Monocytic-Myeloid Derived Suppressor Cells of HIV-Infected Individuals With Viral Suppression Exhibit Suppressed Innate Immunity to Mycobacterium tuberculosis

Tuberculosis can occur during any stage of Human Immunodeficiency virus 1 (HIV) -infection including times when CD4+ T cell numbers have reconstituted and viral replication suppressed. We have previously shown that CD11b+CD33+CD14+HLA-DR-/lo monocytic myeloid-derived suppressor cells (MDSC) persist in HIV-infected individuals on combined anti-retroviral therapy (cART) and with virologic suppression. The response of MDSC to Mycobacterium tuberculosis (Mtb) is not known. In this study, we compared the anti-mycobacterial activity of MDSC isolated from HIV –infected individuals on cART with virologic suppression (HIV MDSC) and HIV-uninfected healthy controls (HIV (-) MDSC). Compared to HIV (-) MDSC, HIV MDSC produced significantly less quantities of anti-mycobacterial cytokines IL-12p70 and TNFα, and reactive oxygen species when cultured with infectious Mtb or Mtb antigens. Furthermore, HIV MDSC showed changes in the Toll-like receptor and IL-27 signaling, including reduced expression of MyD88 and higher levels of IL-27. Neutralizing IL-27 and overexpression of MyD88 synergistically controlled intracellular replication of Mtb in HIV MDSC. These results demonstrate that MDSC in fully suppressed HIV-infected individuals are permissive to Mtb and exhibit downregulated anti-mycobacterial innate immune activity through mechanisms involving IL-27 and TLR signaling. Our findings suggest MDSC as novel mediators of tuberculosis in HIV-Mtb co-infected individuals with virologic suppression.


INTRODUCTION
Infection with Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV) constitute major burdens of infectious disease in resource-limited countries (1,2). Co-infection with HIV increases the risk of developing tuberculosis (TB) between 16-27 times (1,3). It is intriguing that TB can occur in the settings of HIV at any disease state and irrespective of CD4 + T-cell numbers (4)(5)(6)(7)(8).

Patient Groups
Blood was obtained after written informed consent from Quantiferon ® -TB negative HIV-uninfected (HIV-) and HIVinfected (HIV+) persons on cART with virologic suppression and CD4 + . All studies were conducted in accordance with the Declaration of Helsinki guidelines and approved by Institutional Review Board of the University of California San Diego and Institutional Review Board of the University of Georgia Athens.

Infection of Cells and Measurement of Intracellular Mycobacterial Growth
MDSC (0.1 x 10 5 /well) were plated in 48-well plates in antibiotic free RPMI 1640 medium and 10% human serum. Cells were infected with Erdman at a multiplicity of infection (MOI) of 1:5 for 3 hrs; subsequently, cells were washed and treated with Gentamycin Sulfate (30 µg/ml; VWR Life Sciences) for additional 2-hours to kill extracellular bacteria, and cultured in RPMI 1640 with 10% human serum. Cells were lysed with 0.1% SDS at day-0 and day -3 or day -5 post-infection, and cellular lysates were serially diluted and plated in triplicate on Middlebrook 7H10 agar supplemented with OADC enrichment. The number of colonies were counted after 3weeks and colony forming units (CFU)/ml determined.

Overexpression of MyD88
MyD88 was overexpressed in MDSC by transfecting pUNO1-hMYD88 (InvivoGen) expression plasmid using Lipofectamine 3000 (from Thermo Fisher Scientific). Briefly, 1µg MyD88 or empty vector plasmid DNA was diluted in P3000 ™ reagent and Lipofectamine 3000 in Opti-MEM medium. Diluted plasmid and Lipofectamine were mixed in 1:1 ratio, and incubated for 15 min at room temperature. The plasmid-lipid complex was added to cells and incubated at 37°C and 5% CO 2 .

