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Co-Infection of ART-treated SIVmac239+ Rhesus Macaques with Plasmodium fragile Results in Elevated Inflammation which Associates with Neutrophil Function

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16 May 2024

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16 May 2024

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Abstract
Human immunodeficiency virus (HIV) and malaria, caused by infection with Plasmodium spp., are endemic in similar geographical locations. As a result, there is high potential for HIV/Plasmodium co-infection, which increases the pathology of both diseases. However, the immunological mechanisms underlying the exacerbated disease pathology observed in co-infected individuals are poorly understood. Here, we used the rhesus macaque (RM) model to characterize the immunopathogenic impact of Plasmodium fragile co-infection during antiretroviral therapy (ART)-treated simian immunodeficiency virus (SIV)-infection. We observed that P. fragile co-infection resulted in parasitemia and anemia, as well as persistently detectable viral loads (VL) and decreased absolute CD4+ T-cell counts despite daily ART treatment. Notably, P. fragile co-infection was associated with increased levels of inflammatory cytokines linked with neutrophil function, including monocyte chemoattractant protein 1 (MCP-1). P. fragile co-infection was associated with increased levels of neutrophil elastase, a plasma marker of neutrophil extracellular trap (NET) formation, but significant decreases in markers of neutrophil degranulation, potentially indicating a shift in neutrophil functionality during co-infection. Finally, we characterized levels of plasma markers of gastrointestinal (GI) barrier permeability and microbial translocation and observed significant correlations between indicators of GI dysfunction, clinical markers of SIV and Plasmodium infection, and neutrophil frequency and function. Taken together, these data indicate that neutrophil-driven inflammation and GI dysfunction may underlie heightened SIV/P. fragile co-infection pathogenesis.
Keywords: 
NHP
;  
P. fragile
;  
SIV
;  
malaria
;  
neutrophils
;  
co-infection
;  
immunology
Subject: 
Biology and Life Sciences  -   Immunology and Microbiology

Introduction

Human immunodeficiency virus (HIV) and malaria, caused by Plasmodium parasites, are two of the world’s most devastating infections. In 2022, over 39 million people were living with HIV (PWH) [1], and there were over 247 million cases of malaria [2]. Despite effective tools and treatments, challenges in prevention and eradication of both infections remain. Indeed, consistent use of antiretrovirals (ART) allows for sustained viral suppression, improving health and quality of life of PWH [3]. However, ART does not eliminate the viral reservoir and plasma viremia rapidly rebounds post-treatment interruption [4,5]. Similarly, antimalarial drugs can prevent and cure Plasmodium infection, but emergence of drug-resistance undermines control efforts and contributes to increased morbidity and mortality [6,7]. For both, the complexities of the infectious agents, combined with an incomplete knowledge of the immunological mechanisms underlying pathogenesis, hampers identification of immune correlates of protection and development of fully efficacious prophylactic vaccines.
HIV and malaria endemicity are geographically overlapped, creating high potential for co-infection. A meta-analysis of studies conducted between 1991 and 2018 found that co-incidence of HIV and severe malaria, or presence of peripheral parasitemia associated with fatal outcomes, was 43% [8]. Prior work has demonstrated reciprocal antagonistic effects that results in increased transmission of both HIV and malaria. For example, Plasmodium increases HIV VL both in vitro and in ART-naïve PWH [9,10,11]. High VLs correlate with increased HIV transmission, suggesting that malaria co-infection of PWH could enhance HIV transmission risk [12,13,14]. Similarly, clinical malaria prevalence, malaria infection severity, and malaria-associated mortality rates are increased in ART-naïve PWH [15,16,17,18]. Additionally, previous in vitro and ex vivo studies have indicated that co-infection with both HIV and Plasmodium exacerbates disease pathogenesis. For example, in vitro infection of monocyte-derived macrophages with a laboratory strain of HIV-1 resulted in inhibited phagocytic capability and cytokine production in response to stimulation with opsonized trophozoites from a laboratory-adapted strain of P. falciparum [19]. Moreover, HIV-infected children in Malawi with cerebral malaria infection had higher rates and more rapid progression to mortality, greater parasite loads in brain and spleen, and greater brain accumulation of monocytes and platelets in the brain, as compared to children without HIV [20,21]. Notably, uncontrolled inflammation underlies disease pathogenesis in separate HIV and malaria infection [22,23]. In PWH, increased inflammation associates with viral persistence, disruptions in intestinal homeostasis, and increased risk of co-infection with other pathogens [24]. Likewise, a pro-inflammatory environment associates with severe malaria and increased malaria-associated mortality [23,25]. Importantly, the impact of Plasmodium co-infection on ART efficacy in PWH, and the link between inflammatory responses and disease pathology during co-infection, has not yet been defined [26].
Neutrophils are granulocytes that constitute up to 70% of all circulating leukocytes [27] and aid in host defense through: 1) exocytosis of anti-microbial molecule-containing granules; 2) phagocytosis and destruction of microbes in phagosomes; and 3) formation of neutrophil extracellular traps (NETs), DNA decorated with granule contents that aid pathogen clearance [28,29,30,31]. Conversely, dysregulated neutrophil activation causes uncontrolled inflammation and collateral host tissue damage [32]. For example, although NET formation associates with protection against HIV infection and impaired replication in vitro [33], increased neutrophil activation was linked with adverse clinical outcomes in PWH [34,35,36] and ART-treated PWH exhibited impaired neutrophil phagocytosis and increased neutrophil apoptosis, as compared to uninfected controls [37,38]. Additionally, increased neutrophil infiltration into the gastrointestinal (GI) mucosa in ART-treated PWH and macaques with chronic simian immunodeficiency virus (SIV) infection associated with loss of GI epithelial barrier integrity and elevated microbial translocation [39,40,41], both of which associate with chronic inflammation, morbidity, and mortality in PWH [42,43,44]. Likewise, neutrophil phagocytosis aids in Plasmodium clearance [45,46,47] and NET formation in children associated with parasite killing [48]. However, NET formation also associated with increased inflammation and severe malaria [49,50,51], and excessive neutrophil degranulation contributed to risk of severe malaria (48, 52, 53). Importantly, the role of neutrophils in disease pathogenesis during HIV/Plasmodium co-infection has not been established.
SIV infection induces pathologies similar to progressive HIV, including high peak and chronic plasma VLs and CD4+ T-cell depletion [54]. Additionally, like P. falciparum, which causes most human malaria cases worldwide [2], P. fragile is capable of endothelial adherence, tissue sequestration, and antigenic variation in rhesus macaques (RMs) [55]. SIV/P. fragile co-infection in RMs mimics HIV/P. falciparum co-infection in humans, including increased SIV VL and innate immune dysfunction in ART-naïve SIV/P. fragile co-infected versus singly infected RMs [56,57]. However, SIV/P. fragile co-infection has not been characterized in the context of ART. In this pilot study, we sought to define the impact of P. fragile co-infection on ART-treated SIV infection. We hypothesized that P. fragile co-infection would result in exacerbated SIV pathology that associated with neutrophil dysfunction despite ART. To test this hypothesis, we infected four adult RMs with SIVmac239, followed by ART-initiation, and P. fragile co-infection, and longitudinally monitored clinical and immune markers.

