Expansion of a subset of CD14(high)CD16(neg)CCR2(low/neg) monocytes functionally similar to myeloid-derived suppressor cells during SIV and HIV infection

Monocytes have been categorized in three main subpopulations based on CD14 and CD16 surface expression. Classical monocytes express the CD14(++)CD16(-) CCR2(+) phenotype and migrate to inflammatory sites by quickly responding to CCL2 signaling. Here, we identified and characterized the expansion of a novel monocyte subset during HIV and SIV infection, which were undistinguishable from classical monocytes, based on CD14 and CD16 expression, but expressed significantly lower surface CCR2. Transcriptome analysis of sorted cells demonstrated that the CCR2(low/neg) cells are a distinct subpopulation and express lower levels of inflammatory cytokines and activation markers than their CCR2(high) counterparts. They exhibited impaired phagocytosis and greatly diminished chemotaxis in response to CCL2 and CCL7. In addition, these monocytes are refractory to SIV infection and suppress CD8(+) T cell proliferation in vitro. These cells express higher levels of STAT3 and NOS2, suggesting a phenotype similar to monocytic myeloid-derived cells, which suppress expansion of CD8(+) T cells in vivo. They may reflect an antiproliferative response against the extreme immune activation observed during HIV and SIV infections. In addition, they may suppress antiviral responses and thus, have a role in AIDS pathogenesis. Antiretroviral therapy in infected macaque and human subjects caused this population to decline, suggesting that this atypical phenotype is linked to viral replication. J. Leukoc. Biol. 91: 803-816; 2012.National Institutes of HealthNational Institutes of Health [MH070306, NS055648, MH085554]Ministry of Health [914/BRA/3014-UNESCO/Kallas]Ministry of HealthSao Paulo City Health Department [2004-0.168.922-7/Kallas]Sao Paulo City Health DepartmentFundacao de Amparo a Pesquisa do Estado de Sao PauloFundacao de Amparo a Pesquisa do Estado de Sao Paulo [04/15856-9/Kallas]Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Brazilian Ministry of EducationBrazilian Ministry of EducationBrazilian Program for STD and AIDSBrazilian Program for STD and AID

Distinct Patterns of Tryptophan Maintenance in Tissues during Kynurenine Pathway Activation in Simian Immunodeficiency Virus-Infected Macaques

Induction of the kynurenine pathway (KP) of tryptophan catabolism has been proposed to contribute to T cell dysfunction during human/simian immunodeficiency virus (HIV/SIV) infection via depletion of local tryptophan levels and production of immunomodulatory KP metabolites. However, while changes in tryptophan and KP metabolites have been observed in plasma, their levels in lymphoid tissues and levels of enzymes downstream of indoleamine 2,3-dioxygenase (IDO1) have been relatively unexplored. We used our SIV-infected pigtailed macaque model to analyze longitudinal changes in KP metabolites and enzymes by gas chromatography/mass spectrometry and NanoString nCounter gene expression analysis, respectively, in spleen and blood compared to changes previously established in brain and CSF. We found that tryptophan levels were remarkably stable in tissue sites despite robust depletion in the circulating plasma and CSF. We also demonstrated that intracellular tryptophan reserves were maintained in cultured cells even in the presence of depleted extracellular tryptophan levels. Kynurenine, 3-hydroxykynurenine, quinolinic acid, and the KP enzymes all displayed highly divergent patterns in the sites examined, though IDO1 expression always correlated with local kynurenine/tryptophan ratios. Finally, we demonstrated by FACS that myeloid dendritic cells (mDCs) and cells of monocytic lineage were the highest producers of IDO1 in chronically infected spleens. Overall our study reveals insights into the tissue-specific regulation of KP enzymes and metabolites and, in particular, highlights the multiple mechanisms by which cells and tissues seek to prevent TRP starvation during inflammation

Platelet factor 4 mediates inflammation in experimental cerebral malaria

Cerebral malaria (CM) is a major complication of Plasmodium falciparum infection in children. The pathogenesis of CM involves vascular inflammation, immune stimulation, and obstruction of cerebral capillaries. Platelets have a prominent role in both immune responses and vascular obstruction. We now demonstrate that the platelet-derived chemokine, platelet factor 4 (PF4)/CXCL4, promotes the development of experimental cerebral malaria (ECM). Plasmodium-infected red blood cells (RBCs) activated platelets independently of vascular effects, resulting in increased plasma PF4. PF4 or chemokine receptor CXCR3 null mice had less severe ECM, including decreased T cell recruitment to the brain, and platelet depletion or aspirin treatment reduced the development of ECM. We conclude that Plasmodium-infected RBCs can directly activate platelets, and platelet-derived PF4 then contributes to immune activation and T cell trafficking as part of the pathogenesis of ECM