Immunolabelling, Flow Cytometry and ROS
Cells were surface stained for CD11b, CD33, CD14, HLA-DR, using cell staining buffer and respective antibodies. Controls for each experiment included unstained cells and fluorescence minus one (FMO) (29).
For ROS, sorted MDSC were infected with Green Fluorescence Protein expressing Mtb Erdman (GFP-Mtb) at MOI of 1:5 for 2-hrs. During the last 30-min of infection, cells were incubated with 1µM of CellROX ™ Deep Red reagent at 37°C/5% CO 2 and subsequently stained with aqua fluorescent LIVE/DEAD fixable dye. All the procedures were performed in Biosafety Level-3 and samples were analyzed on flow cytometer following the institutional biosafety guidelines. Dead cells were excluded and expression of CellROX was analyzed in GFP + Mtb cell gate; Net ROS expression= [Mean fluorescence intensity of ROS by GFP + Mtb MDSC -Mean fluorescence intensity of ROS by uninfected MDSC] (34).

Quantification of Cytokines
IL-12p70, TNFa, IL-6, IL-23 and IL-1b was determined in the culture supernatants at 24 hrs-post infection using LUMINEX multiplex system (R&D Systems) and custom designed kit. The fold change in cytokine was determined in M tuberculosis treated cells as: cytokine by Erdman infected or WCL treated cells/ cytokine by uninfected or unstimulated controls. The quantity of IL-27 in the plasma was measured using Duoset ELISA kit (R&D Systems catalog DY2526).

Quantitative Real Time PCR (qRT PCR)
Total RNA was isolated from MDSC using TRIzol ™ reagent (Thermo Fisher Scientific) according to manufacturer's protocol; 100-250 ng RNA was used in 20 µl of reverse transcription reaction using SuperScript ™ IV VILO ™ with ezDNAse Reverse Transcription kit (Thermo Fisher Scientific). TaqMan Gene expression Assay (Thermo Fisher Scientific) were used for qPCR analysis ( Table 1). The changes in the threshold cycle (C T ) were calculated by the equation D C T = C T,target -C T18S for control and WCL stimulated cells, DDC T = DC T,WCL -DC T, control . The fold change was calculated as 2 −( △ △ C T ) .

Statistical Analysis
Data are expressed as mean values ± standard error mean (SEM). Paired Student's t-tests were used to determine the statistical significance for in vitro experiments. Comparisons between HIV (-) and HIV (+) using Mann-Whitney U test. Statistical analysis was performed using Graphpad Prism 8 (La Jolla, CA). P-values of <0.05 were considered statistically significant.

Innate Immune Activity of MDSC in Response to M tuberculosis
We have previously established that HIV-infected person with virologic suppression as a result of successful cART exhibit increased circulating numbers of MDSC which regulate immune response to HIV-associated opportunistic pathogen (30). Herein we sought to investigate and compare the response of HIV (-) MDSC and HIV MDSC to M tuberculosis. For this, in the initial set of experiments we isolated HIV (-) and HIV MDSC, in vitro infected them with M tuberculosis or stimulated with M tuberculosis whole cell lysate antigen (WCL) and measured cytokines in the culture supernatants; the fold change in response to M tuberculosis or WCL was determined. The sorted MDSC were >95% pure (Supplemental Figure 1). Similar to a previous report, we found HIV MDSC produced inflammatory cytokines in response to M tuberculosis; however, compared to HIV (-) MDSC, fold increase in IL-12p70 and TNFa quantities produced by HIV MDSC infected with live M tuberculosis was less (1.2 ± 0.1 vs 2.2 ± 0.4; p=0.001 for IL-12p70, and 248.5 ± 166 vs 556.4 ± 265.4; p=0.04 for TNFa). However, the fold increase in IL-1b produced by HIV MDSC as compared to HIV (-) MDSC was more (61.7±32 vs 29.4±6.5; p=0.02). IL-6 produced by HIV MDSC was also higher than HIV (-) MDSC, but was not significant (15.3 ± 9.6 vs 7.4 ± 4; p=0.6) ( Figure 1A). Importantly, similar pattern was observed when these cells were cultured with WCL ( Figure 1B) which further suggests that HIV MDSC are responsive to M tuberculosis antigens and do not require active mycobacterial replication for downregulated cytokine production. Of note, quantities of anti-mycobacterial cytokines TNFa and IL-12p70 produced by HIV MDSC was significantly less than that produced by HLA DR hi monocytes (Supplemental Figures 2A, B). Collectively, these findings suggest that despite preservation of CD4 + T cells and virologic suppression, HIV MDSC exhibit dysregulated anti-M tuberculosis cytokine response.
Reactive oxygen species (ROS) is critical for the control of M tuberculosis early in infectious process, and ROS production by

