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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Cell Immunol. 2021 Feb 10;362:104304. doi: 10.1016/j.cellimm.2021.104304

Residual Immune Activation in HIV-Infected Individuals Expands Monocytic- Myeloid Derived Suppressor Cells

Ritesh Singh a, Mouli Chakraborty b, Anuradha Gautam b, Suman K Roy c, Indranil Halder d, Jamie Barber e, Ankita Garg e,*
PMCID: PMC8112256  NIHMSID: NIHMS1672375  PMID: 33610024

Abstract

HIV-infected individuals on combined antiretroviral therapy (ART) with virologic suppression exhibit sustained immune dysfunction. Our recent work has highlighted that monocytic myeloid derived suppressor cells (M-MDSC) are elevated in these individuals and suppress immune responses. Factors responsible for M-MDSC expansion in vivo are unknown. Here we compared circulating frequency of M-MDSC in HIV-infected persons from the US and India where HIV subtype-B or –C predominate, respectively. We further investigated soluble mediators of residual immune activation in two cohorts and determined their correlation with M-MDSC expansion. Our findings show that M-MDSC are elevated and correlate with plasma levels of IL-6 in both cohorts. Chemokines CXCL10, CCL4 and CXCL8 were also elevated in HIV-infected individuals, but were not correlated with M-MDSC. These findings support that IL-6 is important in M-MDSC expansion which is independent of HIV subtype.

Keywords: Myeloid derived suppressor cells, HIV, IL-6, Immune activation, People Living with HIV

1. INTRODUCTION

Human immunodeficiency virus type 1 (HIV-1) infection causes profound immune suppression leading to progressive destruction of the immune system in untreated patients. An early initiation of combined anti-retroviral therapy (ART) inhibits viral replication to undetectable levels and reconstitutes CD4+ T cell count, resulting in a substantial decrease in morbidity and AIDS-related mortality [1–3]. Despite virologic suppression with ART, HIV-infected individuals only partially restore immune function. Moreover, they continue to exhibit residual immune activation (RIA) characterized by the continued elevation of IL-6, IL-1, TNF-α, C-reactive protein, D-dimers, fibrinogen and soluble (s)CD14 (a marker of microbial translocation) and CD163 [4–9].

Recent studies establish expansion of myeloid derived suppressor cells (MDSC) in HIV-infected individuals, and correlate with disease progression [10–12]. MDSC are a heterogeneous cell population comprising myeloid cell progenitors and immature cells, which suppress both innate and adaptive immune responses by utilizing multiple mechanisms and expanding regulatory T cells (Tregs) in both HIV and other pathologic conditions [10, 13–19]. MDSC expansion is regulated by various cytokines, including interleukin (IL)- 6, IL-10, prostaglandins, stem-cell factor, granulocyte macrophage colony-stimulating factor, transforming growth factor β, vascular endothelial growth factor, and tumor necrosis factor α (TNF-α) [15, 20, 21]. In humans, MDSC are identified as cells expressing common myeloid markers (CD11b+CD33+HLA DR−/lo) and, depending on the presence of CD15+/CD66b+ or CD14+, are divided into granulocytic (G-MDSC) or monocytic (M-MDSC) subsets, respectively [22–24]. We and others have shown that MDSC frequency and numbers decline during course of ART but when compared to HIV-uninfected healthy controls, remain elevated upon virologic suppression and CD4+ T cell recovery [10–12, 25, 26]. Our in vitro studies establish that replication incompetent HIV can expand M-MDSC in an IL-6 dependent manner, suppress T-cell functions and induce regulatory T-cell expansion [10].

In the present study, we performed a cross-sectional analysis of the circulating frequency of M-MDSC and its association with pro-inflammatory cytokines in HIV-infected individuals with virologic suppression, and compared it to HIV-uninfected healthy controls. Additionally, we compared M-MDSC and cytokine levels in two geographically distant HIV-infected cohorts, predominantly infected with HIV subtype B (US cohort) and subtype C (India cohort). Our findings indicate M-MDSC expand irrespective of geographical location and HIV subtype. Additionally, although there are differences in the levels of proinflammatory cytokines between the two cohorts studied, M-MDSC exhibited a positive correlation with plasma IL-6 levels in both groups.

