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HiACT - HIV immune activation and signals through ITAM
thesisposted on 09.02.2017, 05:09 by Lichtfuss, Gregor Frederik
Introduction: HIV-1 infection is associated with generalized immune activation that persists despite suppression of viremia with combination antiretroviral therapy (cART). Chronic immune activation is thought to be the consequence of persistent low-level viral replication, cART toxicity and translocation of microbial products through an impaired gut mucosa. A number of immune defects remain in T-cells, natural killer (NK) cells and monocytes and dysfunction persists despite cART. Immune cell dysfunction is characterized by alterations in the response to activating stimuli. Such stimuli are processed through signal transduction via a variety of immune receptors specific to each cell type, including the T-cell receptor (TCR) on T-cells, and Fc gamma receptor (FcγR) CD16 expressed on NK cells and CD16 and CD64 expressed on monocytes. These surface receptors stimulate similar signaling pathways, which are dependent on adaptor proteins that bear the characteristic immunoreceptor tyrosine activation motif (ITAM). ITAM proteins include TCR zeta (TCRζ) expressed in T-cells and NK cells and FcR gamma (FcRγ) expressed in NK cells and monocytes. These proteins are critical both for receptor surface presentation, acting as chaperones for the immune receptors, and for receptor function, acting as docking sites for kinases that transduce signals between receptor and downstream pathways. The loss of ITAM proteins, especially TCRζ in T-cells, is the hallmark of chronic inflammatory conditions like cancers and autoimmune diseases. Loss of TCRζ in HIV-infected subjects has been reported previously, but the underlying mechanism for this loss is not known. Our workgroup previously observed loss of FcRγ expression in NK cells purified from the blood of HIV-infected subjects receiving cART. These subjects were a heterogeneous convenience sample of patients with largely controlled viremia in which loss of FcRγ was not correlated with viral load. Based on these preliminary findings, and the reports of loss of TCRζ in other chronic inflammatory conditions, we hypothesized that ITAM protein expression is reduced by persistent immune activation driven by microbial translocation in the setting of HIV infection, and that this leads to inhibition of ITAM-dependent signal transduction pathways and loss of cell function in T-cells, NK cells and monocytes. Methods: We established a cross sectional study of cART-naive HIV-infected subjects and subjects currently receiving cART, and age/sex-matched HIV-uninfected controls (the HIV immune activation: signals through ITAM (HiACT) study). Highly purified, FACS-sorted T-cells, NK cells and monocytes were analyzed for mRNA and protein expression of TCRζ in T-cells and NK cells, FcRγ in NK cells and monocytes and DAP12, a functional homologue of FcRγ, which is important for natural cytotoxicity signaling in NK cells using real-time PCR and quantitative immunoblotting. Cellular activation was measured by assessing CD38 and HLA-DR expression on the surface of T-cells and NK cells. In addition, plasma was tested for markers of myeloid cell activation (soluble CD14 (sCD14), neopterin) and translocation of microbial products (prokaryotic DNA coding for 16S rRNA, 16S DNA, and lipopolysaccharide, LPS). TCRζ and FcRγ-dependent signaling were measured in whole blood by cross-linking of CD3 (T-cells) and CD16 (NK cells) surface receptors coupled with Phosflow analysis measuring tyrosine phosphorylation of Syk/ZAP70 (in both T-cells and NK cells) and CD107a mobilization (in NK cells) by flow cytometry. Results: Seventeen cART-naive subjects (ART-), 17 subjects currently receiving cART (ART+), and 20 HIV-uninfected subjects (HIV-) were recruited into HiACT. Subjects receiving cART had a median time of virologic suppression prior to recruitment of 1270 days (range 218-2817 days). Measurement of ITAM protein expression showed firstly, that TCRζ mRNA was reduced in CD3+ T-cells of HIV-infected subjects (ART- and ART+; p<0.05), secondly, that expression of FcRγ mRNA (p<0.01) and protein (p<0.01) along with CD16 surface expression was reduced in NK cells from cART-naive subjects and subjects receiving cART compared to HIV-uninfected subjects (p<0.01), and thirdly, that FcRγ mRNA and protein showed trends towards increased expression in monocytes of cART-naive subjects, which returned to control values in subjects receiving cART (mRNA: p<0.01). Measurement of ITAM-dependent signaling and cell functions showed firstly, that TCRζ-dependent phosphorylation of ZAP70 (pZAP70) was reduced in T-cells of cART-naive subjects and subjects receiving