fcs	83.3		15.8	0.904		BDL		n/a		n/a	BDL		40
77: S D_CD14-PE+Cd16-AF647.fcs	24.9		70.7	4.36		BDL		1131		485	BDL		40
81: sp jo_CD14-PE+CD16AF647.fcs	75.2		22.6	2.18		BDL		907		734	BDL		40
83: Th_CD14-PE+CD16-AF647.fcs	52.7		46.3	1.01		BDL<40		450		868	BDL		40
85: TO_CD14-PE+CD16AF647.fcs	49.4		47.4	3.28		BDL<40		n/a		n/a	BDL		40
91: VAR_CD14-PE+CD16-AF647.fcs		18.8	70.5	10.6	BDL		934		759			BDL		40 40 40
97: YU_CD14-PE+CD16AF647.fcs		3.7	84.3	11.9	BDL		n/a		n/a			BDL
101: 15893 pl lo_CD14-PE+CD16-AF647.fcs		40.9	48.8	10.3	BDL		834		1607			BDL
1: ANCR_CD14-PE+CD16AF647.fcs		24.2	62.2	13.6	2.0:107		194		1642			Viremic		107
5: Be Ra_CD14-PE+CD16-AF647.fcs		34.5	63.9	1.6	4.4:22900		320		1109			Viremic		22900
17: DIJA_CD14-PE+CD16AF647.fcs		8.67	55.9	35.5	84		222		1112			Viremic		84
21: EP_CD14-PE+CD16_AF647.fcs		40.4	53	6.62	1.9:91		n/a		n/a			Viremic		91
25: GTP_CD14-PE+CD16AF647.fcs		20.3	65.1	14.6	231000		7		318			Viremic		231000
33: GWJ_CD14-PE+CD16AF647.fcs		31.8	66.2	2.02	4.1:13800		n/a		n/a			Viremic		13800
45: JU_Cd14-PE+CD16-AQF647.fcs		26.2	64	9.88	2.0:104		850		975			Viremic		104
53: LE(2)_CD14-PE+CD16AF647.fcs		23.4	62.2	14.4	4.5:33600		156		1672			Viremic		33600
55: MS_CD14-PE+CD16-AF647.fcs		54.6	44.5	0.92	2.1:121		597		760			Viremic		121
57: mR_CD14-PE+CD16-AF647.fcs		50.9	47.4	1.64	2.1:120		597		688			Viremic		120
67: ok_CD14-PE+CD16AF647.fcs		42.9	52.8	4.29	1.6:40		520		1378			Viremic		40
87: TK_CD14-PE+CD16-AF647.fcs		14.4	75.2	10.4	2.3:192		n/a		n/a			Viremic		192
95: YF_CD14-PE+CD16AF647.fcs		56.2	42	1.82	2.1:121		235		706			Viremic		121
99: YY_CD14-PE+CD16-AF647.fcs		43.4	50.6	6	3.5:2820		487		502			Viremic		2820
69: PH_CD14-PE+CD16AF647.fcs		6.34	39.2	54.4	BDL		660		1200			LNTP		40
79: SD_CD14-PE+CD16AF647.fcs		0.226	39	60.3	BDL		669		491			LNTP		40
1: C122_Double +.fcs		4.9	17.1	78			664		920			LNTP		40
3: C13_Double +.fcs		1.14	12.5	86.3			760		1110			LNTP		40
5: C53_Double +.fcs		5.48	33	61.5			854		900			LNTP		40
7: HIPE_Double +.fcs		4.59	36	59.4			710		1900			LNTP		40
9: S24_Double +.fcs		7.8	42.7	49.5			590		1720			LNTP		40
7: BW_CD14-PE+CD16AF647.fcs		4.35	19.4	76.2	Normal control							Negative
43: JO_CD14-PE+CD16-AF647.fcs		4.24	7.59	87.9								Negative
73: PG 2 retake_CD14-PE+CD16AF647.fcs		7.45	21.2	71.2	normal control							Negative
93: VC_CD14-PE+CD16AF647.fcs		3.82	11.7	84.3	Normal control							Negative

Figure 1: The representative flow cytometry dot pots showing the gating strategy of discriminating the CD14+CD16high, CD14+CD16-medium and CD14+CD16-low populations in HIV patients at different stages of plasma viremia compared to the HIV negative healthy donors.

Figure 2: Histogram showing comparative analysis of CD14+CD16-high (blue), CD14+CD16- medium (brown) and CD14+CD16-low (green) populations in HIV patients at different stages of viremia compared to the normal donors. * Indicates P value <0.05 compared individual CD16 populations in HIV patients to the negative controls accordingly. # Indicates P value

<0.05 compared individual CD16 populations in BDL and Viremic patients to the LTNP patients accordingly.

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