Put them to bed, and “do not disturb” brain microglia in SIV infection

Abstract

In the current issue of The Journal of Leukocyte Biology, Trease and colleagues have presented a unique study with a perspective on the fluidity of the status of brain myeloid cell sub‐populations (microglia and macrophages) within the SIV‐infected brain, and the implications for the cognitive health of people with HIV (PWH). Those implications for more fully understanding the role of myeloid cells in the pathogenesis of HIV‐associated neurocognitive disorders (HAND) are indeed significant. Their study attempts to capture the state of brain myeloid cells in combination ART (cART)‐suppressed, SIV‐infected rhesus macaques, through analyses of myeloid cells isolated from whole‐brain hemisphere preparations, using scRNA seq, IPA and bioinformatics. The goal was to profile the transcriptomic pattern of myeloid homeostasis during virus suppression and compare that profile to those of resting, uninfected microglia and SIV‐infected microglia in states of uncontrolled infection. The later includes active infection in non‐encephalitic and encephalitic states, the precursor and end‐stages of SIV/HIV infection of the brain, which are relevant in untreated individuals. The state of virus suppression represents the status of PLWH on suppressive cART, which is of particular interest. The homeostatic state of microglia/macrophages under viral suppression currently dominates discussions dealing with treated patient populations, which emphasizes the importance of this study. Defining the differences in the homeostatic state might identify the neuropathogenic potential of microglia to induce brain injury even without active SIV replication to reveal new therapeutic targets.

Graphical Abstract

Discussion on the distinct brain myeloid cell response pathways in a study of SIV infection reveals a variable return of microglial homeostasis with virus suppression.

In the current issue of The Journal of Leukocyte Biology, Trease and colleagues have presented a unique study with a perspective on the fluidity of the status of brain myeloid cell subpopulations (microglia and macrophages) within the SIV‐infected brain, and the implications for the cognitive health of people with HIV (PWH). Those implications for more fully understanding the role of myeloid cells in the pathogenesis of HIV‐associated neurocognitive disorders (HAND) are indeed significant. Their study attempts to capture the state of brain myeloid cells in combination ART (cART)‐suppressed, SIV‐infected rhesus macaques, through analyses of myeloid cells isolated from whole‐brain hemisphere preparations, using scRNA seq, IPA, and bioinformatics. The goal was to profile the transcriptomic pattern of myeloid homeostasis during virus suppression and compare that profile to those of resting, uninfected microglia, and SIV‐infected microglia in states of uncontrolled infection. The later includes active infection in nonencephalitic and encephalitic states, the precursor and end‐stages of SIV/HIV infection of the brain, which are relevant in untreated individuals. The state of virus suppression represents the status of PWH on suppressive cART, which is of particular interest. The homeostatic state of microglia/macrophages under viral suppression currently dominates discussions dealing with treated patient populations, which emphasizes the importance of this study. Defining the differences in the homeostatic state might identify the neuropathogenic potential of microglia to induce brain injury even without active SIV replication to reveal new therapeutic targets.

1. WHY IS THIS SO IMPORTANT?

Understanding the “neurotoxic” role of microglia and macrophages in the HIV‐infected brain is a major challenge for determining whether they can be effectively targeted by neuroprotective agents in cART‐suppressed PWH to afford optimal neuroprotection. cART suppression will always be the cornerstone of neuroprotection, and there are no established effective adjunctive (to cART) therapies for neuroprotection against HIV. Therefore, studies in the nonhuman primate model, such as this one, are critical for understanding the state of the brain during viral suppression and its potential evolution. ¹ Although a state of virus suppression in PWH is associated with measurable cognitive stability over years in a majority of patients (∼70%), a significant proportion of patients do show cognitive instability over time. ² So, we have fallen short of the goal of complete neuroprotection against HIV.

A central assumption in the neuroHIV field is that there is a risk for ongoing neuronal injury in the “virally‐suppressed” patient who may have episodic and/or low‐level virus expression, and that this contributes to progressive cognitive decline in some individuals. Thus, more consistent virus suppression is a goal. A major controversy in the field is the role of brain myeloid populations as the sole driver of neuropathogenic processes, which may be amplified by other cell types, and whether trafficking or resident HIV‐infected CD4+ T lymphocytes also play a role throughout the lifetime of PWH. ³ HIV infection results from the transmission of a single virus strain (transmitted/founder virus) that is nearly always T lymphocyte tropic and not macrophage tropic. ⁴ The possibility of a CNS reservoir of HIV‐infected CD4+ T lymphocytes has been recognized, but this is not confirmed. ⁵ Nonetheless, virus evolution and adaptation within the CNS after initial entry results in emergence of macrophage‐tropic strains that can persist within the brain parenchymal myeloid cell populations, and that may be poorly accessible to cART.