Methods and Materials:

Study Animals and Approval

Four adult (aged 6–12 years) male Indian-origin RMs were housed and cared for at the Tulane National Primate Research Center (TNPRC) under an Institutional Animal Care and Use Committee (IACUC; Office of Laboratory Animal Welfare Assurance Number A4499-01) approved protocol (P0477-3564). Animal housing, care, and procedures were performed in Association for Assessment and Accreditation of Laboratory Animal Care accredited facilities (AAALAC Number 000594), compliant with United States Department of Agriculture regulations, including the Animal Welfare Act (9 CFR) and the Animal Care Policy Manual, with guidelines established by the National Research Council in the Guide for the Care and Use of Laboratory Animals and the Weatherall Report. All animals were naïve for both SIV and Plasmodium prior to study assignment. In addition, animals were negative for MHC class I alleles associated with SIV control, including Mamu-A*01, Mamu-B*08, and Mamu-B*17 [58,59,60]. Animals were singly housed indoors under climate-controlled conditions, a 12-hour light/12-hour dark cycle and were monitored daily to ensure welfare. Abnormalities were recorded and reported to a veterinarian. Water was available ad libitum and animals were fed commercial monkey chow (Purina LabDiet; PMI Nutrition International, Richmond, IN), supplemented with fruits, vegetables, and foraging treats as a part of the TNPRC environmental enrichment program. At week 2 post-SIV infection (p.i.), one animal (LN07) received a topical antibiotic for a surface wound. At weeks 10 and 13 p.i., all RMs received Kefzol (6.25mg/kg) during surgical procedures. At week 14 p.i. one animal (LE96) received a blood transfusion. At weeks 14 (LC40) and 16 p.i. (LC40, LE96), two animals received a dose of the antibiotic Excede (200mg/ml). Procedures were performed under direction of TNPRC veterinarians. Anesthesia was used in accordance with TNPRC policy and the Weatherall Report. Euthanasia at study endpoint was performed using methods consistent with recommendations of the American Veterinary Medical Association and per the recommendations of the IACUC.

SIV Inoculation, Monitoring, and ART Treatment

RMs were intravenously inoculated with 50 TCID50 SIVmac239 [61]. Plasma VLs were monitored via RT-qPCR [62]. Starting at week 8 p.i. and continuing until endpoint, RMs received daily ART, administered subcutaneously, consisting of tenofovir disoproxil fumarate (TDF; 5.1mg/kg), emtricitabine (FTC; 30mg/kg; both from Gilead, Foster City, CA), and dolutegravir (DTG; 2.5mg/kg; ViiV Healthcare, London, England, UK), formulated in Kleptose (15% in 0.1 N NaOH, Roquette, Lestrem, France), a formulation selected for effectiveness in suppressing SIV replication in RMs [63].

P. fragile Inoculation, Monitoring, and Anti-Malarial Treatment

RMs were intravenously inoculated with 20x10^6 P. fragile-infected erythrocytes (Sri Lanka strain) [64,65,66]. Briefly, cryopreserved erythrocytes were thawed and resuspended in 12% NaCl (Thermo Fisher Scientific, Waltham, MA) for 5min at room temperature (RT). Next, 1.6% NaCl was added dropwise, followed by centrifugation for 10min, RT, 1400 revolutions per minute (RPM). The pellet was resuspended in 0.9% NaCl and 2% dextrose (Thermo Fisher Scientific), centrifuged for 10min, RT, 1400 RPM, and resuspended in 0.9% of NaCl for inoculation. Anemia was monitored via hematocrit (HCT), calculated as the ratio of erythrocytes to total blood volume. Parasitemia was monitored via Giemsa staining of thin blood smears collected from sedated animals via venipuncture or tail sticks from non-sedated animals using positive reinforcement, three days a week starting in Week 12, delineated as week A, B, or C. Smears were fixed in methanol for 5min, followed by staining in 5% Giemsa solution (pH=7.2) for 45-60min and washed in distilled water. Parasitemia was calculated as the average number of parasitized erythrocytes among all erythrocytes in 10 randomized fields of view. Post week 14A p.i., RMs received anti-malarial drugs via oral gavage consisting of one administration of quinine sulfate (150mg; Archway Apothecary, NDC: 51927-1588-00), followed by four daily administrations of chloroquine (20mg/kg; Health Warehouse, NDC: 64980-0177-50).

Sample Collection and Processing

EDTA and serum gel vacutainer tubes (Starstedt, Newton, NC) were used to collect peripheral blood. Complete blood counts (CBCs) were performed using EDTA blood on a Sysmex XN-1000v (Sysmex, Kobe, Hyogo, Japan). Blood chemistry was performed using fresh serum and C-reactive protein (CRP) levels were quantified using frozen serum on a Beckman AU480 (Beckman, Brea, CA). For experimental procedures, EDTA blood was centrifuged for 10min, 2000 RPM, RT to isolate plasma, which was stored at -80°C. After plasma removal, whole blood was reconstituted with PBS. Aliquots of 250µL of blood-PBS were set aside for flow cytometric staining. Peripheral blood mononuclear cells (PBMCs) were isolated from remaining blood via density-gradient centrifugation using Ficoll-Paque Plus (Sigma-Aldrich, St. Louis, MO) and Accuspin tubes (Sigma-Aldrich). PBMCs were cryopreserved in freezing media (5mls dimethyl sulfoxide (DMSO) [Sigma-Aldrich] + 45mls heat inactivated fetal bovine serum [Thermo Fisher Scientific]) and stored in liquid nitrogen.