2. RESULTS

2.1. Participants, Clinical Characteristics, and HIV and TB status

Samples were collected from HIV (+) and HIV (−) subjects. All HIV (+) subjects were on anti-retroviral therapy for over a year and had achieved suppression of HIV RNA (defined as <50 copies/mL) with ART. None of the participants reported of any systemic infections. We did not perform CB NAAT on samples of USA cohort. Clinical and demographic data are summarized in Table 1.

TABLE 1.

India Cohort US Cohort p-value p-value
HIV (−) (N=15) HIV (+) (N=30) HIV (+) (N=30) HIV (−) vs HIV (+) India Cohort HIV (+) India vs US Cohort
Age in years Median (Range) 45 (23-60) 40.5 (30-62) 27 (20-46) 0.882 0.0001
Absolute CD4 (cells/mm3) Median (Range) 938 (341-1720) 585.5 (268-1089) 730 (262-1548) 0.0003 0.1
LTBI (QFT) (%) 5 (33) 8 (29) 0 (0) NS -
CB-NAAT + (%) 0 (0) 0 (0) ND - -
Viral Load - ND UD§ - -
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*

Non-significant

Not determined

§

Undetectable

2.2. Increased M-MDSC in HIV-infected individuals despite prolonged treatment with anti-retrovirals:

We and others have previously demonstrated that M-MDSC persist despite virologic suppression in HIV-infected individuals [10–12, 25]. We sought to determine if M-MDSC persist in individuals infected with unrelated HIV-subtype and on ART for over a year. For this, we measured the frequency of M-MDSC in the whole blood of HIV patients enrolled in the ICTC at a primary health care hospital of India, and compared it with healthy controls. The frequency of M-MDSC in HIV (+) was higher than age matched healthy controls (0.54±0.08 vs 0.05±0.01; p=<0.0001, Figure 1A). Since India constitutes a high burden of latent tuberculosis infection (LTBI), in order to study the effect of subclinical M tuberculosis infection in persistence of M-MDSC, we determined LTBI in HIV (+) and HIV (−) group by Quantiferon and CB-NAAT. All study participants were negative for M tuberculosis infection by CB-NAAT and there was no difference in the incidence of QFT positivity between HIV (+) and HIV (−) study group (Table 1). Next we sought to compare the frequency of M-MDSC in HIV-infected individuals in India and US cohort. Despite comparable absolute CD4 cell count, we found higher frequency of M-MDSC in US cohort as compared to India cohort (0.95±0.11 vs 0.54±0.08; p=<0.01, Figure 1B).

Figure 1: Increased M-MDSC in HIV-infected individuals despite virologic suppression:

Figure 1:

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Heparinized blood obtained and stained with anti-CD3,-CD19,-CD11b,-CD33,-CD14, -HLA DR Abs, cells were analyzed as CD3−CD19−CD11b+CD33+CD14+HLA DR−/lo by flow cytometry. (A) Percentages of M-MDSC was compared between HIV (−) and HIV (+) on ART with virologic suppression in Indian cohort. (B) Percentages of M-MDSC was compared between HIV (+) US and India cohort. Each dot in the plots depict data of each individual donor, the plots include observations from 25th to 75th percentile. The horizontal line represents the median value. **p<0.001, ****p<0.0001.

Collectively, these findings suggest that M-MDSC remain elevated in HIV-patients infected with subtype C. However, differences exist in M-MDSC numbers between the two populations.

2.3. Monocyte driven immune activation in HIV-infected individuals as a result of ART:

Innate immune activation is a critical mediator of MDSC expansion in various microbial and cancer pathologies [27–31]. Successfully treated people living with HIV continue to experience monocyte activation driven inflammation, which is largely due to the gut barrier dysfunction [32–34]. In order to determine monocyte activation and gut dysfunction, we initially measured sCD163 and sCD14 in the plasma of HIV-infected individuals of US and India cohorts. The levels of sCD14 and sCD163 were comparable in HIV-infected and healthy controls from India (850±82.4 vs 837±80.3; p=0.9 and 178.2±14.9 vs 210.5±25.9 ng/ml; p=0.5, respectively, Figures 2A and B). HIV-infected US cohort demonstrated an increased level of sCD163 when compared to HIV-uninfected control group (115.5±12.8 vs 159.6±6.6 ng/ml; p=0.001, Figure 2E) and comparable level of sCD14 (176.3±3.6 vs 187.2±5.1 ng/ml; p=0.19, Figure 2D).

