HIV-1 stimulates dynamic and complex responses within macrophages through alteration of expression or modification of cellular genes or proteins. While virus modulates multiple signaling pathways over the course of infection, HIV-1 has a selective, rather than global, impact on macrophages. Our analysis identified a complex interplay, through molecular hubs, to establish spatial-temporal relationships across signaling pathways that organize virus-specific activation programs into an HIV-1 response phenotype for macrophages.
Pathway analysis provides a rational and unsupervised statistical algorithm for integrating large transcriptome and proteome data sets and quantifying viral impact on the pathways. High-throughput transcriptome discovery across multiple pathways enhanced by proteomic approaches reduces the significance of outlier data points, such as increased IRF-3 gene expression by HIV-1 in a single donor, which may be difficult to interpret on an individual pathway basis by a single method. Our study was designed to discern physiological responses in a population of macrophages exposed to virus, rather than to distinguish between the direct and indirect effects of virus on cells. Yet, even though only a minority of macrophages was infected by HIV-1 within 2 days, the initial round(s) of virus replication produced the greatest differences in gene expression raising the possibility that the macrophage response to HIV-1 involved bystander cells.
Viral impact on signaling is frequently studied by investigating discrete pathways in cells treated with an isolated viral component, for example Env gp120 
, Tat 
, Nef 
, Vpr 
, or synthetic RNA corresponding to subgenomic regions of HIV-1 
. While these studies have provided a framework for network analysis, our global strategy builds insight through the use of whole virus replication to assess the coordinate impact on multiple cell signaling pathways by viral gene expression during spreading infection. Several pathways in macrophages, including calcium, MAPK, and apoptosis, which are impacted by individual viral components, were also modulated at the level of gene and protein expression over the course of viral replication. In contrast, Env gp120 treatment of macrophages induces the production of TNF 
, while our studies, unexpectedly, failed to detect TNF secretion in response to HIV-1, which could reflect differences in signaling between monomeric Env gp120 and virion-associated trimeric Env complexes, quantitative difference between envelope glycoproteins in soluble or virion form, or activity by viral proteins post-entry to suppress signaling. The fact that multiple signaling pathways can result in similar cytokine responses argues that a global analysis of all pathways simultaneously is important in interpreting cellular response.
Changes in macrophage cell cycle or pathways can be critical for productive HIV-1 infection 
. Our studies demonstrate that changes in cell proliferation pathways by virus are independent of DNA doubling or cell proliferation. Therefore, transitions within compartments of G1, prior to DNA replication during S phase, may be important for establishing an optimal intracellular milieu for viral infection 
. Alternatively, cell cycle factors may be essential for HIV-1 replication in macrophages independent of their role in cell cycle.
Human macrophages express a repertoire of TLRs with functional responses to natural ligands or agonists. Nonetheless, our studies indicated that TLR recognition of HIV-1 replicating in macrophages is impaired, not only during initial infection, but over the course of viral spread, ruling out the possibility that infection of small numbers of cells limited detection of TLR signaling. Manipulation of TLR signaling by a variety of mechanisms is a survival technique evolved by a number of viruses, including human cytomegalovirus, influenza virus, and hepatitis C virus 
. Cytosolic pattern recognition receptors, RIG-I and MDA5, cross-talk through molecular hubs, such as NF-κB and IRF-3, result in proinflammatory cytokine responses similar to responses through TLRs. Our results indicate that HIV-1 infection involves global evasion of molecular pattern recognition receptors within macrophages. HIV-1 might passively evade pattern recognition, actively inhibit or suppress recognition and signaling, or require dynamic interactions between macrophages and other cells, such as lymphocytes or endothelial cells.
Immune activation and inflammatory responses correlate with HIV-1 disease pathogenesis 
, and macrophages are central regulators of systemic immune activation and targets of HIV-1 infection 
. Macrophage activation is a multistep process resulting in at least two major categories of activation, M1 or classical activation and M2 or alternative activation 
. Classical macrophage activation involves two steps, priming and triggering, which modulate gene or protein expression 
. HIV-1 can stimulate macrophages through a variety of signaling pathways, including calcium, MAPK, and apoptosis, which are connected with TLR through the NF-κB molecular hub. Nonetheless, HIV-1 fails to induce TLR-mediated activation indirectly through cross-talk or as a direct trigger. The TLR signaling cascade, which can be activated in pDC by HIV-1 RNA, failed to promote activation in macrophages in response to HIV-1, indicating that viral evasion of TLR may be a cell-type and/or virus-specific survival strategy.
Even though virus was inadequate to provide classical activation of macrophages through TLR signaling, HIV-1 clearly primed macrophages to be hypersensitive to TLR agonists. While the viral doses applied to macrophages in our studies were insufficient to result in detectable levels of TNF, IL-1β, or IL-6 secretion, cells primed by HIV-1 produced enhanced cytokine levels following TLR ligand treatment. HIV-1-induced priming of macrophages is distinct from traditional IFN-γ priming signals, based on STAT1 phosphorylation and multimerization as well as expression of interferon-stimulated genes, in particular Mx1 
. The novel activation program induced macrophages by HIV-1 can be designated M1HIV
. TLR stimulation results in a state of tolerance, due to degradation of activated signaling members within the TLR pathway, such as IRAK1, which prevents cells from mounting a response upon subsequent challenge. M1HIV
-activated macrophages can undergo TLR tolerance upon stimulation with TLR ligands, indicating viral proteins do not mimic TLR signaling. Macrophage sensitization to microbial components may contribute to disease progression in HIV-1-positive individuals who have compromised gut-associated lymphoid tissues and increased microbial translocation into the plasma 
Several possible mechanisms may account for macrophage sensitization to TLR ligands during HIV-1 infection. While activation of pattern recognition receptors, such as TLRs, can induce positive-feedback loops resulting in increased expression of TLRs and proinflammatory cytokines, HIV may selectively activate specific components of this response in macrophages through signaling cross-talk, independent of TLR recognition, resulting in increased TLR expression levels 
in the absence of proinflammatory cytokine production. For example, activity of NF-κB during HIV infection may contribute to TLR-independent enhanced responsiveness of macrophages. Consequently, novel therapeutics might selectively suppress exaggerated innate immune responses induced by HIV-1 infection, without inhibiting protective TLR responses.
Macrophages regulate HIV-1 disease progression through their secretory products 
. HIV-1-induced macrophage sensitization may correlate with poor clinical outcome if priming in vivo
increases disease sequelae by coinfecting pathogens. Viral tropism and coreceptor-specificity change over the course of disease 
, raising the possibility that viruses may evolve in their potential to prime macrophages concomitant with the evolution of coreceptor use from CCR5 to CXCR4 
or increased tropism for macrophages by CCR5-using viruses 
. Macrophage priming potential by quasispecies of HIV-1 may provide a novel biomarker for assessing the course of HIV-1 disease progression within infected individuals.