Understanding the MIND phenotype: macrophage/microglia inflammation in neurocognitive disorders related to human immunodeficiency virus infection
Macrophages were first identified in 1841 as inflammatory corpuscles in damaged brain,
as foam cells by Virchow in 1846, stained by Weigert and Golgi in 1873, and its key
function as a mediator of phagocytosis was reported by Metchnikoff in 1882 4]. The central nervous system, and particularly the brain, represents the farthest
reaches of the frontier, as any future translational therapeutic approaches must,
based on our current understanding, surmount the blood–brain-barrier, penetrate into
the brain parenchyma, and localize to the affected site in order to have a beneficial
effect.
It is now well established, based on a series of recent elegant studies using mouse
genetics, lineage fate mapping techniques, and transcription profiling, that embryonic
monocytes give rise to the majority of tissue macrophages in an adult animal except
for in the intestine, heart, and skin 1]-3]. Moreover, these tissue macrophages are capable of self-renewal. However, in the
context of neuronal injury and infection, patrolling monocytes/macrophages from the
bone marrow will invade the brain. In this regard, trafficking of CCR2 expressing
monocytes to the brain in a model of Alzheimer’s disease, plays a key role in ameliorating
the toxic effect of amyloid accumulation 5]. In the adult animal, myeloid cells develop in the bone marrow from a CD34+ hematopoietic
stem cell that gives rise to a common myeloid progenitor that can migrate into tissues
6],7]. Turnover studies in mice suggest that monocytes circulate in the blood for 1–3 days
before entering tissues where, depending on signals received from local resident macrophages
1], they differentiate into mature cells with distinct morphology and function. Whether
different subpopulations of monocytes give rise to specific types of tissue macrophages
is not currently fully understood, but there is agreement that the presence of the
fractalkine receptor CX3CR1 marks inflammatory monocytes that migrate into tissues in response to its ligand
8],9].
At least three distinct populations of monocytes have been identified in human blood
based on the expression of the cell surface receptors: 1) CD14hiCD16? (classical) which also express CCR2hi, CX3CR1lo and represent 80-90% of total monocytes, 2) the CD14+CD16hi (non-classical), and 3) the CD14hiCD16lo (intermediate) proinflammatory monocytes that also express CX3CR1, and low levels of CCR2, and are found in the blood at 5-10% 10]. This latter population is preferentially infected by in HIV 11] and infiltrates the brain during infection 12],13]. Moreover, transcriptional profiling suggests that distinct genetic programs distinguish
the three subsets 14],15]. What we do not yet know is whether these subpopulations can be reprogrammed into
different subtypes. In vitro, and likely in microenvironments within tissue compartments, specific cytokines can
polarize monocytes to develop along different effector pathways that have been called
M1 or M2, analogous to the nomenclature used for T-cell subsets 16]-18]. Several excellent reviews discuss the discovery and spectrum of phenotypes and functional
characteristics of these subtypes of macrophages 18],19].
In addition to inflammatory-directed polarization, the determinants of macrophage
morphology and function may in part, be governed by the cells in the microenvironment
with which macrophages interact. For example, a subtype of macrophage found in the
intestine, the muscularis macrophages, associate very tightly with enteric neurons
to help regulate intestinal peristalsis 20]. Analogous to the microglia-neuron regulatory signaling mechanism using fractalkine
ligand on neurons and fractalkine receptor on microglia, muscularis macrophages secrete
bone morphogenetic protein 2 (BMP2), which activates the BMP receptor on enteric neurons
20]. Microglia, the resident macrophages in the brain, assume an ameboid shape when engaged
in phagocytic functions. Microglia display an extensive ramified morphology under
normal homeostatic conditions during which they continually make contact through extended
finger-like projections to neurons in their vicinity 21],22]. Microglia also play critical roles in shaping neuronal networks during development,
and in the adult animal by modulating synaptic transmission 21],23].
Within the brain, macrophage phenotype varies with their location in this tissue.
Perivascular macrophages, as the name implies, are located intimately with vessels
and enter from the blood circulation into the brain at a low level during normal conditions,
and at higher frequency in the context of damage or invasion of the brain by a pathogen.