MDSC Exhibit Truncated TLR Signaling
To determine if the reduced anti-mycobactericidal activity and cytokine response of HIV MDSC observed in Figures Figures 3A, B) Despite a higher expression of TLR-2, HIV MDSC produced less anti-M tuberculosis cytokines, we questioned if the differences occur in the expression of cytoplasmic adapter protein MyD88 (42,43). Since our cytokine studies ( Figures  1A, B) demonstrate similar anti-mycobacterial activity in response to infectious or non-infectious M tuberculosis. Therefore, for gene expression assays, we cultured these cell types with WCL for 24-hrs and quantified messenger RNA (mRNA) for MyD88 by qRT PCR. HIV (-) MDSC cultured with WCL exhibited a 3.3-fold increased expression of MyD88 as compared to unstimulated controls; the expression of MyD88 in HIV MDSC cultured with WCL was less than HIV (-) MDSC (1.22 ± 0.5 vs 3.3 ± 1.5; p= 0.04) ( Figure 2E) and HIV HLA DR hi monocytes (Supplemental Figure 3C). Downstream of TLRsignaling and pivotal for the synthesis of proinflammatory cytokines and ROS, is the activation and nuclear transport of the transcription factor NF-kB, which is regulated by the degradation of NFkB-inhibitor-a (NFkBIa) encoded protein IkBa (42,43). An increase in NFkBIa expression inhibits the degradation of IkBa thus sequestering NF-kB in the cytoplasm and inhibition in proinflammatory cytokine production (37)(38)(39). In order to establish that reduced quantities of cytokine produced by HIV MDSC is due to insufficient NF-kB activation, we quantified mRNA for NFkBIa in response to WCL by RT PCR in HIV MDSC and HIV (-) MDSC. HIV MDSC compared to HIV HLA DR hi monocytes (Supplemental Figure 3D), and HIV (-) MDSC exhibited a 5-fold increased expression of NFkBIa in response WCL (4.8 ± 0.8 vs 2.1 ± 0.7; p= 0.05) ( Figure 2F). This was accompanied by less phosphorylation of NF-kB p65 in HIV MDSC cultured with WCL ( Figures 2G, H).
Next, we overexpressed MyD88 by transfecting expression plasmid containing open reading frame of human MyD88 in HIV MDSC and measured the intracellular replication of M tuberculosis at 72-hrs post-infection. Compared to MDSC transfected with control plasmid, MyD88 transfection decreased the intracellular replication of M tuberculosis, but this was not significant (50 x 10 4 ± 17 x 10 3 vs 28 x 10 4 ± 14 x 10 3 CFU/ml, p=0.06) ( Figure 2I). Collectively, these studies establish that the impaired antimycobacterial innate immune response of HIV MDSC is due to the reduced expression of MyD88, and reconstituting it partially restores the anti-mycobacterial function of HIV MDSC.

