This study delineates the effects of in vivo HIV-1 infection on the signaling behavior of single immune cells interacting within a complex cellular milieu. Interrogation of phospho-epitopes within pathogen-altered intracellular signaling networks at the primary cell level allowed linkage of network states with virologic and clinical parameters. In this cross-sectional study, our comparison of the Jak/STAT pathways within prominent peripheral blood immune cell populations revealed a specific defect in GM-CSF-stimulated Stat5 phosphorylation within monocytes associated with HIV-1 infection. Expansion of the phospho-flow screen to include other phospho-proteins activated by GM-CSF led to the observation that LPS-stimulated ERK phosphorylation was enhanced in HIV-1 infection. Reduced signaling potential of “antiviral” STAT pathways, and enhanced signaling of “inflammatory” MAPK pathways are consistent with long-standing observations of monocyte dysfunction in HIV-1 infection, including reduced antigen uptake and hyperactive inflammatory cytokine secretion (5
). These results provide a specific molecular basis for the prior observations of dysfunction.
In order to put these results into context, it may be helpful to organize the complex body of literature on HIV-1 and Jak/STAT signaling into studies of pathway activation state (i.e., activated or depressed signaling) and studies of pathway responsiveness (i.e., the ability to activate in response to an upstream stimulus). Activation state studies have shown increased phospho-Stat5 after in vitro HIV-1 infection or exposure of both CD4+
T-cell and monocytic cell lines (24
) and constitutive activation of a truncated form of Stat5 (Stat5Δ) in a majority of HIV-1-infected donors (4
). In contrast, studies of Jak/STAT responsiveness in HIV-1 have shown a reduced ability to phosphorylate Stat5 after IL-2 stimulation of CD8+
T cells from HIV-1-infected adults (26
), as well as after GM-CSF stimulation of monocyte-derived macrophages infected with HIV-1 in vitro (43
). Preexisting activation through viral antigen stimulation or immune activation may desensitize the pathway to further stimulation. This could occur through negative feedback mechanisms, for instance, as mediated by the suppressor of cytokine signaling (SOCS) family (17
), among others. Except for donors with advanced HIV-1 infection, our results were not consistent with a systemic decrease in Jak/STAT responsiveness across all cell types and pathways but rather a specific inhibition of Jak2/Stat5 signaling in response to related βc
cytokines (GM-CSF and IL-3) in monocytes. Since our results are based on blood samples obtained from HIV-1-infected donors, we both validate and extend the significance of similar observations based on in vitro model infection systems (43
), providing here a physiologic and in vivo characterization of the effect.
By studying a cohort of perinatally HIV-1-infected pediatric subjects and their exposed, uninfected controls, we were able to take advantage of closely matched socioeconomic status and environment. In addition, the drawing, processing, and storage of the blood samples were performed completely in parallel, providing ideal controls for sample handling conditions. Our results are not limited to pediatric disease but bear significance to HIV-1 in general. In separate experiments, we found that GM-CSF induced Stat5 phosphorylation was significantly reduced in HIV-infected adults compared to uninfected controls (67% of control, n = 8 [HIV+], n = 4 [HIV−], P = 0.02).
In addition to the advantage of monitoring cell signaling in primary immune cells ex vivo, phospho-flow screening allowed the integration of signaling results with the clinical and virologic parameters of the HIV-1-infected donors under study. In the initial comparison of treated HIV-1+ pediatric subjects (low VL and high CD4s) versus those with advanced HIV-1 disease (high VL and low CD4s), STAT activation was normal in the former group, whereas the latter group could not activate multiple STATs, suggesting a correlation between severe disease and nonresponsive STATs. However, repeat testing in a larger cohort of HIV-1-infected pediatric subjects did not reveal a clear relationship between low Stat5 activation and HIV-1 disease state, as measured by CD4% or HIV-1 viral burden. We also noted improvement in low Stat5 responses associated with HAART, as seen with other HIV-1-related immune dysfunctions, although the difference was not statistically significant (Fig. ).
We also tested our HIV-1-infected pediatric subjects for differences in signaling among different ethnic groups, taking advantage of the ethnic diversity reflected in this urban cohort. We did not find significant differences in ex vivo-stimulated STAT phosphorylation between Hispanics and African-Americans, despite an earlier demonstration of variable HIV-1-specific immunity in this cohort (35
). Our results also show that reduced Stat1 responses to IFN stimulation in blacks, compared to whites, in the context of HCV infection (18
) does not extend to other viruses such as HIV-1. As such, it is obvious the different disease contexts, as well as different ethnic groups, preclude a direct comparison of studies.
Testing the idea that a GM-CSF signaling block at Stat5 might lead to enhanced signaling of other GM-CSF-stimulated pathways led to a second significant finding, namely, that MAPK signaling in monocytes is potentiated in HIV-1 in response to specific natural ligands of innate immunity, but not to broadly active, small molecule inducers of cell signaling. As with the STAT activation observations, we found that the HIV-1-related increase in MAPK activity was specific, i.e., only in response to LPS or the combination of LPS and GM-CSF. MAPK signaling is essential for inflammatory cytokine secretion, and monocytes play a key role in secretion of these molecules. The data supports a hypothesis that HIV-1 increases the ERK response in monocytes specifically, in order to promote aberrant inflammatory cytokine secretion in response to Toll-like receptor (TLR) signals.
