Monocyte-derived macrophages contain persistent latent HIV reservoirs, Veenhuis, et Al, 2023

[darrellpf]

https://www.nature.com/articles/s41564-023-01349-3

The development of persistent cellular reservoirs of latent human immunodeficiency virus (HIV) is a critical obstacle to viral eradication since viral rebound takes place once anti-retroviral therapy (ART) is interrupted. Previous studies show that HIV persists in myeloid cells (monocytes and macrophages) in blood and tissues in virologically suppressed people with HIV (vsPWH). However, how myeloid cells contribute to the size of the HIV reservoir and what impact they have on rebound after treatment interruption remain unclear. Here we report the development of a human monocyte-derived macrophage quantitative viral outgrowth assay (MDM-QVOA) and highly sensitive T cell detection assays to confirm purity. We assess the frequency of latent HIV in monocytes using this assay in a longitudinal cohort of vsPWH (n = 10, 100% male, ART duration 5–14 yr) and find half of the participants showed latent HIV in monocytes. In some participants, these reservoirs could be detected over several years. Additionally, we assessed HIV genomes in monocytes from 30 vsPWH (27% male, ART duration 5–22 yr) utilizing a myeloid-adapted intact proviral DNA assay (IPDA) and demonstrate that intact genomes were present in 40% of the participants and higher total HIV DNA correlated with reactivatable latent reservoirs. The virus produced in the MDM-QVOA was capable of infecting bystander cells resulting in viral spread. These findings provide further evidence that myeloid cells meet the definition of a clinically relevant HIV reservoir and emphasize that myeloid reservoirs should be included in efforts towards an HIV cure.
.....
Despite these limitations, we provide evidence that monocytes from long-term vsPWH contain persistent latent HIV that upon reactivation is replication-competent and capable of viral spread. This study provides direct evidence that monocyte reservoirs should be included in HIV cure efforts.

https://doi.org/10.1038/s41564-023-01349-3

 

[Hutan]

HIV DNA has been detected in highly purified monocytes1,2,3,4,5 and macrophages isolated from the urethra6, gut7, liver8 and brain9,10 of vsPWH.

Data from the Last Gift cohort show that infected brain can repopulate viral reservoirs during rebound11.

 

[SNT Gatchaman]

Furthermore, we provide evidence that the MDM [monocyte-derived macrophages] reservoir is stable and persistent in long-term vsPWH [virally-suppressed people with HIV], as we measured the MDM reservoir in the same participants over a period of 9 months to 4 yr. Given that circulating monocytes have a lifespan of 72 h in blood, these data support two possible hypotheses of myeloid reservoir maintenance.

First, that the bone marrow contains latent virus that seeds blood monocytes.

The second hypothesis is that ongoing replication occurs in an unknown tissue, potentially the spleen or lymph node, leading to consistent infection of circulating monocytes. This hypothesis is supported by the finding that CD16+ monocytes are preferentially infected in vivo and ex vivo, as these are the subset of monocytes thought to traverse tissues and return to circulation. However, this is not supported by our data, since the monocytes require differentiation and substantial activation to produce virus in culture, and we did not detect HIV RNA in these cells, suggesting the virus is latent.

Of potential relevance to LC given the finding of spike protein in (non-classical) monocytes out to 16 months from infection. SARS-CoV-2 in myeloid precursors sounds like something worth checking for.

 

[Jonathan Edwards]

There is something peculiar about the way this group describes the cell populations - as if they don't really understand the cell life history.

As far as I can see their 'monocyte-derived-macrophages are monocytes that they turn into macrophages in their assay system. So the population in vivo is monocytes. There is no 'MDM reservoir'. I am not aware that monocytes ever travel into tissues and then return to blood as monocytes. So their second hypothesis seems to be a misunderstanding.

The spleen has no real vascular barrier so in a sense is part of the blood compartment, but lymph node is more similar to other tissues.

Monocytes are cells designed to pick up rubbish so it is not surprising they contain some viral material. I can see that it might be important if been marrow myeloid precursors were a reservoir of viral DNA, but why not make that the topic of the paper?