Choroid plexus macrophages and meningeal macrophages, which are closely associated
with the meninges, the thin blood vessels that line the brain, express MHC class II
and costimulatory molecules. Parenchymal macrophages include the microglia population
and cell surface markers such as CD68, Iba1, and CD163 stain both cell types 24]. Moreover, it is possible that infiltrating macrophages that move deeper into the
parenchyma are able to do so because the appropriate transcriptional program has been
initiated and recognized within the local microenvironment. Indeed, blood monocytes
can home to the brain when microglia are experimentally depleted in mice 25]. However, the bone marrow-derived microglia are not able to penetrate deeply into
the brain parenchyma, which suggests the possibility that they lack the genetic instructions
to do so and/or that they do not receive the proper secondary signals perhaps because
they are not in the correct location 25].
Traffic across the blood–brain-barrier is strictly regulated, but many pathogens are
able to gain entry through monocytes, which are able to extravasate across. HIV enters
the body through mucosal sites where it infects resident macrophages, dendritic cells,
and CD4+ T-cells at these sites leading to dissemination throughout the lymphatic
system. HIV enters the brain rapidly after infection. Entry into the brain was shown
in a case of human iatrogenic transmission, and to occur within three to fifteen days
depending on the experimental monkey model examined 26]-29]. In the absence of treatment, encephalitis occurs in ~20-30% of infected individuals,
which is characterized pathologically by the presence of multinucleated giant cells,
microglia nodules, microgliosis, astrocytosis, and abundant CD68+ macrophages staining
for HIV antigens 30]. As not all HIV-infected individuals develop CNS dysfunction, there is a role for
host genetics in conferring susceptibility or resistance to the development of cognitive
impairment 31].
The simian immunodeficiency virus (SIV) non-human primate models of HIV infection
have been invaluable in adding to our understanding of the central role of macrophages
as initiators of an inflammatory cascade, which ultimately results in neuronal damage
and dysfunction 32],33]. Analyses of blood monocytes from infected individuals with HIV-associated dementia
detected an expansion of the CD14?+?CD16+ subset 12], which was later confirmed in SIV-infected monkeys. Indeed, monocyte turnover in
the bone marrow is a better predictor of progression to AIDS than CD4+ T-cell count
and plasma viral load 34],35]. A very recent set of studies suggests a mechanism for CCR2/CCL2 signaling in the
recruitment and trafficking of CD14?+?CD16+ monocytes into the brain in HIV-associated
cognitive impairment 36],37]. The CD14?+ CD16+ monocytes that crossed an artificial blood–brain-barrier model
as well as those found in the CSF of individuals with HIV-associated cognitive impairment,
were found to preferentially express CCR2 37]. Moreover, antibody against the tight junction protein JAM-A and adhesion molecule
ALCAM was able to block the accumulation of CD14?+?CD16+ monocytes 38]. The macrophage marker CD163 is present on CD14?+?CD16+ blood monocytes expanded
in SIV encephalitis and these cells are believed to be the bone marrow derived CD34+
precursors of the CD163+ perivascular macrophages seen in the brain 39]-41]. Moreover, CD68?+?CD163+ macrophages accumulate in the CNS in SIV-infected monkeys
and in human brain tissue from HIV-infected individuals 34],39]-42]. In SIV encephalitis, productively infected CD14?+?CD16?+ CD163?+?CD45hi perivascular
macrophages are abundant. The myeloid marker MAC387 is found on BrdU+ monocytes/macrophages
in SIV-infected animals in the early stages of CNS invasion only when inflammation
is abundant 43]. In contrast, in chronic lesions CD68?+?CD163+ macs are most highly represented in
SIV-infected animals and in HIV-infected human with encephalitis 34],39]. Moreover, MAC387?+?CD163-CD68-CCR2- macrophages do not appear to be productively
infected with HIV. Multinucleated giant cells, which are the hallmark of fulminant
HIV replication in macrophages, expressed CCR2 and CD68 43]. Collectively, these data suggest the possibility that HIV-infected and uninfected
M1-type macrophages may be present early, and as the host attempts to downregulate
the immune response, M2-type macrophages become more abundant 44]. In in vitro monocyte-derived macrophages (MDM), HIV replication in M1 and M2 macrophages is reduced,
however the extent of the inhibition varies with the stimuli used 45],46]. Microarray analyses on HIV-infected MDM have shown that the production of proinflammatory
cytokines is increased through a TLR-independent pathway suggesting that HIV infection
induces an M1-type milieu 47]. A proteomic study suggested that M1-HIV infected macrophages cocultured with T-regulatory
cells can shift to an M2 phenotype, which was associated with neuroprotection 48]. Further study of the potential role played by M1 and M2 type macrophages in HIV
CNS infection is required.