Figure 2: Monocyte driven immune activation in HIV-infected individuals:

Figure 2:

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Plasma obtained from heparinized blood was stored at −80°C until further use. The quantity of sCD14, sCD163 and IL-22 was measured in plasma by ELISA, as in Methods. Quantities were compared in the study groups of (A-C) India cohort, (D-F) US cohort and (G-I) HIV (+) US and India cohort. Each dot in the plots depict data of each individual donor, the plots include observations from 25th to 75th percentile. The horizontal line represents the median value. ***p=0.001, ****p<0.0001.

Cytokine IL-22 has gut epithelial regenerative properties and provides innate immune protection against bacterial and fungal infection [35–38]. We compared plasma level of IL-22 in HIV-infected and healthy controls from US and India. The quantity of IL-22 in HIV-infected with virologic suppression was comparable to healthy controls both in US and India cohort (51.84±6.7 vs 41.5±4.3 pg/ml; p=0.15 and 58.6±4.6 vs 73.2±6.5 pg/ml; p=0.5, respectively, Figures 2C and F).

Next, to determine if the increased M-MDSC frequency in HIV-infected US cohort is due to aberrant gut associated cytokines, we compared sCD14, sCD163 and IL-22 levels among HIV-infected from US and India. sCD14 level was comparable among the 2-cohorts, however both sCD163 and IL-22 quantities were higher in HIV-infected individuals with prolonged treatment with anti-retrovirals in India (Figures 2G–I). Of note, despite the levels of sCD163 being more in HIV-uninfected controls or HIV-infected in Indian cohort, the M-MDSC frequency in Indian cohort was low. Collectively, these findings suggest that M-MDSC expansion in these individuals is independent of dysbiosis in mucosal barriers.

2.4. Cytokine and chemokine levels in HIV-infected with virologic suppression:

Although early initiation of ART causes significant reduction in immune activation, many immune activation pathways remain abnormal despite virologic suppression. Independent studies have established sustained immune activation in individuals infected with unrelated HIV subtype and residing in distant geographical location [1, 4, 39]. Here, we sought to determine and compare residual immune activation (IA) in HIV-infected individuals with healthy controls from US and India. For this, we measured proinflammatory cytokines and chemokines in the plasma of the study groups using multiplex assay. We found HIV-infected individuals as compared to healthy controls in US cohort exhibited an increased plasma levels of CXCL2, CX3CL1, CCL2, CCL5 and IL-18; the quantities of these inflammatory mediators were comparable in HIV-infected and –uninfected individuals from India. However, CXCL-10, CCL4, CXCL8 and IL-6 quantities were increased in HIV-infected individuals both in US and India (Table 2 and Supplementary Figures 1 and 2).

TABLE 2.