IL-27 and MDSC
IL-27 is an immune regulatory cytokine that inhibits phagosomal activity of macrophages in response to bacterial infection including Mycobacterium (44,45). In HIV-infection, we previously showed that the plasma IL-27 level correlates positively with CD4 + T-cell count and negatively with HIV-viral load (30). We sought to determine if IL-27 inhibits antimycobacterial activity of HIV MDSC. To this end, initially we quantified the amount of IL-27 in the plasma of HIV-infected individuals and in healthy controls. Compared to healthy controls, the quantity of IL-27 in the plasma of HIV-infected individuals was high (4.6 ± 1.7 vs 17.7 ± 7 ng/ml; p= 0.03) ( Figure 3A). Next, we measured IL-27 in the culture supernatants of HIV MDSC and HIV (-) MDSC infected with M tuberculosis or stimulated with WCL as in Figures 1A, B; IL-27 in these culture supernatants was undetectable. However, we observed increased expression of IL-27 in the cellular lysates of HIV MDSC in vitro stimulated with WCL ( Figures 3B, C).
Additionally  Figure 3E). IL-27R is indispensable for IL-27 signaling, which induces phosphorylation of STAT1 and STAT3 in myeloid cells (46,47).  Figure 1D. Consistent with our findings of Figure  1D that down-regulate T-cell response to cytomegalovirus (30,31). In this research, we demonstrate that MDSC isolated from HIV (+) individuals with virologic suppression as compared MDSC isolated from HIV (-) controls exhibit suppressed cytokine production in response to M tuberculosis or WCL. We also demonstrate that M tuberculosis infected HIV MDSC produce less ROS and exhibit a higher intracellular replication. Additionally, we show that the impaired anti-mycobacterial activity of HIV MDSC is through mechanisms involving cytokine IL-27 and truncated TLRsignaling. These support the previous findings demonstrating suppressed innate immune function and increased bacillary load in HIV-M tuberculosis co-infected individuals (10,17,18,20). MDSC expand during various pathological conditions as a result of acute or chronic inflammation and are important mediators of immune suppression (21, 23-26, 29, 31, 32, 51). In murine TB, MDSC are present at the disease site, harbor mycobacteria, produce cytokines TNF-a, IL-1 a and IL-6, and suppress anti-mycobacterial T-cell IFN-g responses (25,33). Accumulation of MDSC in lungs heightens TB lethality, and MDSC depletion by all-trans-retinoic acid ameliorates primary progressive TB (25,52,53). MDSC also expand in peripheral blood and pleural fluid of TB patients and HIV-M tuberculosis co-infected children, and suppress T-cell effector functions through mechanisms involving suppressed TNF-a, IL-2 and IL-10 (25,(54)(55)(56). These findings collectively establish downregulated innate immune responses mediated by MDSC contribute to the failure to control M tuberculosis. However, we provide the first evidence that MDSC present in HIV-infected individuals with virologic suppression are defective in innate immune mediated control of M tuberculosis and are potential mediators of tuberculosis in these individuals. Similar to the previous findings, we also observed that MDSC isolated from  (33). A limitation of our study is that we isolated peripheral MDSC from HIV-infected individuals to determine their antimycobacterial activity. Nonetheless, presence of arginase-I and nitric oxide co-expressing MDSC like cells in the necrotic granulomas of M tuberculosis infected macaques (53,57), and accumulation of MDSC in blood at advanced stage of M tuberculosis infection establish that MDSC are critical mediators of tuberculosis disease pathogenesis and suppress both innate and adaptive immune mediated control of M tuberculosis (25,33,53). The increased bacillary load observed in HIV MDSC conceivably is due to higher infectivity of these cells.
Owing to a small sample size we were unable to statistically determine this aspect. We are currently investigating the interaction and trafficking of M tuberculosis inside MDSC of HIV-uninfected and -infected individuals. Here we propose that MDSC in virologically suppressed HIV-infected individuals are permissive to M tuberculosis and contribute to increased bacillary load observed in these individuals. TNF-a and IL12p70 are indispensable to control M tuberculosis; these cytokines enhance phagosome-lysosome maturation, antigen presentation and mobilization of activated T cells (58)(59)(60). Asymptomatic HIV-infection is associated with reduced release of TNF-a by alveolar macrophages and peripheral