There are currently two approaches for investigating the contribution of human macrophage
phenotype as it relates to HIV infection: 1) with in vitro culture models based on the isolation and differentiation of monocytes isolated from
the blood (monocyte-derived macrophages or MDM) and 2) immunophenotyping using brain
tissue obtained at autopsy. Monocyte isolation methods vary from low purity using
adherence to plastic, to density gradient centrifugation, elutriation, or the use
of positive or negative immunomagnetic bead selection for example for CD14+ cells.
Each of these methods has their advantages and drawbacks, which relate to the level
of purity, yield, and the inadvertent induction of cellular activation. Culture conditions
can also vary widely and may make comparisons between laboratories difficult. This
topic has been recently addressed and recommendations made 19].
We have used a standardized culture model for several years in conjunction with a
recombinant HIV that encodes the enhanced green fluorescent (GFP) gene in a portion
of the viral genome that is expressed early during infection 49]-51]. Using flow cytometric analyses, we identified a consensus surface activation marker
signature (SAMS), CD14+CD69+CD86+CD68lo on a subpopulation of MDM in which HIV replication was active 52]. Interestingly, the presence of CD69, but not CD14 or CD86 on the cell surface was
dependent on the expression of the viral protein Nef in the infected macrophage. Nef
is essential for disease pathogenesis in vivo, for robust replication in primary T-cells and macrophages in vitro, and helps HIV to evade the immune response through multiple mechanisms 53]-57]. Induction of CD69 expression on murine macrophages treated with LPS, TNF-? or IFN-?
and LPS has been reported, however relatively little is currently known regarding
the function of CD69 on this cell type 58]. One study suggests that CD69 may have a role in downregulating immune responses
through TGF-? 59]. CD86 is a marker for M1-type macrophages and its presence together with CD69 suggests
that these cells are highly activated. We understand what may at first seem paradoxical,
that HIV would prefer to replicate in an activated cell, is that the virus has evolved
to use host proteins as cofactors in the essential steps of reverse transcription,
and transcription of the integrated proviral DNA. These cofactors are present at sufficient
concentrations only in stimulated cells. At least in this in vitro model, both HIV-dependent and –independent mechanisms are involved in the regulation
of expression of these innate inflammatory surface proteins. It remains to be determined
whether the phenotype identified will be recapitulated in human tissues in vivo, however CD14?+?CD16?+?CD69+ monocyte/macrophages were previously reported to be
expanded in the brain during HIV infection 12].
In regions of the world where therapy is widely available, the incidence of HIV encephalitis
and full-blown dementia has greatly diminished 60],61]. However, the prevalence of milder forms of HIV-associated cognitive impairment has
increased as people live longer with the infection. Importantly, comorbidities including
aging, illicit drug use, exposure to antiretrovirals, cardiovascular disease, and
insulin resistance could potentially contribute to cognitive impairment 62],63]. A major goal of the field is to identify the neuropathogenic mechanisms leading
to the persistence of neuronal injury and dysfunction in HIV infection despite complete
suppression of viral replication in the periphery. Much of the macrophage phenotype
data that currently exists utilized autopsy tissue that predated the widespread use
of antiretroviral inhibitors in the developed world.
Hence, there is a need to identify biological markers that could help predict risk
for the development of cognitive impairment, and would serve a predictive function
in assessing the efficacy of current treatments and for novel therapeutics in development.
In this regard, a recent study suggests that individuals with asymptomatic neurocognitive
impairment (ANI) have a 2-6-fold increased risk of progressing to further cognitive
decline 64]. One recurring and prominent feature is the persistence of immune activation. Proinflammatory
markers including IL-6, sCD14, sCD163 remain elevated in those on successful anti-viral
therapy 63],65]. In part, the persistence of these factors is due to early HIV-mediated damage to
the gastrointestinal tract in which microbial cell wall components are released into
the circulation 66]. Moreover, the presence of these latter inflammatory mediators is associated with
cognitive impairment in HIV 67].