India Cohort US Cohort
Analytes HIV (−) (n=15) HIV (+) (n=30) p-value HIV (−) (n=15) HIV (+) (n=30) p-value
sCD14 (ng/ml) (Range) 171.1 (93.1-354.2) 139.5 (42.4-376.4) 0.4 173.3 (156.1-201.5) 182.9 (149.9-269.7) 0.2
sCD163 (ng/ml) (Range) 826.8 (345.4-1450) 889.6 (126.8-1920) 0.9 98.66 (66.35-220.5) 159.2 (113.7-234.1) 0.001
IL-22 (pg/ml) (Range) 54.5 (31.7-88.6) 62.1 (29.7-155) 0.5 55.1 (1.8-95.3) 44.4 (1.1-87.22) 0.15
CXCL2 (pg/ml) (Range) 516.5 (204.5-1160) 707.7 (73.43-2584) 0.21 354.3 (83.31-717.7) 1276 (293.3-3717) <0.0001
CX3CL1 (pg/ml) (Range) 1011 (252-1564) 1288 (252-1917) 0.13 16.28 (8.28-453) 1265 (905.4-1886) <0.0001
CXCL10 (pg/ml) (Range) 77.62 (32.77-160.4) 100.9 (22.18-786.3) 0.01 27.48 (8.48-59.74) 69.42 (39.14-368.9) <0.0001
CCL4 (pg/ml) (Range) 80.78 (37.01-198.6) 172 (37.01-725.3) 0.006 79.89 (59.12-117.4) 142.7 (79.89-678.2) <0.0001
CCL2 (pg/ml) (Range) 151.5 (61.03-257) 102.6 (38.43-254.9) 0.07 134.9 (57.12-353) 215 (95.6-392.3) 0.04
CCL5 (ng/ml) (Range) 569.5 (315.4-818.5) 571.2 (248-2400) 0.48 14.37 (2.46-42.49) 51.48 (6.09-178.8) <0.0001
CXCL8 (pg/ml) (Range) 15.15 (3.94-46.78) 34.58 (3.72-370.9) 0.02 0.03 (0.03-7.7) 26.24 (0.72-1165) <0.0001
IL-18 (pg/ml) (Range) 192.1 (62.97-339.6) 168.1 (44.38-602.1) 0.5 244.9 (16.59-2420) 395.3 (194.9-817) 0.04
MBL (ng/ml) (Range) 23.57 (21.18-31.77) 21.41 (11.45-74.1) 0.33 26.7 (22.17-31.67) 27.19 (8.6-55.12) 0.66
IL-6 (pg/ml) (Range) 0.14 (0.1-3.49) 0.93 (0.1-8.55) 0.003 5.51 (2.57-7.84) 7.95 (2.64-46.98) 0.006
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Since US and India differ not only in their geographical location, but also HIV subtype circulating in these regions, with subtype -B and –C dominant in US and India, respectively [40–43], we sought to determine if these differences potentially affect the IA status of HIV-infected individuals. For this, we compared the chemokine/cytokines levels in HIV-infected individuals from US and India, and found higher quantities of CXCL2, CCL2, CCL5, IL-18 and IL-6 in HIV-infected US cohort (1679±182.7 vs 953.8±131.7 pg/ml, p=0.002; 228±15 vs 115±11.24 pg/ml, p=0.0001; 61.5±8 vs 0.8±0.1, p=0.0001; 415±24 vs 175.6±21.6 pg/ml, p=0.0001 and 13.6±2.3 vs 1.6±0.3 pg/ml, p=0.0001, respectively) (Figures 3A, E, F, H and I). Compared to the US cohort, levels of CXCL10 remained high in the HIV-infected India cohort (91.3±13.3 vs 159.8±28.7, p=0.001) (Figure 3C), and MBL was marginally high in the US cohort (31.9±2.3 vs 28±3 pg/ml, p=0.05) (Figure 3J); CX3CL1, CCL4 and CXCL8 were comparable in the two study cohorts (Figures 3B, D and G). These findings collectively suggest sustained hyper inflammation in HIV-infected individuals irrespective of virus subtype and geographic location; however, differences occur in immune activation pathways in different population resulting in differential inflammatory outcome(s).

Figure 3: Proinflammatory mediators in HIV-infected individuals from US and India cohort:

Figure 3:

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Plasma obtained from heparinized blood was stored at −80°C until further use. (A-J) The quantity of proinflammatory mediators was measured by Luminex® Human XL Cytokine assay and compared between HIV (+) US and India cohort. Each dot in the plots depict data of each individual donor, the plots include observations from 25th to 75th percentile. The horizontal line represents the median value. *p=0.05, **p=0.001, ****p<0.0001.

2.5. M-MDSC expansion depends on proinflammatory cytokines:

We found compared to healthy controls, a higher frequency of M-MDSC is present in HIV-infected individuals in US and India, irrespective of dominant HIV subtype in these geographical locations. We also found, of all the inflammatory mediators, levels of CXCL10, CCL4, CXCL8 and IL-6 (Table 2) are consistently elevated in HIV-infected individuals of both cohorts. Therefore, we sought to determine if these soluble factors are associated MDSC expansion in HIV-infected individuals with virologic suppression. We found IL-6 quantity positively correlated with the frequency of M-MDSC in both cohorts separately (Spearman correlation r=0.45, p=0.03 and r=0.4, p=0.04, respectively; Figure 4), and also when combined (Spearman correlation r=0.7, p=<0.0001). CXCL8 quantity correlated with M-MDSC frequency in India but not in the US or when combine together; CXCL10 and CCL4 did not show any correlation with MDSC (Figure 4). These data demonstrate, even though differences may occur between the cohorts, proinflammatory cytokine IL-6 seems to be an important mediator of M-MDSC expansion in HIV-infected individuals.