cART in comparison to HIV-uninfected subjects (p<0.05). Secondly, CD16-dependent Syk phosphorylation (p<0.01) and CD107a mobilization (p<0.05) were reduced in NK cells of cART-naive subjects, and increased towards, but did not reach, uninfected control levels in NK cells of subjects receiving cART. Flow cytometry profiles from the Phosflow experiments revealed a bimodal distribution of pZAP70 within CD4+ and CD8+ T-cells following CD3 cross-linking, suggesting the existence of two distinct subpopulations with respect to TCR signaling. The presence of the unresponsive population was most prominent in cART-naive subjects, present to a lesser degree in subjects receiving cART, and observed infrequently in HIV-uninfected subjects. Measurement of immune activation showed that T-cells and NK cells from cART-naive subjects expressed high levels of HLA-DR/CD38 co-expression. T-cell activation was reduced in subjects receiving cART, but not to HIV-uninfected control levels (CD4+ T-cells, CD8+ T-cells: p<0.01). By contrast, activation was not reduced in NK cells from subjects receiving cART compared to cART-naive subjects. In addition, NK cells showed high rates of baseline degranulation in both cART-naive and cART-receiving study groups. Plasma sCD14 and neopterin levels were also elevated in both cART-naive and cART-receiving study groups (p<0.05). Markers of myeloid cell activation and inflammation were increased in cART-naive subjects (neopterin, sCD14 p<0.0001) and remained elevated in subjects receiving cART (neopterin p<0.019, sCD14 p<0.0001). Increased plasma sCD14 and neopterin levels correlated with increased CD8+ T-cell activation (p<0.05). Plasma sCD14 and HLA-DR/CD38 expression on CD8+ T-cells correlated with reduced ZAP70 phosphorylation in CD8+ T-cells (p<0.05). HLA-DR/CD38 expression on T-cells and NK cells and plasma levels of sCD14 inversely correlated with both FcRγ levels (p<0.05) and Syk phosphorylation in NK cells (p<0.05). Increased transcription of FcRγ in monocytes correlated with activation of CD4+ T-cells and plasma levels of neopterin (p<0.05). Bacterial 16S DNA and LPS levels did not correlate with markers for cell activation nor with inflammation, nor with alterations in ITAM proteins in any cell type, including the study endpoint of altered expression of FcRγ in NK cells. Discussion: These data support the hypothesis that chronic immune activation is associated with down-regulated expression of TCRζ and FcRγ signaling adaptor proteins in T-cells and NK cells respectively, and with concomitant reduction in cell signaling and function. However, comparison of data from the three cell types studied in HiACT suggest that the underlying mechanisms of altered ITAM protein expression are complex and cell type specific. As an example, monocytes show increased FcRγ mRNA expression in cART-naive subjects which contrasts with the reduced FcRγ mRNA expression in NK cells. Furthermore, normalization of ITAM mRNA expression was observed in monocytes from subjects receiving cART, whereas levels remained low in NK cells from subjects receiving cART. The stimuli, which cause activation of different cell types are also likely to be different. Thus, cART is associated with reduced activation of CD8+ T-cells, which correlated with reduction of viremia, but had little impact on activation of NK cells, which was associated with elevated markers of myeloid cell activation, suggesting that NK-specific stimulatory factors persist in subjects receiving cART. The finding that NK activation is not decreased in subjects receiving cART and that this is associated with persistent decreased FcRγ expression is a major novel finding in this study. The reduced expression of FcRγ and CD16 in NK cells may be the direct result of the high NK cell activity that was observed. Due to the lack of association of markers of microbial translocation with any of the measured markers of immune activation or ITAM expression, we conclude that such NK-specific activity may be driven by residual viral replication of HIV or co-infection with other viruses, including Cytomegalovirus, a potent trigger of NK cell activation. Future studies will be required to establish the actual molecular mechanisms leading to alterations in ITAM expression in each cell type. In conclusion, we show that despite similarities in ITAM mediated activating signaling pathways, T-cells, NK cells and monocytes are differentially affected during HIV infection and after viral suppression with cART. The factors that cause such altered ITAM expression in each cell type will need to be defined in future studies. Discovering such factors might lead to the identification of targets for immunotherapy to improve immune cell function in HIV-infected patients receiving cART.