Functional proteomic analysis for regulatory T cell surveillance of the HIV-1-infected macrophage

Abstract

Regulatory T cells (Treg) induce robust neuroprotection in murine models of neuroAIDS, in part, through eliciting anti-inflammatory responses for HIV-1-infected brain mononuclear phagocytes (MP; macrophage and microglia). Herein, using both murine and human primary cell cultures in proteomic and cell biologic tests, we report that Treg promotes such neuroprotection by an even broader range of mechanisms than previously seen including inhibition of virus release, killing infected MP, and inducing phenotypic cell switches. Changes in individual Treg-induced macrophage proteins were quantified by iTRAQ labeling followed by mass spectrometry identifications. Reduction in virus release paralleled the upregulation of interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity. Treg killed virus-infected macrophages through caspase-3 and granzyme and perforin pathways. Independently, Treg transformed virus-infected macrophages from an M1 to an M2 phenotype by down- and up- regulation of inducible nitric oxide synthase and arginase 1, respectively. Taken together, Treg affects a range of virus-infected MP functions. The observations made serve to challenge the dogma of solitary Treg immune suppressor functions and provides novel insights into how Treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection.

Figure 1

Treg inhibition of virus release from infected BMM is interferon-stimulated gene 15 pathway associated. (A) Western blotting showed increased STAT1 phosphorylation in HIV-1/VSV infected BMM cocultured with Treg group. Data shown are representative of three independent experiments. (B) Western blotting showed dramatically increased free ISG15 and ISGylated proteins in HIV-1/VSV infected BMM cocultured with Treg group. Data shown are representative of three independent experiments. (C) Western blotting showed no change of HIV-1 p24 expression between HIV-1/VSV infected BMM group, HIV-1/VSV infected BMM group cocultured with Tcon group and HIV-1/VSV infected BMM group cocultured with Treg group. In this study the pseudotyped virus we used could enter the mouse BMM once, so the level of p24 staining is dependent only on the infective dose. Data shown are representative of three independent experiments. (D) Both Tcon and Treg could inhibit HIV-1 release determined by HIV-1 p24 ELISA. Data shown are representative of three independent experiments. ***p < 0.001. (E) Proposed pathways involved in initiating antiviral immune response in HIV-1-infected macrophages, which was enhanced by Treg and Tcon. In macrophages, engagement of TLR3 by HIV derived dsRNA in endosomes mediates IFN-α/β/λ production. TLR3 induces a Trif-dependent pathway, which recruits kinases (TBK1, IKKϵ and IKKαβγ) that mediate activation of the transcription factors IRF3 and NF-κB. Together with IRF7, IRF3 and NF-κB translocate into the nucleus and bind to positive regulatory domains on the Ifn genes promoter, leading to Ifn transcription. Secreted IFNs signal through binding to their cognate receptors (IFNAR1 and IFNAR2) and then recruit kinases (TYK2 and JAK1) leading to translocation of phosphorylated transcription factors (STAT1 and STAT2) and IRF9 to the nucleus where binding to the enhancer region of the ifn promoter and isg promoter occurs, which leads to the activation and nuclear transport of ISGF3 and induction of ISRE to produce IFNs and ISGs. Both IFNs and ISGs could inhibit ubiquitination of HIV Gag and Tsg101, which results in less virus release. With unknown mechanisms, Treg exhibit much greater capacity to enhance the antiviral immune response than Tcon.