Dual Role of Novel Ingenol Derivatives from <i>Euphorbia tirucalli</i> in HIV Replication: Inhibition of <i>De Novo</i> Infection and Activation of Viral LTR

<div><p>HIV infection is not cleared by antiretroviral drugs due to the presence of latently infected cells that are not eliminated with current therapies and persist in the blood and organs of infected patients. New compounds to activate these latent reservoirs have been evaluated so that, along with HAART, they can be used to activate latent virus and eliminate the latently infected cells resulting in eradication of viral infection. Here we describe three novel diterpenes isolated from the sap of <i>Euphorbia tirucalli,</i> a tropical shrub. These molecules, identified as ingenols, were modified at carbon 3 and termed ingenol synthetic derivatives (ISD). They activated the HIV-LTR in reporter cell lines and human PBMCs with latent virus in concentrations as low as 10 nM. ISDs were also able to inhibit the replication of HIV-1 subtype B and C in MT-4 cells and human PBMCs at concentrations of EC₅₀ 0.02 and 0.09 µM respectively, which are comparable to the EC₅₀ of some antiretroviral currently used in AIDS treatment. Control of viral replication may be caused by downregulation of surface CD4, CCR5 and CXCR4 observed after ISD treatment <i>in vitro</i>. These compounds appear to be less cytotoxic than other diterpenes such as PMA and prostratin, with effective dose versus toxic dose TI>400. Although the mechanisms of action of the three ISDs are primarily attributed to the PKC pathway, downregulation of surface receptors and stimulation of the viral LTR might be differentially modulated by different PKC isoforms.</p></div

ING-B blocks HIV <i>de novo</i> replication through downregulation of surface receptors.

<p>CD4+ T cells from three healthy donors were stimulated with PHA/IL-2 for 5 days and then treated with different concentrations of ING-B for 24 h. A portion of the cells was used for cytometry evaluation (A), and the graph depicts the mean and standard deviation of three independent experiments. The remaining cells were infected with HIV NL4-3-Luc for 24 h and cell lysates were analyzed by luciferase activity (B). Results are shown as relative light units (RLU). ZVD 1 µM was used as positive control and DMSO 1% as vehicle control. Results are representative of mean and standard deviation of triplicates for each blood donor.</p

ISDs reduce HIV replication in a dose-dependent manner.

<p>Lymphocytic cell line MT-4 (A and B) and human PBMCs (C and D) were infected with HIV subtype B NL-4.3 (A and C) or HIV subtype C ZM247Fv-1 (B and D) and treated with different concentrations of ISDs. After six days, cell viability was evaluated using the Cell Titer blue kit. Curves were derived by non-linear regression (dose-response curve by Hill for 3 parameters), and dotted lines represent EC₅₀. Experiments were done in triplicate for PBMCs and sextuplicate for MT-4. ZVD was used as positive control.</p

CC₅₀ and EC₅₀ of ISDs and ZVD in MT-4 cells and primary CD4+ T lymphocytes infected with different HIV subtypes (see Figure 5).

<p>CC₅₀ and EC₅₀ values are presented in µM. Therapeutic index (TI) was defined as the ratio CC₅₀/EC₅₀. NA: non applicable. (*) Value estimated by extrapolation.</p

ISDs modulate surface expression of CD4, CXCR4, CCR5 and activation markers.

<p>MT-4 cells (A) and CD4+ T lymphocytes (B) were stimulated with ISDs (ING-A, ING-B, ING-C), bryostatin (bryo), prostratin (prost) and PMA for 48 h. Surface marker expression was evaluated by cytometry. CD4+ T cells were also evaluated for the activation markers CD25, CD38, HLA-DR (C) and CD69 (D). Results are representative of the mean and standard deviation of three independent experiments. DMSO 1% was used as vehicle control.</p

Baseline characteristics of study subjects.

<p>(*) Time on HAART with documented continuous suppression of plasma viremia (< 50 copies/mL); (**) Drug abbreviations: ZDV, zidovudine; 3TC, lamivudine; EFV, efavirenz; TDF, tenofovir disoproxil fumarate.</p

ISDs upregulate HIV-LTR-driven GFP expression in J-Lat clones.

<p>GFP expression (A) and viability (B) in J-Lat clone 9.2 were measured after cells were treated with ISDs, prostratin and PMA in different concentrations for 48 h. Similar experiment was done in J-Lat clone 10.6 (C and D). E) Comparison of GFP expression in four different J-Lat clones, all treated with 1 µM of each compound for 48 h. J-Lat clone 15.4 is depicted in a different graph (F). Graphs show mean and standard deviation of 3 separate tests. In all experiments, TNF-α 10 ng/mL was used as positive control.</p