blood mononuclear cells in response to M tuberculosis infection or immunogenic M tuberculosis specific proteins (61,62). This is a result of impaired nuclear translocation of NF-kB, and HIV Nef mediated destabilization of TNF-a mRNA (61,62). Accordingly, we found that M tuberculosis upregulates NFkBIa in HIV MDSC which masks the nuclear localization signals of NF-kB by stabilizing IkBa. Additionally, both TNF-a and IL12p70 play an important link between innate and adaptive immune responses. Although yet to be investigated, it is plausible that the reduced M tuberculosis specific cytokines produced by HIV MDSC results in the decreased expression of chemokines CCL5, CXCL9 and CXCL10 thus restricting the migration of CXCR3 + , CCR1 + and CCR5 + activated T cells to granuloma or secondary lymphoid tissues, resulting in loss of immunity in HIV-M tuberculosis coinfected individuals (63,64). We previously reported increased mRNA expression of p47phox subunit of ROS by HIV MDSC, which mediates down regulation of T cell function (29). Consistently, HIV MDSC produced profound ROS at basal level, but not in response to M tuberculosis infection. Here we show that the reduced release of anti-mycobacterial cytokines by HIV MDSC directly affects the mycobacterial load through mechanism involving ROS suppression. ROS generated by NADPH oxidase is a vital component of M tuberculosis containing mature phagosome, facilitating bacterial killing; ROS also regulates NF-kB and MAP kinase signaling in TLR-dependent manner (36). Our study affirms that truncated TLR-MyD88 axis in HIV MDSC increases mycobacterial load, which potentially amplifies the risk of tuberculosis in HIV patients with virologic suppression; reconstituting MyD88 partially recovers mycobactericidal activity of HIV MDSC. It is possible that TNF-a and IL12p70 produced by macrophages compensates the suppressed anti-mycobacterial activity of HIV MDSC, but inhibition of monocyte/macrophage function by MDSC observed in tumors cannot be ruled out in HIV-M tuberculosis co-infection (28,65).
IL-6 is a pleiotropic proinflammatory cytokine produced by multiple cell types in response to inflammatory stimuli including IL-1b, TNF-a, TLRs, prostaglandins and stress responses (66,67). IL-6 deficiency leads to impaired innate and adaptive immunity to viral, bacterial and parasitic infection. Even though, the importance of IL-6 during M tuberculosis infection is not well understood, its neutralization increases susceptibility to infection and mycobacterial load, while delaying T-cell accumulation and IFNg expression, both during primary infection, and vaccination with BCG and a subunit vaccine (68)(69)(70). Additionally, IL-6 is critical for differentiation and maintenance of IL-23 dependent T H 17 cells-important for recruitment of neutrophils to infection site, and IFNg mediated control of M tuberculosis (71)(72)(73). IL-23 treatment of M tuberculosis infected animals reduces mycobacterial burden and augments cellular responses; its absence increases mycobacterial burden, and decreases the expression of IL-17, IL-22 and CXCL13 resulting in accumulation of lymphocytes around the vessels rather than within granulomas (74,75). While we did not evaluate T-cell responses in this study, we found comparable level of IL-6, but lower level of IL-23 produced by HIV MDSC as compared to HIV (-) MDSC in response to M tuberculosis ( Figure 1A and Supplemental Figures 4A, B). Further studies are needed to determine the exact role of HIV MDSC in suppressing adaptive immunity to M tuberculosis. Chronic immune activation driven by IL-6 and TNF-a are major predictors of HIV disease progression. Elevated level of IL-6 present in serum, mucosal and lymphoid tissues augment HIV replication through C/EBPb mediated binding to the HIV long terminal repeats and inhibition of APOBEC3G (76)(77)(78)(79)(80)(81). We and others have established that IL-6 mediates MDSC expansion, and even though its level decline following successful administration of cART, but still remain elevated as compared to healthy controls thus maintaining increased numbers of MDSC in HIV patients with virologic suppression (29)(30)(31). These MDSC increase the risk of tuberculosis in HIV-M tuberculosis co-infection. IL-27 and IL-27R are expressed by activating myeloid cells including dendritic cells and macrophages, and modulate both macrophage and T-cell activity during M tuberculosis infection (45,82,83). We and others have observed that IL-27 directly inhibits HIV replication (Supplemental