Osteopontin (OPN) is a proinflammatory cytokine first described as early T-cell activation
marker 1 that is expressed in several cell types including T-cells and tissue macrophages
68]. OPN was detected as a cytokine that was highly upregulated in the brains of monkeys
with SIV encephalitis and later shown to also be elevated in the CSF and brain of
individuals with HIV-associated neurocognitive disorder 69],70]. Interestingly, OPN enhances HIV replication in macrophages by 50% through mechanisms
that involve activation of the NF-?B responsive viral promoter, perhaps through integrin
receptors and also by promoting cell-to-cell adhesion, which facilitates viral spread
69],71]. Interestingly, OPN levels in the plasma remain high despite undetectable HIV viral
load, supporting other observations that inflammatory processes remain active 72]. Both HIV-infected and uninfected inflammatory monocytes have been shown to be key
contributors to the inflammation seen in the brain 73]. We hypothesized that these cells were the major source for OPN in the brain of HIV-infected
individuals. However, a recent immunohistochemical study on autopsy brain tissue suggests
that astrocytes are also a source of OPN, although no significant differences in expression
were seen between the HIV-infected and HIV-infected with cognitive impairment groups
74]. Unexpectedly, however, significant expression of OPN was detected in cortical neurons
of brain tissue from those with HIV-associated cognitive impairment 74].
HIV-associated neurocognitive disorders (HAND) is an umbrella term to describe three
levels of neurocognitive dysfunction: asymptomatic neurocognitive impairment (ANI)
in which individuals show deficits (greater than one standard deviation) in two or
more cognitive domains, but no impairment of activities of daily living (ADL); minor
neurocognitive disorder has the same level of impairment as ANI, but ADLs are mildly
affected and HIV-associated dementia (HAD) represents the most severe form of impairment
in which deficits in two or more cognitive domains is greater than two standard deviations
and there are a severe impact on ADLs 75],76]. In tissue from an HIV-infected individual with ANI, with very low viral load in
the CSF (19 RNA copies/ml) or plasma (249 RNA copies/ml) who had been on antiretrovirals,
an abundance of double-stained Iba-1/OPN perivascular macrophages and microglia and
parenchymal microglia were detected (Figure 1). In a case with MND, the microglia appear to be predominantly of the ameboid type
with few ramified processes (Figure 2). This patient at death despite being on therapy had a plasma viral load of 48,000
copies/ml, a CSF load of 148 copies/ml and was severely immunosuppressed with a CD4
T-cell count of 10. A case with HAD displayed microgliosis with abundant ameboid and
ramified microglia in the parenchyma, and a high level of OPN expression (Figure 3). This individual at death had a high plasma viral load of ~40,000 copies/ml, a CSF
load of 2747 copies/ml but a CD4 T-cell level of 299, not indicative of immunosuppression.
Another study also found, as detected by the markers CD16, CD163, HLA-DR, and GFAP,
that for macrophages/microglia and astrocytes, elevated levels of inflammation in
the brain remained a common feature in HIV-infected individuals without evidence of
encephalitis or productive viral replication in the brain 77]. Together these results highlight the variation in microglia activation that persists
in the brain at the individual level and the need for reliable plasma and/or CSF markers
that would allow clinicians to track smoldering CNS inflammation.
Figure 1. Abundant Iba1/AIF-1 positive parenchymal and perivascular macrophage/microglia (red
color) costain for osteopontin (OPN) (brown color) expression in tissue from the occipital
lobe of an HIV-infected individual with asymptomatic neurocognitive impairment (ANI). Paraffinembedded human autopsy tissue from the occipital lobe (National NeuroAIDS
Tissue Consortium). Antigen retrieval was performed in citric acid buffer pH 6.0 and
slides were stained sequentially with rabbit polyclonal antisera against Iba1/AIF-1
(SIGMA) overnight at 4°C followed by incubation with goat-anti-rabbitalkaline phosphatase
(AP) secondary for 1 hr at room temperature and developed with permanent FastRed Quanto
(ThermoFisher) (red color). Slides were then incubated with mouse monoclonal antibody
to OPN (MAB194, Maine Biotechnology) at room temperature for 2 hrs followed by goat
anti-mouse-horse radish peroxidase for 1 hr and developed with 3,3’-diaminobenzidine
(brown color). Images were taken on an Axio Observer A1 inverted microscope (Zeiss)
at 20x magnification. Adjustment of the image brightness, contrast and sharpness was
performed with Adobe Photoshop 5.5 using the same settings for each image.
Figure 2. Iba1/AIF-1 positive parenchymal macrophage/microglia (red color) costain for osteopontin
(OPN) (brown color) expression in tissue from the occipital lobe of an HIV-infected
individual with minor neurocognitive disorder (MND). Microglia with ameboid morphology are more abundant than cells with a ramified phenotype.
Figure 3. Abundant expression of osteopontin (brown color) in Iba1/AIF-1 positive parenchymal
and perivascular macrophage/microglia (red color) in tissue from the occipital lobe
of an HIV-infected individual with HIV-associated dementia (HAD). Osteopontin in HIV-infected HAD cases were significantly elevated compared to normal
controls 74].