 

[Hutan]

I am not aware that monocytes ever travel into tissues and then return to blood as monocytes. So their second hypothesis seems to be a misunderstanding.

This paper cites
Circulatory and maturation kinetics of human monocyte subsets in vivo, 2017, Tak et al
That paper, while acknowledging that these ideas need further testing, suggest that the classical monocytes come out of the bone marrow and last about 2.5 days in the blood. About 90% turn into macrophages in the skin and dendritic cells in the gut and they stay there. About 10% of the classical monocytes develop into intermediate monocytes which last less than a day in the blood. They suggest that most of the intermediate monocytes go into tissue before coming back into the blood as non-classical monocytes. They seem to be suggesting that not all of these NC monocytes are in the blood, some can be in the bone marrow, attached to blood vessels.

So, the Tak paper therefore does suggest that the intermediate monocytes do go into the tissue before coming back into the blood as non-classical monocytes. And the NC monocytes can turn into macrophages if needed.

Monocytes are cells designed to pick up rubbish so it is not surprising they contain some viral material.

The paper talks about finding a slightly different form of the virus in the monocytes as compared to the t-cells in one person. The authors seemed to be fairly confident that it wasn't just the monocytes picking up the virus from t-cells.

I freely admit I am at the edge of, and beyond my understanding here, and may have stuff wrong. I need to read the paper again later.

 

[SNT Gatchaman]

Additional to Hutan's comment above.

I was confused by their use of the term ''monocyte-derived macrophages". The first Google hit was from Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro (2020, Immunology). The first line said —

Monocyte‐derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies.

Which suggests the term might be confined to the investigative technique. But this paper uses the term for in vivo macrophages. Presumably the term has been broadened in order to draw a distinction with tissue-resident macrophages.

From Tissue-specific macrophages: how they develop and choreograph tissue biology (2023, Nature Reviews Immunology) —

For decades, it was thought that macrophages are a relatively homogeneous population of mononuclear phagocytes arising from bone-marrow haematopoietic stem cells (HSCs), with their primary function being to protect organs during infections. However, the development of novel fate-mapping mouse models allowed for longitudinal tracking of macrophages from their progenitors into their mature cellular state within their organ of residence. Combined with systems biology approaches, these methodologies have changed our view of macrophage biology entirely in the past decade.

Macrophages arise early during embryogenesis and colonize developing organs, forming a 3D network within every tissue. These tissue-resident macrophages have a high self-renewal capacity and generally do not require input from HSCs.

See also —

Tissue-Resident Macrophage Ontogeny and Homeostasis (2016, Immunity)
The development and maintenance of resident macrophages (2016, Nature Immunology)

 

[Jonathan Edwards]

This paper cites
Circulatory and maturation kinetics of human monocyte subsets in vivo, 2017, Tak et al

I am pretty sceptical about all this 'non-classical monocyte' stuff. People love to invent these terms but often with very limited knowledge of both histology and haematology. I published on the distribution of CD16+ macrophages in human tissues with Ajay Bhatia back in 1998. At that time some other subsetting was popular.

I haven't looked at it in detail but there is difference between cells wandering through spleen, where there are no real vascular boundaries, and setting up in other tissues like skin and pericardium. CD16+ cells in these other tissues take on an extended macrophage cytoplasm and position themselves in very specific microenvironments. I find it hard to believe that they then get rid of all the cytoplasm and return to 'monocyte' morphology in which much of the cell is nucleus. Anything bigger than that would not go through capillaries - which is why all white blood cells are compact.

 

[Jonathan Edwards]

From Tissue-specific macrophages: how they develop and choreograph tissue biology (2023, Nature Reviews Immunology) —

That looks like a typical promotional review by people who haven't bothered to read literature from forty years earlier! The complexity of macrophage behaviour was known to people like Wally Spector and Ralph van Furth in 1980. And mice are often very different from men.