Figures 4C, D) in PBMC. Although the mechanism of IL-27 mediated viral suppression is not fully established, it appears that the inhibition of spectrin b nonreythrocyte 1 (SPTBN1) by IL-27 plays a critical role (84). In HIV-infected individuals serum IL-27 levels correlate negatively with viral load and positively with CD4 + T-cell counts (30), and IL-27 induced IL-6 and TNF-a production is downregulated in HIV infection (85). This is of significance in the settings of HIV-M tuberculosis co-infection where IL-27 supports clinical recovery from HIV but is detrimental to control M tuberculosis. Previously, Egidio et al. also found increased expression of IL-27 in TB patients coinfected with HIV as compared to latent TB infection in south and southeast African cohorts (82). In this study, we provide the first evidence of the direct effect of IL-27 on M tuberculosis in HIV-M tuberculosis co-infection. Our findings that neutralizing IL-27 augments the expression of ROS in response to M tuberculosis and controls intracellular replication of M tuberculosis corroborate with the findings that, IL-27 negatively regulates macrophage response by inhibiting the expression of phagosomal vacuolar ATPase (V-ATPase) and lysosomal integrated membrane protein-1 (CD63), resulting in suppression of phagosomal acidification and cathepsin D maturation, all these lead to increased bacillary load (44,45). With regard to the expression of IL-27, unlike Egidio et al. and Cory et al, IL-27 transcripts were undetectable in our study (45,82). The potential reasons for this could be: 1) difference in the samples used, Egidio et al. measured IL-27 gene expression in whole blood RNA (82) and Cory et al. utilized macrophages (45), and 2) the amount of RNA used by Cory et al. for reverse transcription and subsequent transcript analysis was 750 ng; given the low numbers of MDSC that could be isolated from HIV-infected individuals, we were unable to obtain such a high quantity of RNA and thus low copies of IL-27 could be present below our detection limit. Of note, p28 subunit specific to IL-27 is produced by activated myeloid cells through TLR-2, -4 and -9 in MyD88 dependent manner (86), and in MyD88 independent manner through TLR-4 -TIR-domain-containing adapter inducing IFNb (TRIF) and IFN regulatory factor 3 (IRF3) pathways (87,88). In this research, we show that HIV MDSC are deficient in MyD88 expression, suggesting that a MyD88independent mechanism is the major pathway involved in IL-27 synthesis in response to M tuberculosis. We are currently investigating the mechanism of IL-27 mediated suppression of innate immunity in response to M tuberculosis with or without co-infection with HIV. The increased plasma IL-27 in HIVinfected individuals in this and other studies suggest multiple cell types produce this cytokine and exhibits anti-HIV activity (30,84). In the present study, we provide the first evidence of increased IL-27 and IL-27R by MDSC in HIV-infected individuals with virologic suppression. We previously showed that IL-27 upregulates B7-H4 expression on MDSC, which regulates T-cell function (30). In this research, we propose that IL-27 in HIV-M tuberculosis co-infection acts on MDSC both in autocrine and paracrine manner, and that IL-27-IL27R axis is a potential mediator of suppressed immunological response to M tuberculosis ( Figure 4D).
In summary, our ex vivo and in vitro data collectively establish that HIV-infected individuals with virologic suppression have increased levels of circulating IL-27 and MDSC. MDSC exhibit truncated TLR-mediated innate immunological function in response to M tuberculosis. Further, these MDSC express increased surface expression of IL-27R which may further downregulate the innate immunity to M tuberculosis. These findings provide a mechanistic model of how MDSC can increase the risk of tuberculosis in HIV-M tuberculosis coinfected individuals. Moreover, our findings suggest that IL-27/ IL-27R and MDSC provide attractive biomarkers to assess tuberculosis prognosis during HIV-infection.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by The University of California San Diego and The University of Georgia Athens. The patients/participants provided their written informed consent to donate blood for this study.

AUTHOR CONTRIBUTIONS
AG developed the study, performed experiments, analyzed data, and wrote the paper. PN, SP and BS performed the experiments, and analyzed the data. All authors contributed to the article and approved the submitted version.