We did not find evidence for reduced GM-CSF receptor levels in HIV-1 as a potential explanation for low phosphorylation of Stat5 (Fig. ). In addition, HIV-1 infection was not associated with lower total Stat5 levels by Western blotting (E. R. Sharp et al., unpublished results), arguing that the low pStat5 response we observed resulted from a reduction in steady-state phosphorylation. Although we considered the possibility that a reduction in the fold increase in pStat5 after stimulation might arise from elevated baseline phosphorylation due to chronic activation in the setting of HIV-1 infection, we did not find any significant difference in basal pStat5 between HIV-1− and HIV-1+ (P
= 0.81, n
= 11 [HIV-1−], n
= 14 [HIV-1+]). The recent observation that HIV-1 Nef induces SOCS, which negatively regulates pStat1 and pStat3 activation in B cells (32
) is potentially relevant to our findings. However, an upregulation in negative feedback mechanisms should result in generalized inhibition of STAT signaling, not a specific inhibition of βc
cytokine-driven Stat5 activation, as we observed. Thus, a simple explanation of upregulation of broadly active SOCS inhibition is not the cause, but the results do not obviate a mechanism involving cell type specific utilization of SOCS.
Exposure to several HIV-1 proteins is known to result in ERK hyperphosphorylation. Nef exposure resulted in ERK activation in CD4+
T cells (33
), increasing target cell activation and infectivity (44
), and endothelial cells (38
). HIV-1 gp120 binding to CCR5 on macrophages resulted in the activation of both ERK and phosphatidylinositol-3 kinase, which were required for gp120-stimulated TNF-α secretion (27
). Extrapolating from the work above, one might predict that HIV-1 infection would result in elevated ERK phosphorylation ex vivo. However, we found that basal pERK levels were nearly equivalent (P
= 0.56, n
= 6 [HIV-1−], n
= 10 [HIV-1+]), and instead we observed significant enhancement of LPS-stimulated ERK phosphorylation by HIV-1 (Fig. ). LPS stimulation of ERK in monocytes signals through TLR4, whereas HIV-1 signals through TLR7/8 (3
), raising the possibility that HIV-1 infection enhances ERK activation by providing additional or complementary TLR signals (40
What are the implications for the altered signaling responses we have demonstrated in this group of HIV-1-infected pediatric subjects? Although full-length Stat5 binds to the HIV-1 LTR and increases viral production in CD4+
T cells (34
), a constitutively activated, truncated form of Stat5 is present in HIV-1-infected individuals and negatively regulates HIV-1 expression (10
). Although we cannot distinguish isoforms with the phospho-specific Stat5 antibody, reduction in pStat5 is likely to counteract the antiviral effect of truncated Stat5. Variability in GM-CSF-mediated pStat5 response in HIV-1+ donors (Fig. ) may have contributed to observed differences in effects of GM-CSF therapy on HIV-1 viral load in clinical trials (7
). Nonresponsiveness to GM-CSF in a proportion of subjects has been observed in these and other studies of HIV-infected and uninfected adults.
MAPKs, including ERK, are integral for inflammatory cytokine secretion in response to a variety of stimuli. Enhancement of LPS-stimulated ERK phosphorylation by HIV-1 represents a possible mechanism for increasing inflammatory cytokine secretion, thus contributing to the development of generalized immune activation. Notably, HIV-1 gp120 may cause TNF-α secretion from macrophages in a CCR5- and Lyn (a member of the src family kinases)-dependent manner (37
). Although “traditional” CD14+
monocytes lack CCR5 (42
monocytes in HIV-1 have elevated CCR5 expression (up to 40% CCR5 in HIV-1+ donors) (16
). It will be of interest to determine whether the enhancement of LPS-stimulated ERK in monocytes we have identified here depends on CCR5.
The potentiation of LPS-stimulated signaling in HIV-1 infection is of special interest, in light of the recent work by Brenchley et al. demonstrating a strong correlation between elevated serum LPS levels (resulting from gut microbial translocation) and HIV-1-related chronic immune activation (6
). Chronic in vivo LPS stimulation also led to reduced ability of monocytes to secrete TNF-α ex vivo. The potentiation of LPS-induced ERK phosphorylation may represent a strategy used by HIV-1 to counter the desensitizing effects of chronic LPS stimulation and maintain persistent immune activation through inflammatory cytokine secretion.
In summary, we have applied phospho-specific flow cytometry to determine the extent of altered cell signaling profiles in HIV-1-infected individuals. In this cross-sectional study, we identified a significant defect in Stat5 activation and enhancement of ERK signaling within the monocytes of HIV-1-infected pediatric subjects, with intriguing implications for the role of monocytes in the immunopathogenesis of HIV-1 infection. These findings could guide further investigations into long-described immune cell dysfunctions that occur in HIV-1 infection but have eluded understanding at a mechanistic level. In the era of “personalized medicine,” one can envision how phospho-epitope-specific analysis could be applied to larger and/or longitudinal studies to further bridge our understanding of how HIV-related signaling abnormalities may affect disease progression and clinical outcomes.