 

[FMMM1]

That looks like a typical promotional review by people who haven't bothered to read literature from forty years earlier! The complexity of macrophage behaviour was known to people like Wally Spector and Ralph van Furth in 1980. And mice are often very different from men.

[Off topic!] Interested in your views re this research -
https://news.cornell.edu/stories/2023/04/95m-fund-chronic-fatigue-syndrome-research

 

[SNT Gatchaman]

Is the idea being proposed that they don't become macrophages with macrophage morphology, but that they are still monocytes (briefly) in tissue, without time to further differentiate and increase cytoplasm and contents?

The reference Hutan noted above is Circulatory and maturation kinetics of human monocyte subsets in vivo (2017) and has only been cited 4 times since. But to (hopefully correctly) summarise the proposed concept —

Monocytes can be subclassified based on their surface markers, particularly CD16 and CD14. Three subtypes seem popular in the literature, eg Nomenclature of monocytes and dendritic cells in blood (2010, Blood).

Classical (CM): CD14Hi CD16Lo
Intermediate (IM): CD14Hi CD16Dim
Non-Classical (NCM): CD14Dim CD16Hi

the currently held view is that at least 3 monocyte subsets exist: classical CD14++ CD16– monocytes (CMs), intermediate CD14++ CD16+ monocytes (IMs), and nonclassical CD14+ CD16++ monocytes (NCMs).

Then they say that these subsets differentiate in a linear fashion:

CM -> IM -> NCM

Their maturation kinetics also differ, because human CMs repopulate the bloodstream first after hematopoietic stem cell transplantation, followed by IMs and later by NCMs. [...] The gradually changing expression patterns, combined with their consecutive repopulation/labeling kinetics has led to the prevailing idea that monocytes differentiate from CMs via IMs to NCMs.

They then propose - by fitting to a model to explain observations relating to deuterium-labelled glucose - that IMs might spend time in tissue.

CM -> IM -> IM (briefly in tissue, but still monocyte morphology) -> NCM

Assuming differentiation from IMs into NCMs directly in the circulation gave reasonable fits to the enrichment data of CMs and IMs but failed to describe the delay with which label was observed in NCMs. We therefore extended the model by assuming that IMs can mature into NCMs only after a delay of δ2 days, Assuming that this maturation step occurs inside the blood gave an improved fit to the data, but an even better fit was obtained when we assumed that this differentiation step occurs outside the blood.

So if the potential for a detour into tissue were valid, what's the purpose?

From Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets (2011, Blood) —

For intermediate monocytes, significant enrichment for genes under major histocompatibility complex (MHC) class II processing and presentation were found. This included 6 of these genes for α- and β-chains of MHC class II, and also 2 genes, CD74 (class II invariant chain) and HLA-DO, which are involved in MHC class II antigen processing. The costimulatory molecule CD40 was also most highly expressed by the intermediate subset.

Perhaps IMs can briefly scavenge from tissue and present antigens back in the blood, where they might affect eg circulating T cells etc?

 

[Jonathan Edwards]

All you need to do to make a CD14+ CD16- monocyte into a CD14low/CD16high monocyte is sit them on plastic for a day or two, as Vicki Abrahams did in my lab. Calling these 'subsets' is popular with people with pigeonhole minds but obscures the dynamics.

 

[SNT Gatchaman]

Thanks for clarifying. So to attempt to summarise this aspect for the thread and I hope I'm following you correctly —

Publications and indeed professional orgs seem to have settled on the idea of monocyte subsets, principally on the basis of the observed CD14/16 surface markers. Further work then tries to demonstrate and explain functional differences and maturation/differentiation based on the idea that there are these subsets.

However, this is simplistic and in the real world of experienced immunology observations, this might be more of a spectrum and it's clear that monos can - let's call it - "swap priority expression of 14 vs 16" (possibly in either direction?). A '14' can be triggered into a '16' pretty easily and even just with time, while leaving them alone in vitro. So, it's not especially important to the understanding of monocyte functions to over-emphasise the 14 vs 16 aspect, and it might be better if we simply look at them as a somewhat heterogeneous group of monocytes.