Figure 4: Proinflammatory cytokines in HIV-infected individuals mediate M-MDSC expansion:

Figure 4:

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The chemokine/cytokine in plasma were quantified by Luminex® Human XL Cytokine assay (CXCL10, CCL4 and CXCL8) or ELISA (IL-6) correlated with circulating frequency of M-MDSC in HIV (+) on ART with virologic suppression in (A-D) US cohort, (E-H) Indian cohort and (I-J) combined. Each dot in the plots depict data of each individual donor.

3. DISCUSSION

The expansion of M-MDSC occurs during HIV infection in human and non-human primates, but we and others have demonstrated an increased frequency of G- and M- MDSC in HIV-infected individuals with virologic suppression as a result of successful ART [11, 12, 25, 44]. Using an in vitro system we demonstrated that IL-6 mediates M-MDSC expansion in the presence of infectious or non-infectious HIV, and suppresses immune responses [10]. In line with these results, herein we show that IL-6 is associated with in vivo M-MDSC expansion in HIV-infected individuals on ART and with virologic suppression, and this is irrespective of geographical location and predominant HIV subtype. We also demonstrate that even though hyper inflammation persists in HIV-infection, the immune activation pathways differ depending on the predominant HIV- subtype.

MDSC are immune suppressive cells that expand during various pathological conditions as a result of acute or chronic inflammation. The expansion of both G-MDSC and M-MDSC has been clearly reported during primary or chronic HIV-infection irrespective of ART regimen, and their frequencies exhibit a positive correlation with viral load and negative correlation with CD4+ T cells [10–12, 45, 46]. We and other have shown that persistent MDSC in HIV-infected individuals on ART with CD4+ T cell recovery and virologic suppression continue to suppress CD4+ and CD8+ T cell function, thus contributing to immune dysfunction in these individuals [10, 11, 25]. Since the phenotypic markers to identify MDSC in peripheral blood are present on other myeloid cell subsets, the hallmark feature of MDSC is the demonstration of their ability to suppress immune activity of other immune cells. We found that depleting M-MDSC in HIV-infected individuals in India increased IFNγ production in response to plate bound anti-CD3 and –CD28 antibodies (unpublished observation). HIV itself or HIV encoded gp120 and Tat proteins induce MDSC expansion by altering the transcription of genes essential for myeloid cell development leading to MDSC accumulation [10, 11]. In this regard, HIV gp120 induces IL-6, which facilitates HIV replication in monocytes/macrophages by regulating transcription factor C/EBPβ and drives the expansion of pSTAT3 expressing MDSC [10, 47–49]. Levels of IL-6 are elevated in serum of HIV-infected persons, which decline following administration of ART but remain high as compared to HIV-uninfected healthy controls [50, 51]. To our knowledge, this is the first demonstration of a direct correlation of plasma IL-6 level with circulating frequency of M-MDSC in HIV-infected individuals infected with subtype B or C. We found HIV-infected from US compared to India, exhibit a higher frequency of M-MDSC with a simultaneous increased quantity of plasma IL-6. These findings further implicate IL-6 as an important mediator of M-MDSC expansion. In addition to IL-6, levels of CXCL10, CCL4 and CXCL8 remain elevated in HIV-infected individuals infected with subtype -B or -C. However, none of these inflammatory mediators correlate with M-MDSC frequency in HIV-infected individuals with the exception of CXCL8, which exhibited a positive correlation with MDSC frequency in HIV-infected individuals from India. Although the mechanism of CXCL8 mediated MDSC expansion is not established, CXCL8 facilitates differentiation and subsequent mobilization of MDSC in patients diagnosed with several types of cancers [52, 53]. Chemokine CCL2 mediated trafficking of MDSC from bone marrow to periphery has been shown in SIV-infected macaques, and it is likely that CXCL8 contributes to MDSC mobilization in HIV-infection [54]. Elevated level of IL-6 is associated with older age, higher body mass index, low nadir CD4+ T cell count. However, because of the retrospective nature of this research, we were unable to determine these parameters for each patient. Future studies designed to correlate IL-6 with these clinical parameters along with M-MDSC will be important to understand their role in M-MDSC expansion in HIV-infection.