Endogenous microglia and monocyte‐derived macrophages have been consistently identified as cellular sites of HIV integration and replication in autopsied brains of individuals infected with HIV; these myeloid reservoirs represent a potential source for both production of neurotoxins and amplification of metabolic processes that mediate neuronal injury and dysfunction. Thus, optimization of cART effectiveness by selection of regimens with increased penetrance into the CNS has been a primary focus of neuroprotection strategies, and this does have some benefit, although it is incomplete. ⁶ , ⁷ Additionally, initiating cART immediately after HIV infection is a concurrent goal for optimizing neuroprotection. This likely to have significant long‐term benefits despite the likelihood of some acute brain injury occurring before suppression is achieved. ⁸ However, one cannot escape the reality that complete neuroprotection against HIV will require adjunctive neuroprotective therapies in addition to cART to preserve brain integrity and function in the “virally‐suppressed” individual. These therapies must address the role of CNS myeloid cell populations.

2. WHAT DOES THIS STUDY SHOW?

The study is unique in its investigation of microglia in several different states of brain infection: none, uncontrolled infection with and without encephalitis, and suppressed infection. These states cover the spectrum of HIV infection in PWH. The focus of early published studies of HIV/SIV encephalitis, most relevant decades ago, has evolved to studies of the virally suppressed state, which apply to the era of effective cART. This study includes these critical comparator groups.

It shows that microglia in SIV‐infected macaques have a range of transcriptomic phenotypes that are associated with the state of ART suppression and the relative severity of infection, and that “resting” (uninfected brain) microglia are transcriptionally similar to those in a cART‐suppressed brain. This is described as returning the brain to its “homeostatic state,” that is, the uninfected state. This basic concept has been generally assumed to be true, but is still unproved. This study is a relatively comprehensive and successful attempt to validate that assumption, and it largely succeeds. The combination of single‐cell RNA sequencing and pathway analyses accomplishes what has not heretofore been as rigorously studied or as finely displayed at a cellular level. This demonstrates the power of the macaque model for understanding both cellular and regional brain responses to infection.

3. HOW MUCH DOES THE STUDY TELL US?

The observation that the transcriptome groupings of certain immune, inflammation, and survival functions or pathways in ART‐suppressed microglia resembles that of uninfected microglia is consistent with the hypothesis that active SIV replication in the brain is the key to altering microglial phenotypes. This can drive microglia from a relatively nonpathogenic state to a pathogenic state. The status of activation of pathways of inflammation and immune activation under cART and in the uninfected state were found to be similar, and one would expect this, based upon much published literature. This could reasonably lead one to conclude that the suppressed and the un‐infected states of microglia are functionally equivalent (“the quiescent state”) in having a low potential to drive progressive neurodegeneration. This is also consistent with the observation that the majority (∼70%) of ART‐suppressed patients diagnosed with HAND do not demonstrate progressive cognitive decline, at least over several years. ² So, this study finding can be considered comforting in that it supports the value of cART in providing a high level of neuroprotection in most patients.

The observation of the greatest microglia/macrophage diversity in SIV‐infected nonencephalitic animals and the lowest diversity in SIV‐infected encephalitic animals requires much additional thought, and its implications are less clear but also intriguing. In the encephalitic brain, are myeloid cell activation and inflammation uniformly triggering a few, possibly “dominating,” mechanisms of neuronal injury? Is this a consequence of a low myeloid diversity that may be expressed throughout the entire encephalitic brain? In the nonencephalitic brain, are a higher myeloid diversity and a “low level” of infection associated with a lower level of expression of the same mechanisms of neuronal injury? Could a low‐level infection drive multiple overlapping injury mechanisms without one or few mechanisms dominating? Such questions are indeed only speculative, but this study suggests many possible consequences of altering myeloid cell population gene expression diversity and cellular homeostasis.

There are some weaknesses in this study, which could unpredictably affect the results, but the methodologic approaches and the data analyses are rigorously executed, and the conclusions are reasonably supported. There are many implications for pathogenesis. Differences in experimental conditions (different SIV swarms used to inoculate animals held in chronic cART‐treated infection status and untreated animals allowed to progress to the encephalitic state), lack of a noninfected cART‐treated group, different epochs of animal testing, and differing viral loads in the untreated groups could confound the results. Would animals without encephalitis, but with a similarly high VL (or just equal numbers of infected microglia) profile similarly to those with encephalitis? This would not change the conclusion that levels of virus replication (high/low) are important in myeloid homeostasis. The use of cryopreserved specimens raises some concerns for ex vivo modeling of in vivo cellular immunologic status, and the harvesting of myeloid cells from a whole brain hemisphere also precludes analyses of regional brain differences in cellular responses. Regional brain variations in cellular responses are certainly important for understanding brain vulnerability to SIV infection. ⁹ In another neuroinflammatory, neurodegenerative disease, multiple sclerosis, regional differences in the microglial transcriptome suggest different regional pathogenic roles for microglia. ¹⁰ So, there clearly are broad implications for this study.

Further examination of the differential responses of brain myeloid populations to infection and concurrent cART in prospective studies in the nonhuman primate model are essential for complementing and extending tissue analyses in PLWH, and such studies are hopefully forthcoming.

Kolson D. Put them to bed, and “do not disturb” brain microglia in SIV infection. J Leukoc Biol. 2022;112:951–953. 10.1002/JLB.3CE0322-165R