Figure 2

Treg Induces Virus-Infected BMM Death. (A) Treg induces HIV-1/VSV-infected BMM death shown by TUNEL assay plus confocal immunofluorescence microscopy and trypan blue staining plus light microscopy. For TUNEL assay, bright field is shown in the top panel and the corresponding fluorescent field is shown in the middle panel. Trypan blue staining is shown in the bottom panel (trypan blue positive cells are indicated by yellow arrows). White scale bar represent 100 μm. (B) Graphs represent the mean percentage of TUNEL positive BMM and trypan blue positive BMM counted and calculated from five different fields (data were pooled from three independent experiments). Each bar represents a mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Treg and Tcon Induced Virus-Infected BMM Apoptosis and Pyroptosis. (A) Western blotting showed HIV-1/VSV infection triggers caspase-1 activation in BMM, which was enhanced by Tcon coculture while attenuated by Treg coculture. Data shown are representative of five independent experiments. (B) Western blotting showed Treg coculture activated caspase-3 pathway in HIV-1/VSV infection BMM. Data shown are representative of five independent experiments. (C) Caspase-1 activation was measured by FLICA assay plus immunofluorescence microscopy. White scale bars represent 50 μm. Caspase-3 and 7 activation was measured by FLICA assay plus immunofluorescence microscopy. White scale bars represent 50 μm. (D) Proposed mechanisms Treg and Tcon use to modulate HIV-1-infected macrophage functions. Upon infection, HIV-1 activates NF-κB and the inflammasome, which results in IL-1β release. Tcon worsened this response by secretion of proinflammatory cytokines. In addition, those proinflammatory cytokines increase mitochondrial activity, which could lead to energy exhaustion and pyroptosis. However, Treg use their anti-inflammatory cytokines to extinguish the inflammation resulting from virus infection. Treg induce infected BMM apoptosis. Compared with apoptosis, pyroptosis causes more ATP release. Excessive ATP is an important inflammatory molecule and could be hydrolyzed by ecto-nucleoside triphosphate diphosphohydrolase CD39 and CD73 expressed on Treg. Moreover, Treg could transport cAMP to infected BMM and contribute to inflammation resolution. The activation status of infected macrophage affects Th0 cell differentiation. For example, M1 induce Th1 and Th17, while M2 induce Th2 and iTreg. (E) Comparison of CD39 expression on Tcon and Treg was determined by flow cytometric analysis.

Figure 4

Treg Uses Granzyme A, B and Perforin to Kill Virus-Infected BMM. (A) Western blotting demonstrating increased p38 phosphorylation (upper panel) and perforin insertion (middle panel), which were normalized to β-actin (bottom panel) in HIV-1/VSV infected BMM cocultured with Treg compared with HIV-1/VSV infected BMM cocultured with Tcon groups. Data shown are representative of three independent experiments. (B) Confocal immunofluorescences of BMM with specific monoclonal antibodies against granzyme A (upper panel), granzyme B (middle panel) and perforin (bottom panel) show cytoplasmic granular staining of granzyme A (red), granzyme B (red) and perforin (red) in Tcon and Treg treated groups. Both control and HIV-1/VSV infected groups showed no staining of those antibodies, which means BMM did not produce granzyme and perforin. Higher intensity of granzyme and perforin were seen in the Treg treated group compared with the Tcon treated group. Data shown are representative of three independent experiments. White scale bars represent 5 μm. (C) Cleaved NDUFS3, the 30 kDa subunit of mitochondrial complex I and the important substrate of granzyme A, was shown in HIV-1/VSV infected BMM cocultured with Treg group by Western blotting. Data shown are representative of three independent experiments. (D) Membrane potential-dependent staining of mitochondria in HIV-1/VSV infected BMM by JC-1 visualized by fluorescence microscopy showed the loss of red JC-1 aggregates fluorescence and cytoplasmic diffusion of green monomer fluorescence following coculture with Treg.

Figure 5

Treg Transforms the Infected BMM. (A) Treg induces HIV-1/VSV infected BMM morphology change from elongation to roundness shown by confocal immunofluorescence microscopy (zoomed part is shown at the bottom panel). White scale bars represent 20 μm. (B) Western blotting showed Treg downregulated iNOS, while upregulated arginase-1. (C) The concentration of NO in supernatant was measured with Griess test. Treg inhibited NO production from HIV-1/VSV infected BMM, ***p < 0.001. (D) Concentrations of cAMP in the lysate (L) and supernatant (S) of Tcon or Treg were detected by ELISA, *p < 0.05 (E) Concentrations of cAMP in the lysate and supernatant of BMM with different treatments. The cAMP in the supernatant was undetectable, while its concentration in lysate increased in Treg treated group. ***p < 0.001.

Figure 6

Treg-induced infected BMM elicits neuroprotection. Tubulin staining shows persevered neuronal dendrites in groups cultured with both CM1 (upper panel) and CM2 (bottom panel) from Treg treated HIV-1/VSV-infected BMM, which was not seen in Tcon derived CM. Moreover, CM2 from the Tcon treated group exhibits more toxicity than the infected group, which suggests that the proinflammatory cytokines are more destructive than viral proteins. White scale bar represents 50 μm.