A major driver of ongoing chronic immune activation in HIV-infected individuals on ART is monocyte driven inflammation and HIV-induced injury of mucosal barrier. CD14 is the receptor for lipopolysaccharide (LPS) - a cell wall component of gram-negative bacteria. A high level of plasma sCD14 shed by monocytes is reflective of LPS exposure released due to gut barrier dysfunction [55]. Additionally, IL-22 provides innate immune protection against microbes as well as enhances the proliferation and reconstitution of gut epithelial cells [35, 38, 56]. Although IL-22 driven mucosal protection during HIV-infection is not well defined, but IL-22 helps goblet cells to produce mucus-associated proteins and synergize with IL-17 to enhance the expression of antimicrobial peptides at the mucosal tissues [57]. IL-22 producing cells dramatically deplete during chronic HIV-infection further contributing to microbial translocation [36]. However, we did not observe aberrant sCD14 or IL-22 in the HIV-infected cohorts in this study, corroborating with the findings that ART initiation early during HIV-infection normalizes immune activation and possibly gut dysbiosis [1, 2]. Activated monocytes release the haptoglobin receptor CD163, expressed exclusively by macrophages and monocytes. Given that sCD163 may be increased by smoking and/or alcohol, its association in HIV mediated immune dysfunction is in part driven by behavioral risk factors [58]. Despite consuming alcohol and/or smoking, sCD163 plasma level was comparable in HIV (+) and HIV (−) Indian cohort, agreeing with the findings of Krishnan et al and suggesting an association of sCD163 with HIV-infection persists despite adjustment for behavioral risk factors [59]. Although we did not record these risk factors for the US cohort, sCD163 was elevated in HIV (+) as compared to HIV (−) healthy individuals. Nevertheless, high sCD163 levels have been associated with surrogate markers of neurologic and cardiovascular disease. Neurological dysfunction is also associated with mannose binding lectin (MBL) protein [60]. We found compared to HIV-uninfected, HIV-infected individuals from the US exhibited sustained elevated levels of both sCD163 and MBL. The levels of these mediators were comparable in HIV-uninfected and HIV-infected individuals in India.

A limitation of our study is the small sample size, but an important finding is the differences found in the mediators of RIA in both the study cohorts. HIV-infected individuals of US cohort as compared to Indian counterpart demonstrated elevated levels of CXCL2, CCL2, CCL5, IL-18, IL-6 and MBL protein. These are involved in inflammation and though better known for their role in mobilizing neutrophils, T cells and myeloid cells, an increased chemokine and MBL protein is associated with HIV associated neurological abnormalities [60, 61]. It will be intriguing to compare HIV associated neurological abnormalities in individuals infected with divergent HIV subtypes, and investigate the role of inflammatory mediators in neuroAIDS pathogenesis. The underlying factors contributing to the difference in immunological outcome between the two cohorts are multifactorial such as: different HIV subtypes, the genetic constitution, socioeconomic status and co-infections, nadir CD4+ T cell count. Due to the retrospective nature of our study, HIV molecular typing could not be performed. However, multiple studies have established prevalence of subtype B and C in the US and India, respectively. The antiretroviral regimen administered also influences the inflammatory response in periphery. To this end, both the cohorts in this study were receiving combination antiretroviral therapy consisting of at least three drugs, thus ART regimen related differences in immune activation were minimized in this study.

In conclusion, this is the first report of an association of M-MDSC with IL-6 in HIV-infected with virologic suppression. This study for the first time compares M-MDSC and mediators of RIA among geographically distant cohorts infected with likely infected with divergent HIV subtypes. As M-MDSC impair T cell function and its expansion is dependent on IL-6, reducing IL-6 levels or MDSC provide attractive new therapeutic targets to restore normal immune function for HIV-infected persons.