Flow Cytometry

Multicolor flow cytometry was performed on whole blood using RM cross-reactive monoclonal antibodies. Samples were first stained with a Live/Dead Fixable Aqua dead cell stain (Thermo Fisher Scientific), then treated with Fc block (BD Biosciences, Franklin Lakes, NJ). Extracellular staining was performed using predetermined fluorochrome conjugated antibody concentrations (Supplemental Table S1), followed by red blood cell lysis using 1x FACS lysing solution (BD Biosciences). Cells were fixed, permeabilized (CytoFix/Perm Kit, BD Biosciences), then intracellularly stained (Supplemental Table S1 and Supplemental Figure S1).
Phagocytosis was evaluated using E. coli bioparticles conjugated to a dye that fluoresces in acidic environments (pHrodo Red E. coli Bioparticles Phagocytosis Kit for Flow Cytometry; Thermo Fisher Scientific). Briefly, pHrodo bioparticles were incubated with plasma from healthy RMs (1:3 plasma:pHrodo ratio) for 30min to allow for bioparticle opsonization. Opsonized pHrodo bioparticles were added to 250µL whole blood aliquots for 2hrs at 37°C, followed by surface staining (Supplemental Figure S2).
All samples were fixed with 1% paraformaldehyde and held at 4°C until acquisition on a BD LSRFortessa using FACSDiva software (v9.0). Single-color controls were acquired in every experiment as compensation. Analysis was performed using FlowJo (v10). In all analysis, individual cell subsets with less than 100 parental gate events were not included in downstream analysis due to the inability to ensure adequate fluorescence separation.
CD4+ T lymphocyte kinetics were monitored by flow cytometric evaluation of absolute counts. Briefly, 50μL whole blood were surface stained (Supplemental Table S2), and incubated for 20min, RT, in the dark. Red blood cells were lysed with 1X BD FACS Lysing Solution for 30-45 minutes. The sample was mixed and volumetrically analyzed on a Miltenyi MACSQuant 16 (Miltenyi, Bergisch Gladbach, Germany).

Detection of Plasma Markers of Neutrophil Function, GI Mucosal Barrier Integrity, and Microbial Translocation

Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to quantify plasma levels of neutrophil granule components, including myeloperoxidase (MPO; Abcam, Cambridge, UK), cathepsin G (MyBiosource, San Diego, CA), proteinase 3 (PR3; MyBiosource); biomarkers of NET formation, including citrullinated histone 3 (CitH3; Cayman Chemicals, Ann Arbor, MI), and neutrophil elastase (NE; LSBio, Lynwood, WA); markers of GI barrier permeability, including Zonulin-1 (MyBiosource) and intestinal fatty acid binding protein (IFAB-P; Novus Biologicals, Centennial, CO); and surrogate markers of microbial translocation, including lipopolysaccharide (LPS) binding protein (LBP; Novus Biologicals) and soluble CD14 (sCD14) ELISA (ThermoFisher), as per the manufacturers’ recommended protocols.

Detection of Plasma Markers of Systemic Inflammation

A BioLegend LEGENDplex™ NHP Inflammation Panel (13-plex) with V-bottom plate was used to quantify plasma levels of inflammation markers IL-6, IL-10, CXCL10 (IP-10), IL-1β, IL-12p40, IL-17A, IFN-β, IL-23, TNF-α, IFN-γ, GM-CSF, CXCL8 (IL-8), CCL2 (MCP-1). Plasma samples were run in duplicate at a 1:4 dilution, as per manufacturer recommendations. Samples were acquired on a Miltenyi MACSQuant 16 in a 96 well plate format. Data were analyzed with the BioLegend LEGENDplex™ online analysis software against a standard curve.

Data and Statistical Analysis

We used the absolute number of neutrophils/µL blood to calculate the number of neutrophils positive for pHrodo bioparticles to characterize phagocytosis (phagocytic score, or the number of neutrophils capable of phagocytosis) [67]. Phagocytic index, representing phagocytic proficiency, was calculated by multiplying the phagocytic score by the Mean Fluorescence Intensity of pHrodo positive neutrophils [68].
Statistical significance between all timepoints was calculated using a mixed-effects analysis of variance with the Geisser-Greenhouse correction. Multiple comparisons between all timepoints were performed using Tukey’s multiple comparisons tests based on individual variances for each comparison. In all figures, multiplicity adjusted significant P values are shown above horizontal black bars. Multivariate analysis of variance (MANOVA) approaches were applied to identify potential relationships between various neutrophil measures, clinical signs of both SIV and P. fragile infection, peripheral markers of inflammation, and peripheral markers of GI dysfunction. The MANOVA approach allowed us to account for temporal dependence, as previously described [69]. MANOVA was used to model 13 parameters (VL, anemia, absolute CD4+ count, peripheral neutrophils, plasma zonulin, sCD4, I-FABP, LBP, NE, cathepsin G, CitH3, IP-10, MCP-1, and CRP) against animal number. Partial correlation coefficients were generated based on the MANOVA error terms adjusted for animal effects. Statistical analyses were performed using GraphPad Prism (Version 10; GraphPad Software, San Diego, CA) and Statistical Analysis System (SAS, Version 9.4; Cary, NC). All reported P values were multiplicity-adjusted and values of <0.05 were considered significant. The JoinPoint Regression Program (NIH, V5.0.2, Bethesda, MD) was utilized to assess longitudinal trends in VLs.

Results:

Experimental Design

Following baseline (BL) sampling, RMs (n=4) were intravenously inoculated with SIVmac239 (Figure 1). At week 8 post-SIV infection (p.i.), RMs initiated ART, which continued until euthanasia at week 20 p.i.. For simplicity, all time points in our analyses are reported as post-SIV infection. At week 12 p.i., RMs were intravenously inoculated with P. fragile. At week 14 p.i., RMs surpassed our treatment threshold of 0.5% parasitemia and received antimalarial drugs via oral gavage. Physical exams, peripheral blood collections, CBCs, and serum chemistries were conducted throughout the study.

P. fragile co-Infection of ART-Treated SIV+ RMs Results in Clinical Signs of Malaria

Parasitemia and anemia are clinical hallmarks of malaria [70,71]. Following P. fragile co-infection, parasitemia was assessed tri-weekly (A, B, C). RMs reached peak parasitemia by week 14A p.i. and had undetectable parasitemia following antimalarial treatment (P=0.0429 for week 14 p.i. versus all other timepoints; Figure 2A). All RMs experienced mild to severe anemia between weeks 14-15 p.i., coinciding with peak parasitemia (Figure 2B). Percent hematocrit (% HCT) was significantly lower at week 14 p.i. compared to weeks 10, 12, and 13 p.i. (P=0.0367, 0.0391, and 0.049, respectively; Figure 2B). Additionally, % HCT was significantly lower at week 15 p.i. compared to BL and weeks 2, 6, 19, and 20 p.i. (P=0.0122, 0.0155, 0.0448, 0.0286, and 0.0366, respectively; Figure 2B). RMs remained mildly anemic at week 17 p.i., demonstrated by significantly lower % HCT compared to week 8 p.i. (P=0.0493; Figure 2B). By week 18 p.i., all RMs were non-anemic and had significantly greater % HCT at week 19 p.i. as compared to week 17 p.i. (P=0.0485; Figure 2B). Taken together, P. fragile co-infection of ART-treated SIV+ RMs induced significant parasitemia and anemia, hallmarks of clinical malaria.

