The study findings were published in the peer reviewed journal: Cell Reports.
https://www.cell.com/cell-reports/fulltext/S2211-1247(22)01548-0
In this study, we analyzed how treatment of monocytes with EVs carrying HIV-1 protein Nef (exNef) induces a pro-inflammatory memory in MDMs that differentiate from treated monocytes.
Our findings demonstrate exNef-induced changes in chromatin composition of MDMs associated with enhanced secretion of two key inflammatory cytokines, TNF-α and IL-6, and increased expression of other pro-inflammatory genes. In contrast to previously demonstrated pro-inflammatory effects of Nef on MDMs,14,42,64,65,66,67,68 the effects reported here are delayed and are maintained for extended periods of time in vitro and in vivo.
These effects of exNef on monocytes are similar to those of β-glucan, as described by Quintin et al.69 and termed “trained immunity.”70 The trained immunity paradigm implies preservation of the trained phenotype in the absence of the initiating factor. Whereas we cannot prove that no Nef remained in the cells, results of bone marrow transplantation experiments, where over 12 weeks and multiple cell divisions separated exNef exposure and BMDM stimulation, make the possibility that residual Nef is the cause of enhanced response to LPS highly unlikely.
Phenotypic analysis of MDMs produced from monocytes exposed to exNef revealed formation of new cell populations characterized by expression of several markers associated with the M1 polarization. None of these populations expressed a classical M1 phenotype, consistent with previous studies that demonstrated that training of monocytes does not induce classical M1 or M2 macrophage phenotypes.22,69
This is particularly true for in vitro differentiation, where polarization may be skewed by growth factors.71 M-CSF used in our study is known to skew monocyte polarization toward the M2 status, whereas exNef stimulated expression of M1-specific markers, counteracting activity of M-CSF. We used ATAC-seq to identify genes whose promoters were associated with open chromatin regions.
Previous studies detected activation of multiple genes belonging to metabolic and inflammatory pathways in β-glucan-trained MDMs.21,27,32,70,72 Consistent with these reports, in exNef-trained MDMs, we found changes in the open chromatin regions of genes associated with inflammation and cholesterol metabolism; activation of these pathways may contribute to the co-morbidities in PLWH.
Consistent with functional manifestations of trained immunity, MDMs trained with exNef responded to LPS stimulation with increased expression levels of a number of genes, including IL-3, IL-6, IL-9, IL-11, CXCL8, CSF3, CSF2, and TNF-α. The mechanism behind exNef-induced trained immunity is consistent with that described for β-glucan:48 it depends on activation of aerobic glycolysis, cholesterol biosynthesis, and IGF1R signaling.
Another important component of exNef-induced trained immunity is an increased abundance of the lipid rafts. This effect is likely caused by exNef-mediated suppression of ABCA1 activity14 and may lead to an increase in IGF1R signaling, which is a key mechanism of training.48,70
Decreased abundance of ABCA1 was maintained in BMDMs for an extended time, suggesting that effects of exNef on cholesterol homeostasis are long lived. Long-lived effects on the cholesterol metabolism pathway would lead to sustained changes in the abundance and properties of the lipid rafts, which need to be taken into consideration in future studies of trained immunity induced by other factors.
The contribution of the observed hyperresponsiveness of myeloid cells to pathogenesis of HIV disease and HIV-associated co-morbidities remains to be investigated. While evolutionarily trained immunity has evolved as a protection mechanism, enhanced responses of monocytes/macrophages of HIV-infected individuals to inflammatory stimuli due to trained immunity may explain sustained low-level inflammation when viral load is reduced to undetectable levels by ART.5,73
Further, in HIV infection, this protective mechanism may be unable to deal with a long-lasting influx of pathogenic factors, such as exNef continuously released from HIV reservoirs7 and bacterial products leaking through incompletely recovered mucosal tissue.74
Thus, contribution of exNef to HIV co-morbidities may be two-fold: induction of long-lasting hyperresponsiveness of myeloid cells and persistent stimulation of these cells.
The half-life of circulating monocytes is relatively short (several days), but a possibility of training by exNef of myeloid progenitor cells, as suggested by our in vivo studies, may provide a long-lasting memory. Thus, even complete cure of HIV infection may still leave infected individuals hyperresponsive to inflammatory stimuli.
This may provide a protective effect for acute infections but may put them at risk for inflammation-associated diseases if chronic stimuli are encountered. How long such memory can persist is an important question, but recent findings by Netea’s group suggested that even a transgenerational transmission of trained immunity is possible.75,76