3. MATERIALS & METHODS

4.1. Study Participants

Blood was obtained after written informed consent from HIV (+) and HIV (−) controls from the USA and India. All HIV (+) subjects were on antiretroviral therapy for over a year. Samples in India were obtained from HIV (+) subjects visiting Integrated Counselling and Testing Center (ICTC) at a primary health care hospital which provides ART in a supervised manner. This study was approved by the University of California, San Diego, Office of Human Research Protections Program (USA cohort), and Institutional Ethics Committee College of Medicine and JNM Hospital, West Bengal University of Health Sciences, Kalyani, West Bengal (India cohort).

4.2. TB-Quantiferon and Latent TB Test

Three tube QuantiFERON®-TB Gold In-Tube test (Qiagen) was performed on HIV (+) and HIV (−) subjects (India cohort) as per manufacturer’s instructions. As a part of national TB surveillance program, CB-NAAT was performed on the saliva of HIV (+) and HIV (−) subjects and the lab report was provided for this study. An annual TB test of USA cohort did not indicate LTBI.

4.3. Antibodies and Other Reagents

The following antibodies were used for flow cytometry: Alexa Fluor488-anti-CD11b, allophycocyanin-anti-CD33, PE/Dazzle594-anti-HLA DR, PE/Cy7-anti-CD14, PercpCy5.5-anti-CD66b, Brilliant Violet 510-anti-CD3, Brilliant Violet 650-anti-CD19 (all from Biolegend).

4.4. Immunolabeling and Flow Cytometry

Whole blood was stained for surface markers using respective antibodies and cell staining buffer (Biolegend) [10, 25]. Flow cytometry was performed using FACS Canto (USA) or FACS Aria FUSION (India), 150 000 cells were acquired and analyzed using FlowJo software (Tree Star). Controls for each experiment included unstained cells and fluorescence-minus one controls.

4.5. Enzyme-Linked Immunosorbent Assay (ELISA)

Plasma was separated from heparinized blood and stored at −80°C until further use. The quantity of CXCL2, CX3CL1, CXCL10, CCL4, CCL2, CCL5, CXCL8 (IL-8), IL-18, sCD14 and MBL in the plasma was measured using Luminex® Human XL Cytokine kit; IL-22 using Quantikine; sCD163 using DuoSet; IL-6 using High sensitivity ELISA kits (all from R&D systems) as per manufacturer’s instructions.

4.6. Statistical Analysis

Nonparametric tests were used to determine associations between 2 variables. M-MDSC frequency and cytokine production was compared between HIV (+) and HIV (−) with nonparametric Mann-Whitney U test and comparisons between different parameters were analyzed using Spearman correlation; p-value of <0.05 was considered significant.

Supplementary Material

1

HIGHLIGHTS.

  • M-MDSC persist in HIV(+) individuals despite prolonged treatment with anti-retroviral

  • M-MDSC expansion is independent of gut dysbiosis in HIV (+) individuals

  • IL-6 drives monocytic-MDSC expansion in HIV (+) individuals

  • Immune activation pathways differ depending on predominant HIV-subtype

Acknowledgement:

We thank Stephen A Spector MD, Department of Pediatrics, Division of Infectious Diseases, University of California, San Diego, La Jolla, for critical reading of the manuscript, and providing with the blood samples from HIV-infected individuals of US cohort. A part of this work was performed with the support of Flow Cytometry Core at the UC San Diego Center for AIDS Research (P30 AI036214), the VA San Diego Health Care System, and the San Diego Veterans Medical Research Foundation. We thank Ms Bhaswati Tarafdar, National Institute of Bomedical Genomics, Kalyani, West Bengal, INDIA, and Ms Tara Rambaldo and Mr Neal Sekia, CFAR, University of California, San Diego for their assistance with flow cytometry.

Financial support

This work was supported by from the National Institute of Allergy and Infectious Diseases [grant number AI127132]; The University of Georgia Research Foundation; and Department of Health Research, GOI [grant number F.V.25011/256 HRD].

Footnotes

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Potential conflict of interest

All authors: No reported conflicts

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