P. fragile co-Infection Results in Clinical Signs of SIV Infection Despite Daily ART

Uncontrolled VL and decreased CD4+ T-cell counts within a few weeks following infection are hallmarks of pathogenic HIV/SIV [72,73,74,75]. Treatment with ART has been shown to suppress viral replication and restore CD4+ T-cell counts in SIV-infected RMs even at end stage disease [63,76]. Here, we observed that peak viremia in RMs inoculated with SIVmac239 occurred by week 3 p.i. (median=1.160 x107 copies/ml; Figure 2C). Lower VLs were observed following ART, with 2/4 RMs (LC40 and LE96) becoming undetectable by week 12 p.i. (Figure 2C). Following P. fragile inoculation, all RMs maintained VLs above the limit of detection from weeks 13-17 p.i.(Figure 2C). Longitudinal assessment by JoinPoint regression demonstrated that following P. fragile co-infection, the VL of all 4 RM remained unchanged, indicating viral persistence (Supplemental Figure 3). Between weeks 18-20 p.i., one RM (LE96) had transiently detectable VLs, two RMs became undetectable by weeks 19 (LN07) and 20 p.i. (LC40), with the remaining RM (JF97) remaining incompletely suppressed. Absolute CD4+T-cell counts were significantly lower at week 3 p.i. compared to BL (P=0.0414) and remained low until ART initiation at week 8 p.i. (Figure 2D). Following P. fragile inoculation, CD4+ T-cell counts were significantly increased at week 13 p.i. compared to week 3 p.i. (P=0.0397; Figure 2D). By week 17 p.i. following antimalarial treatment, CD4+ T-cell counts returned to pre-ART levels (Figure 2D). These data suggest that co-infection of ART-treated SIV+ RMs with P. fragile results in persistently detectable VLs and fluctuations in CD4+ T cell counts.

Increased Levels of Inflammatory Markers Associated with Neutrophil Function during ART-Treated SIV/P. Fragile co-Infection

CRP is an acute phase plasma marker of inflammation that has been shown to be elevated in ART-naïve PWH and is associated with HIV disease progression even despite ART [77,78,79,80]. Additionally, elevated CRP has been observed in individuals with complicated and uncomplicated malaria and can be used as a biomarker for monitoring of malaria severity [81,82]. Here, we observed that serum CRP was significantly increased at week 14 p.i. as compared to BL and weeks 2, 6, 8, and 10 p.i. (P=0.0455 for all), as well as compared to weeks 12, 15, and 20 p.i. (P=0.0435, 0.0424, and 0.0428, respectively; Figure 3A). These data suggest that there is an increase in systemic inflammation following P. fragile co-infection of ART-treated, SIV+ RMs that coincides with peak parasitemia.
Both HIV and Plasmodium infection result in elevated levels of pro-inflammatory cytokines and chemokines [23,83]. Here, we observed some longitudinal and inter-animal variation in plasma levels of interleukin (IL)-8 and interferon gamma-induced protein 10 (IP-10), but no statistically significant changes in these analytes throughout P. fragile co-infection of ART-treated SIV+ RMs (Figure 3B and C). Plasma monocyte chemoattractant protein-1 (MCP-1) was significantly increased at week 14 p.i. compared to BL, and weeks 16 and 20 p.i. (P=0.0223, 0.0406, and 0.0412, respectively; Figure 3D). All other inflammatory analytes remained unchanged (Supplementary Figure 4). These findings suggest that neutrophil-associated chemokines are altered after P. fragile co-infection of ART-treated, SIV+ RMs.

Stable Peripheral Neutrophil Frequencies and Apoptosis during ART-Treated SIV/P. fragile co-Infection

We next characterized peripheral neutrophil dynamics and apoptosis via flow cytometry. Neutrophils were identified as viable, CD45+ HLA-DR- CD11b+ CD66abce+ CD14+ CD49d- cells, as previously described (Supplemental Figure S1) [84,85,86,87,88,89]. As previously demonstrated [90], total peripheral neutrophil frequency was consistent throughout acute SIV and ART-treatment as compared to BL (Figure 4A). Neutrophil frequencies were significantly decreased following anti-malarial treatment as compared to the start of anti-malarial treatment (week 17 vs. 14 p.i., P=0.0136), followed by stable neutrophil frequencies until study endpoint in week 20 p.i. (Figure 4A). Percentages of apoptotic peripheral neutrophils (Caspase3+) fluctuated across all timepoints (Figure 4B). These data indicate that neutrophil frequencies decrease following clearance of P. fragile, while neutrophil apoptosis is unchanged throughout co-infection.

Minimal Disruption in Neutrophil Phagocytosis during ART-Treated SIV/P. Fragile co-Infection

Impaired phagocytosis has been observed in separate HIV and Plasmodium infection [37,38,45,46,47]. Here, we calculated peripheral blood neutrophil phagocytic score (capability) and index (proficiency) throughout ART-treated SIV/P. fragile co-infection. Although inter-animal variations in neutrophil phagocytic score (Figure 5A) and index (Figure 5B) were observed, no statistically significant changes in either phagocytosis parameter were detected over time.

Decreased Plasma Levels of Neutrophil Granule Components during ART-Treated SIV/P. fragile co-Infection

Plasma levels of MPO, PR3, and CATG, three extracellular neutrophil degranulation secreted components [91,92], were quantified throughout ART-treated SIV/P. fragile co-infection. Consistent with prior work [93], plasma MPO levels were elevated in all four RMs at week 2 p.i., but no significant differences were observed throughout SIV infection, ART-treatment and P. fragile co-infection (Figure 6A). Likewise, plasma levels of PR3 were stable throughout acute SIV infection, ART-treatment, and P. fragile co-infection (Figure 6B). Finally, a statistically significant decrease in CATG was observed at week 14 p.i., compared to BL and weeks 6 and 10 p.i. (P=0.032, 0.0038, and 0.0331, respectively), followed by a return to BL levels following antimalarial treatment (Figure 6C). These data indicate that P. fragile co-infection of ART-treated SIV+ RMs resulted in lowered plasma levels CATG.

Increased Plasma Biomarker of NET Formation during ART-Treated SIV/P. Fragile co-Infection

Excessive NET formation contributes to inflammation in separate HIV and Plasmodium infection (33, 48, 52, 53). We assessed plasma levels of NE and CitH3, biomarkers of NET formation [94], throughout ART-treated SIV/P. fragile co-infection. Plasma levels of NE were significantly increased at week 14 p.i. compared to BL and weeks 2, 10, 12, and 13 p.i. (P=0.0354, 0.0237, 0.0450, 0.0406, and 0.0066, respectively; Figure 7A), but significantly reduced at weeks 16 and 20 p.i. as compared to week 14 p.i. (P=0.0137 and 0.0096, respectively) and week 15 p.i. (P=0.0359 and 0.0216, respectively; Figure 7A). Plasma NE was significantly lower at week 20 p.i. compared to week 16 p.i. (P=0.0341; Figure 7A). Plasma levels of CitH3 were elevated in all four RMs at week 14 p.i., but no significant differences were observed throughout SIV infection, ART-treatment, and P. fragile co-infection (Figure 7B). These findings suggest that P. fragile co-infection of ART-treated SIV+ RMs resulted in increased expression of NE, a marker of NET formation.

