Casp8p41 expression in primary T cells induces a proinflammatory response

Abstract

Objective: HIV infection of CD4 T cells can lead to HIV protease-mediated cleavage of procaspase 8 generating a novel, HIV-specific peptide called Casp8p41. Casp8p41 has at least two biologic functions: induction of cell death via mitochondrial depolarization and release of cytochrome C, as well as activation of nuclear factor kappa B (NFkappaB). We have previously shown that Casp8p41-induced NFkappaB activation enhances HIV LTR transcription and consequently increases HIV replication. Herein, we questioned whether Casp8p41-induced NFkappaB activation impacts the cytokine profile of cells expressing Casp8p41.

Design: Analysis of cells expressing Casp8p41 and HIV-infected T cells.

Methods: We assessed whether host genes are transcriptionally activated following Casp8p41 production, using microarray analysis, cytokine quantification, followed by western blot and flow cytometry.

Results: Microarray analysis identified 259 genes significantly upregulated following expression of Casp8p41. Furthermore, Casp8p41 expression in primary CD4 T cells results in increased production of interleukin (IL)-2, IL-15 and tumor necrosis factor (TNF), as well as IL-1RA; whereas levels of granulocyte macrophage colony-stimulating factor and interferon (IFN)-gamma were reduced in the Casp8p41 expressing cells. Intracellular flow cytometry confirmed the co-association of Casp8p41 with elevated TNF in HIV-infected cells.

Conclusion: These data indicate that the expression of Casp8p41 in HIV-infected CD4 T cells in addition to promoting apoptosis and enhancing HIV replication also promotes a proinflammatory cytokine milieu, which is characteristic of untreated HIV infection.

Fig. 1. Casp8p41 expression in T cells alters transcription

(a) Peripheral blood mononuclear cell (PBMC) from an HIV-infected patient were analyzed by flow cytometry for CD3, CD4, CD8, CD27, CD45 and Casp8p41 (representative of >50 patients). (b) Affymetrix HG-U133 plus 2.0 GeneChip analysis of Jurkat T cells transfected with control vector, FL caspase 8, procaspase 8 or Casp8p41 for 2 or 3 h with 54 613 transcripts analyzed. After filtering, 13 666 transcripts were differentially regulated. Samples Casp8p41 (2 h) and Casp8p41 (3 h) were similar. Two hundred and fifty-nine transcripts were upregulated (P <0.05) in the Casp8p41 samples compared to control or procaspase 8, with the top 50 upregulated genes shown. Experiments were performed in triplicate (c). Primary CD4 T cells were transfected with green fluorescent protein (GFP) empty vector, GFP Casp8p41 or GFP Casp8p41ΔDED and analyzed for GFP content by flow cytometry to confirm fusion protein expression (d).

Fig. 2. Casp8p41 expression in primary CD4 T cells alters cytokine production

Primary CD4 T cells were transfected with either empty vector (HA), Casp8p41 (HACasp8p41) or with Casp8p41 missing the amino terminal tandem death effector domain (HACasp8p41ΔDED) and analyzed for cytokine production as indicated. Cytokines significantly upregulated or downregulated by Casp8p41 expression are shown. Each point is a separate blood donor.

Fig. 3. Casp8p41 expression increases tumor necrosis factor

(a) Primary CD4 T cells were transfected with HA, HACasp8p41 or HACasp8p41ΔDED and analyzed by western blot for tumor necrosis factor (TNF) [recombinant TNF (rTNF)]. Control blotting with anti-HA and antitubulin was performed. (b) Primary human CD4 T cells were infected with HIVIIIb and 3 days after infection were analyzed for Casp8p41 and TNF by flow cytometry. Isotype control antibodies were used to determine TNF positivity. (c) The specificity of TNF production in Casp8p41-positive cells was assessed by simultaneous analysis of TNF and Casp8p41. Results are representative of four separate experiments.

Fig. 4. Tumor necrosis factor production is specifically increased in cells where HIV protease has created Casp8p41

The caspase 8-deficient cell line I9.2 was stably transfected with either wild-type caspase 8 or caspase 8 containing an FF:RN mutation at positions 355:356, which render it noncleavable by HIV protease. These cells were either transfected with HIV protease or with protease containing the active site dead mutation, D25G. Following transfection, the cells were analyzed for intracellular Casp8p41 content and for TNF by intracellular flow cytometry.