Increased Plasma Markers of Gut Permeability and Microbial Translocation during ART-Treated SIV/P. Fragile co-Infection

Loss of GI epithelial barrier integrity leads to microbial translocation in both HIV and Plasmodium infection [39,43,44,95]. Here, we examined plasma levels of zonulin, a protein that modulates tight junctions [96], I-FABP, a circulating biomarker of intestinal injury [97,98], sCD14, which is released from monocytes upon lipopolysaccharide (LPS) stimulation, and LBP, which assists in LPS recognition by interacting with LPS receptors [99]. Inter-animal variation in plasma zonulin levels were observed throughout the study (Figure 8A). Plasma I-FABP was significantly increased at week 14 p.i. compared to week 2 p.i. (P=0.0436; Figure 8B), sCD14 was significantly greater at week 16 p.i. compared to week 6 p.i. (P=0.0377; Figure 8C) and LBP was significantly increased at week 14 p.i. compared to weeks 2, 15 and 20 p.i. (P=0.0441, 0.0305 and 0.0027, respectively; Figure 8D). These findings indicate that P. fragile co-infection may exacerbate GI epithelial barrier disruption, resulting in microbial translocation, in ART-treated SIV+ RMs.

Markers of SIV and P. fragile Infection, Neutrophil Frequency and Function, Inflammation, GI Permeability, and Microbial Translocation Are Correlated in P. fragile co-Infected ART-Treated SIV+ RMs

To identify links between markers of SIV and P. fragile infection, neutrophil frequency and function, inflammation, GI barrier permeability, and microbial translocation, we conducted a MANOVA, controlling for time (Figure 9). SIV VLs were positively associated with plasma zonulin, a marker of gut dysfunction ([ρ=0.620, P<0.0001], Figure 9). Absolute CD4+ T-cell counts were positively associated with markers of NET formation (NE and CitH3, both ρ=0.389, P=0.006), gut dysfunction (I-FABP [ρ=0.342, P=0.017]), and systemic inflammation (CRP [ρ=0.369, P=0.001]), but negatively with degranulation (CATG [ρ=-0.434, P=0.002]; Figure 9). HCT was positively associated with markers of degranulation (CATG [ρ=-0.576, P<0.0001]), but negatively associated with markers of NET formation (NE [ρ=-0.785, P<0.0001] and CitH3 [ρ=-0.549, P<0.0001]), chemokine production (MCP-1 [ρ=-0.349, P= 0.015]), GI dysfunction (sCD14 [ρ=-0.694, P<0.0001], I-FABP [ρ=-0.464, p=0.001], LBP [ρ=-0.424, P=0.003]), and systemic inflammation (CRP [ρ=-0.681, P<0.0001]). Neutrophil frequency was positively associated with chemokine production (MCP-1, [ρ=0.313, P=0.030]) and systemic inflammation (CRP [ρ=0.342, P=0.017]), but negatively associated with markers of degranulation (CATG [ρ=-0.514, P<0.0002]). Chemokine production (MCP-1) was positively associated with markers of NET formation (NE [ρ=0.357, P=0.013], CITH3 [ρ=0.403, P=0.005]), as well as with other markers of inflammation (IP-10 [ρ=0.845, P<0.0001] and CRP [ρ=0.353, P=0.014]; Figure 9). Markers of GI dysfunction (sCD14 and IFABP) were positively correlated with markers of NET formation (NE [ρ=0.648, P<0.0001] and ρ=0.537, P<0.0001], respectively; CITH3 [ρ=0.553, P<0.0001] and [ρ=-0.277, P=0.057], respectively), but negatively correlated with markers of degranulation (CATG [ρ=-0.404, P=0.004] and [ρ=-0.460, P=0.001], respectively; Figure 9). Markers of GI dysfunction (sCD14, IFABP, and LBP) were also positively associated with a marker of systemic inflammation (CRP [ρ=0.598, P<0.0001], [ρ=0.504, P=0.0002], and [ρ=0.487, P=0.0004], respectively; Figure 9). Finally, a marker of systemic inflammation (CRP) was positively associated with markers of NET formation (NE [ρ=0.797, P<0.0001] and CitH3 [ρ=0.783, P<0.0001], respectively), but negatively correlated with markers of degranulation (CATG [ρ=-0.561, P<0.0001]; Figure 9). These data indicate that P. fragile co-infection of ART-treated SIV+ RMs was associated with markers of NET formation, CD4+ T-cell proliferation, inflammation, and chemokine production, which may have allowed for viral persistence and exacerbation of GI barrier disruption and microbial translocation.

Discussion:

Here, we characterized the impact of P. fragile co-infection on ART-treated, SIV+ RMs. Pathogenic SIV infection in RMs has been well-characterized, with RMs exhibiting uncontrolled VLs and decreased CD4+ T-cell counts within two weeks following SIV infection, similar to pathogenic HIV infection in humans [73,74,75]. Additionally, previous work has shown that ART-initiation during SIV infection results in decreased VLs within two weeks of starting treatment [63] and that ART treatment rapidly restores CD4+ T-cells and T-cell functionality, even at end stage disease [76]. Consistent with these prior findings, all four RMs in our study exhibited elevated VLs and depleted CD4+ T-cell counts during acute SIV infection, followed by decreased VLs and elevated CD4+ T-cell counts post ART-initiation, indicating that all RMs followed the expected progression of acute SIV infection and response to suppressive therapy.
Following P. fragile co-infection, SIV-infected RMs maintained detectable VLs and decreased CD4+ T-cell counts for several weeks. Although SIV/P. fragile co-infection has been examined previously [56,57], to the best of our knowledge, this is the first assessment of SIV/P. fragile co-infection during ART. Our observation of detectable VLs during P. fragile co-infection despite ART agrees with prior reports noting detectable VLs during co-infection of ART-naïve RMs [56]. Additionally, Plasmodium co-infection resulted in increased HIV replication sans ART in vitro and in vivo in humans (9-11, 100-102). Of note, one RM (JF97) had persistently high VLs despite ART, as well as higher levels of inflammatory cytokines and chemokines. Previous work has shown associations between host genetics, including expression of particular MHC alleles, and high VLs and rapid disease progression in both humans [103] and macaques [104,105]. Recent work suggests an association between Mamu-B*012 and high VLs in RM, but only with specific KIR alleles [104]. Here, one animal (LE96) did express Mamu-B*012, but KIR genotyping was not performed and this animal did not exhibit exceptionally high VLs. In addition, there were no marked differences in clinical history, such as values reported in weekly CBCs or blood chemistries, or physical exams, between JF97 and the other three RMs assessed here. Additional work is needed to understand JF97’s inability to virally control despite ART. Importantly, there is no expected interaction between the ART regimen (TDF/FTC/DTG) and anti-malarial drugs (chloroquine and quinine sulfate) used, indicating that drug-drug interactions are unlikely to be the cause of persistent SIV VLs despite ART treatment [106].
Systemic inflammation is a hallmark of HIV infection, even with consistent ART [24]. Indeed, CRP, an acute-phase marker of inflammation, has been shown to be elevated in ART-naïve and ART-treated PWH [77,78,79,80]. Additionally, increased CRP has been used as a biomarker of malaria severity [81,82]. Here, we observed increased levels of serum CRP that coincided with peak parasitemia in ART-treated, SIV+ RMs, indicating increased inflammation during co-infection. We also observed that serum CRP was significantly positively correlated with not just neutrophil frequency, but also markers of NE and CitH3, biomarkers of NET formation [94]. Notably, neutrophil-associated inflammation contributes to pathogenesis during separate HIV and Plasmodium infection (50, 84, 107-109). Prior work has also identified links between residual viral replication during ART and uncontrolled inflammation [110,111]. Thus, neutrophil-associated inflammation could constitute a mechanism underlying continued SIV replication during P. fragile co-infection despite ART. Here, ART-treated SIV/P. fragile co-infection resulted in increased MCP-1, a potent monocyte chemoattractant produced by many cells including neutrophils [112]. Additionally, we observed significant correlations between plasma MCP-1 with NE and CitH3. Previously, increased MCP-1 was associated with NET release in individuals with myocardial infarction, which in turn stimulated further MCP-1 production [113]. Taken together, increased MCP-1 production during ART-treated SIVP. fragile co-infection could result from increased NET formation [114], and these processes may cooperatively contribute to heightened inflammation, allowing for persistent viral replication. Supporting this, plasma MCP-1 was correlated with SIV VL, indicating a potential association between neutrophil-mediated inflammation and SIV reactivation during P. fragile co-infection.
Notably, although neutrophil frequency was minimally altered throughout ART-treated SIV/P. fragile co-infection, a significant shift in NE, a peripheral marker of neutrophil function, was detected. We identified that the increase in this marker of NET formation was significantly inversely correlated with anemia, a clinical marker of Plasmodium infection. Conversely, we noted decreased expression of the neutrophil degranulation marker, CATG, and unchanged neutrophil phagocytosis. Neutrophil selection between defense mechanisms appears to be size-dependent; phagocytosis of smaller microbes inhibits NET release but inhibition of phagocytosis due to microbe size prompts NETosis [115]. Notably, previous studies have shown that both opsonized and non-opsonized monocyte/macrophage phagocytosis of P. falciparum-infected erythrocytes are impaired in PWH in vitro and in vivo [115,116,117], although non-opsonized parasite phagocytosis was restored after 6 months of ART [117]. Our data could indicate that an insufficient phagocytic response during SIV/P. fragile co-infection skews neutrophils towards NET formation, providing a potential mechanism by which neutrophils contribute to systemic inflammation during SIV/P. fragile co-infection.
GI dysfunction is a major pathogenic process in separate HIV and malaria infection (42-44, 118). Plasmodium parasite sequestration in the GI tract causes barrier permeability [95,119], while HIV-associated GI mucosal dysfunction is linked with loss of barrier integrity and elevated microbial translocation [43,99]. Our data indicating that SIV VL is positively correlated with plasma zonulin levels is in agreement with this. Importantly, GI neutrophil infiltration and survival has previously been correlated with HIV-associated GI mucosal dysfunction [39,40,41], thus GI neutrophil activity in response to parasite sequestration could further exacerbate SIV-associated GI dysfunction. Our data indicating that plasma markers of GI barrier permeability (plasma sCD14, iFABP, LBP) were associated peripheral markers of NET formation (plasma NE and CitH3) in ART-treated SIV/P. fragile co-infected RMs supports this. Moreover, our observation of an inverse correlation between sCD14, LBP, and anemia, a hallmark of clinical malaria infection further supports a potential relationship between malaria-induced GI dysfunction, possibly mediated by GI neutrophil infiltration and inflammation, during ART-treated SIV co-infection. A caveat of these data is that they are currently limited to plasma markers of neutrophil function, GI barrier integrity, and microbial translocation. Future studies will focus on assessing the relationship between these factors in mucosal tissues to fully define mechanistic relationships between neutrophil-associated GI dysfunction and SIV/malaria co-infection pathogenesis.
A major strength of our work is the utilization of an NHP model which mimics HIV and P. falciparum infection (54, 55, 64, 65). Additionally, our longitudinal assessments provided an opportunity to identify how Plasmodium co-infection could influence SIV pathology in the setting of viral suppression. A caveat of our study is the short duration of ART and lack of complete viral suppression for an extended period prior to P. fragile co-infection. Additional work characterizing P. fragile co-infection in RMs with long-term SIV suppression is needed. Another caveat to this study is the usage of chloroquine and quinine sulfate as our anti-malarial treatment. Both quinine sulfate and chloroquine have been shown to influence various immune parameters, including NET formation and phagocytosis [120,121,122,123,124]. Given that our data indicate that neutrophil phagocytosis was unchanged throughout anti-malarial drug treatment, and the significant increase we observed in NE occurred prior to quinine sulfate and chloroquine administration, the use of these drugs in our study likely did not impact our observations on the effects of P. fragile co-infection on neutrophil responses in ART-treated SIV-infected RMs. Nonetheless, we cannot completely discount the possibility that quinine sulfate and/or chloroquine administration may have affected these parameters, and future studies should include anti-malarial drug treatment only groups to control for this possibility. Previous work has found that although neutrophil frequency and function are unchanged during the pre-erythrocytic stage of Plasmodium infection, total leukocytes are significantly increased [125]. Future studies that incorporate Plasmodium transmission through infected mosquito bites will allow for assessment of the pre-erythrocytic stage. Another limitation is the small number of RMs used, particularly since inter-animal variation was observed in some parameters, which may have contributed to the lack of statistical significance observed at some time points. Additional work with more animals, with the addition of SIV-only, P. fragile-only, and anti-malaria treatment only control groups, will be necessary to fully characterize the kinetics and impact of co-infection, particularly in critical tissues, including the GI mucosa.
In summary, ART-treated SIV/P. fragile co-infected RMs displayed clinical signs of SIV and malaria, which were associated with shifts in neutrophil function and increased markers of GI mucosal dysfunction. These observations could have implications for HIV and malaria co-endemic areas; Plasmodium co-infection in PWH may lead to viral reactivation, creating a scenario in which rates of HIV transmission are sustained even despite widespread use and adherence to ART. Indeed, PWH who have viral loads greater than 1000 copies/ml, regardless of ART, are at increased risk of transmitting HIV [126]. It is important to note, however, that little work has been done to examine whether sustained SIV VL correlates with increased risk of SIV transmission, thus additional work will be needed to extend our observations using an NHP model to ART-treated PWH with Plasmodium co-infection. Moreover, our observations of a link between neutrophil function with clinical signs of SIV/P. fragile and GI dysfunction highlight the need for additional research to define the role of this cellular subset during co-infection and supports the rationale for examining the potential for neutrophil-targeted interventions to reduce the burden of HIV and malaria, separately and in the context of co-infection.

Supplementary Materials

The following supporting information can be downloaded at the website of this paper posted on Preprints.org

Author Contributions

SMN performed and analyzed experiments and prepared the manuscript. HCG, JEP and SLF assisted with tissue processing, flow cytometry and ELISA/LegendPLEX. KG, NJM, and PAM assisted with viral inoculum selection, preparation, and administration. LAR performed MHC genotyping. KDP provided the original P. fragile stock used to propagate the parasite stock used in these studies. MV, KD, NG and MJM assisted with flow cytometry acquisition and expertise. CT, MBB, and CA supported qPCR for viral load assessments. CM and ME supported parasitemia assessments. BG provided veterinary support for animals. JGS provided statistical support. RVB, AK, THM, and BLR assisted with data interpretation and paper preparation. JAM conceived of the study and oversaw the planning and direction of the project, including data interpretation and writing of the paper. All authors read and approved the final manuscript.

Data Availability

All data are available withing the manuscript or in its supplemental materials.

Acknowledgements

We thank all the veterinary staff at the Tulane National Primate Research Center (TNPRC) for their aid with this animal study. We thank Gilead and ViiV for their generous contribution of ART drugs for these studies. Flow Cytometry analysis and expertise were provided by the Flow Cytometry Core, Research Resource Identifier (RRID) SCR_024611. The Flow Cytometry Core is funded by TNPRC base grant OD011104 from NIH. Blood chemistry and CBC were provided the Clinical Pathology Cre (RRID: SCR_024609). The Pathogen Detection and Quantification Core provided viral load data and analysis (RRID: SSCR_024614). Parasitemia data and analysis was provided by the Vector-Borne Infectious Disease and Diagnostic Parasitology Core (RRID: SSC_024680). Virus, virus preparation, and inoculation were given and performed by the Virus Characterization, Isolation, Production, and Sequencing Core (SCR_024679) This work was supported by R21OD031435 to JAM. Research reported in this publication was additionally supported in part by TNPRC (RRID:SCR_008167) NIH core grants P51OD011104, U42OD010568, and U42OD024282. The funders had no direct role in study design, data collection, analysis, or preparation and publication of the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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Figure 1. Experimental Timeline. Experimental timeline depicting sample collection from adult male rhesus macaques (RM, n=4). RMs were inoculated with SIVmac239, TCID50=50, intravenously (i.v.) at week 0. Daily antiretroviral treatment (ART) was given subcutaneously, began at week 8, and continued until the end of the study (TDF/FTC/DTG; 5.1/30/2.5 mg/kg). RMs were inoculated with Plasmodium fragile, 20x106 infected erythrocytes, via i.v.. Anti-malaria treatment occurred over one week, at week 14, and consisted of one oral gavage of quinine sulfate (150mg) followed by four daily oral gavages of chloroquine (20mg/kg). .
Figure 1. Experimental Timeline. Experimental timeline depicting sample collection from adult male rhesus macaques (RM, n=4). RMs were inoculated with SIVmac239, TCID50=50, intravenously (i.v.) at week 0. Daily antiretroviral treatment (ART) was given subcutaneously, began at week 8, and continued until the end of the study (TDF/FTC/DTG; 5.1/30/2.5 mg/kg). RMs were inoculated with Plasmodium fragile, 20x106 infected erythrocytes, via i.v.. Anti-malaria treatment occurred over one week, at week 14, and consisted of one oral gavage of quinine sulfate (150mg) followed by four daily oral gavages of chloroquine (20mg/kg). .
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Figure 2. P. fragile co-infection results in clinical signs of SIV infection despite persistent, daily ART. Peripheral P. fragile parasitemia, anemia, SIVmac239 peripheral viral load (VL), and blood CD4+ T cell count were assessed in adult male rhesus macaques (RMs; n=4). A) Following P. fragile inoculation at week 12 post-SIV infection (p.i.), parasitemia was assessed tri-weekly, indicated as week p.i. A, B, and C. % Parasitemia was assessed via Giemsa staining of thin blood smears and was defined as the percentage of erythrocytes infected by a parasite among all erythrocytes. B) Anemia was assessed by characterizing % hematocrit, defined as the ratio of red blood cells to total blood. C) Plasma VL (RNA copies/ml plasma) was assessed by qPCR. D) Absolute number of CD4+ T cells per µL of blood was assessed via flow cytometry. In all panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -4, -2, and 0 p.i.. Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
Figure 2. P. fragile co-infection results in clinical signs of SIV infection despite persistent, daily ART. Peripheral P. fragile parasitemia, anemia, SIVmac239 peripheral viral load (VL), and blood CD4+ T cell count were assessed in adult male rhesus macaques (RMs; n=4). A) Following P. fragile inoculation at week 12 post-SIV infection (p.i.), parasitemia was assessed tri-weekly, indicated as week p.i. A, B, and C. % Parasitemia was assessed via Giemsa staining of thin blood smears and was defined as the percentage of erythrocytes infected by a parasite among all erythrocytes. B) Anemia was assessed by characterizing % hematocrit, defined as the ratio of red blood cells to total blood. C) Plasma VL (RNA copies/ml plasma) was assessed by qPCR. D) Absolute number of CD4+ T cells per µL of blood was assessed via flow cytometry. In all panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -4, -2, and 0 p.i.. Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
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Figure 3. Variable levels of inflammatory markers throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. CRP, cytokine and chemokine levels were measured throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4). A) CRP levels were measured in serum by a Beckman au480. B-D) IL-8 (B), IP-10 (C), and MCP-1 (D) levels were measured in plasma by LegendPlex. In all panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
Figure 3. Variable levels of inflammatory markers throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. CRP, cytokine and chemokine levels were measured throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4). A) CRP levels were measured in serum by a Beckman au480. B-D) IL-8 (B), IP-10 (C), and MCP-1 (D) levels were measured in plasma by LegendPlex. In all panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
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Figure 4. Minimal disruption of peripheral neutrophil frequencies and percentage of neutrophils undergoing apoptosis during P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. Total neutrophil frequencies and frequencies of neutrophils undergoing apoptosis were assessed in whole blood before and after P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4) by flow cytometry. A) Neutrophil (HLA-DR-CD11b+CD66abce+CD14+) frequency of live CD45+ cells were assessed throughout co-infection. B) The frequency of neutrophils undergoing apoptosis (caspase3+) was assessed throughout co-infection. In both panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -4, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
Figure 4. Minimal disruption of peripheral neutrophil frequencies and percentage of neutrophils undergoing apoptosis during P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. Total neutrophil frequencies and frequencies of neutrophils undergoing apoptosis were assessed in whole blood before and after P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4) by flow cytometry. A) Neutrophil (HLA-DR-CD11b+CD66abce+CD14+) frequency of live CD45+ cells were assessed throughout co-infection. B) The frequency of neutrophils undergoing apoptosis (caspase3+) was assessed throughout co-infection. In both panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -4, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
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Figure 5. Nominal alterations in neutrophil phagocytosis during P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. The frequency of phagocytic neutrophils and neutrophil phagocytic capacity was assessed in whole blood throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4) by flow cytometry. Phagocytosis was determined by assessing uptake of pHrodo Red E. coli Bioparticles. A) Neutrophil phagocytic score was calculated by multiplying the absolute number of neutrophils/μL of whole blood by the percentage of neutrophils positive for uptake of pHrodo bioparticles. B) Phagocytic index was calculated by multiplying the phagocytic score in A by the Mean Fluorescence Intensity (MFI) of pHrodo positive neutrophils. In both panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -4, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
Figure 5. Nominal alterations in neutrophil phagocytosis during P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. The frequency of phagocytic neutrophils and neutrophil phagocytic capacity was assessed in whole blood throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4) by flow cytometry. Phagocytosis was determined by assessing uptake of pHrodo Red E. coli Bioparticles. A) Neutrophil phagocytic score was calculated by multiplying the absolute number of neutrophils/μL of whole blood by the percentage of neutrophils positive for uptake of pHrodo bioparticles. B) Phagocytic index was calculated by multiplying the phagocytic score in A by the Mean Fluorescence Intensity (MFI) of pHrodo positive neutrophils. In both panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -4, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
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Figure 6. Decreased plasma levels of neutrophil degranulation markers during P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. Products of neutrophil degranulation were measured throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4) via ELISA. A-B) Myeloperoxidase (A), Proteinase 3 (B), and Cathepsin G (C) levels were measured in plasma by ELISA. In all panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
Figure 6. Decreased plasma levels of neutrophil degranulation markers during P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. Products of neutrophil degranulation were measured throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4) via ELISA. A-B) Myeloperoxidase (A), Proteinase 3 (B), and Cathepsin G (C) levels were measured in plasma by ELISA. In all panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
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Figure 7. Increased plasma levels of neutrophil extracellular trap markers during P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. Markers of neutrophil extracellular trap formation were measured throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4) via ELISA. A) Neutrophil elastase and B) Citrullinated histone 3 levels were measured in plasma by ELISA. In both panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
Figure 7. Increased plasma levels of neutrophil extracellular trap markers during P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. Markers of neutrophil extracellular trap formation were measured throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4) via ELISA. A) Neutrophil elastase and B) Citrullinated histone 3 levels were measured in plasma by ELISA. In both panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
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Figure 8. Increased levels of microbial translocation and gastrointestinal (GI) barrier permeability markers P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. Markers of microbial translocation and GI barrier permeability were measured throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4) via ELISA. A-D) Zonulin (A), Intestinal fatty-acid binding protein (I-FABP; B), Soluble CD14 (sCD14; C), and LPS binding protein (LBP; D) levels were measured in plasma by ELISA. In all panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
Figure 8. Increased levels of microbial translocation and gastrointestinal (GI) barrier permeability markers P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques. Markers of microbial translocation and GI barrier permeability were measured throughout P. fragile co-infection of ART-treated SIVmac239-infected rhesus macaques (RMs; n=4) via ELISA. A-D) Zonulin (A), Intestinal fatty-acid binding protein (I-FABP; B), Soluble CD14 (sCD14; C), and LPS binding protein (LBP; D) levels were measured in plasma by ELISA. In all panels, each RM is represented by a different symbol and color. Baseline (BL) is an average of data collected at weeks -6, -2, and 0 post-SIV infection (p.i.). Inoculation with SIVmac239 at week 0 p.i. is indicated by a purple dashed arrow. Inoculation with P. fragile at week 12 p.i. is indicated by a blue dashed arrow. Antiretroviral therapy (ART) was initiated at week 8 p.i., indicated by the dark grey bar. Anti-malarial treatment occurred throughout week 14 p.i., indicated by the light grey bar. Statistical significance between all time points was calculated using a mixed-effects analysis with the Geisser-Greenhouse correction and a Tukey’s multiple comparisons test, with individual variances computed for each comparison. Multiplicity adjusted significant P values are shown above horizontal black bars.
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Figure 9. Multivariate ANOVA (MANOVA) reveals significant correlations between clinical markers of SIV and malaria infection, as well as neutrophil frequency, function, and peripheral markers of GI dysfunction. Pearson’s partial correlation coefficients were generated using a MANOVA for 13 different parameters (VL, anemia, absolute CD4+ count, peripheral neutrophils, plasma zonulin, sCD4, I-FABP, LBP, NE, cathepsin G, CitH3, IP-10, MCP-1, and CRP). The correlation coefficients were adjusted against animal number. Boxes highlighted in light green represent positive correlations trending towards significance (0.05<p<0.07) and boxes highlighted dark green or red represent statistically significant positive or negative correlations, respectively (p<0.05).
Figure 9. Multivariate ANOVA (MANOVA) reveals significant correlations between clinical markers of SIV and malaria infection, as well as neutrophil frequency, function, and peripheral markers of GI dysfunction. Pearson’s partial correlation coefficients were generated using a MANOVA for 13 different parameters (VL, anemia, absolute CD4+ count, peripheral neutrophils, plasma zonulin, sCD4, I-FABP, LBP, NE, cathepsin G, CitH3, IP-10, MCP-1, and CRP). The correlation coefficients were adjusted against animal number. Boxes highlighted in light green represent positive correlations trending towards significance (0.05<p<0.07) and boxes highlighted dark green or red represent statistically significant positive or negative correlations, respectively (p<0.05).
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