Inhibition of human macrophage activation via pregnane neurosteroid interactions with toll-like receptors: Sex differences and structural requirements.

We recently discovered that (3α,5α)3-hydroxypregnan-20-one (allopregnanolone) inhibits pro-inflammatory toll-like receptor (TLR) activation and cytokine/chemokine production in mouse macrophage RAW264.7 cells. The present studies evaluate neurosteroid actions upon TLR activation in human macrophages from male and female healthy donors. Buffy coat leukocytes were obtained from donors at the New York Blood Center (http://nybloodcenter.org/), and peripheral blood mononuclear cells were isolated and cultured to achieve macrophage differentiation. TLR4 and TLR7 were activated by lipopolysaccharide (LPS) or imiquimod in the presence/absence of allopregnanolone or related neurosteroids and pro-inflammatory markers were detected by ELISA or western blotting. Cultured human monocyte-derived-macrophages exhibited typical morphology, a mixed immune profile of both inflammatory and anti-inflammatory markers, with no sex difference at baseline. Allopregnanolone inhibited TLR4 activation in male and female donors, preventing LPS-induced elevations of TNF-α, MCP-1, pCREB and pSTAT1. In contrast, 3α,5α-THDOC and SGE-516 inhibited the TLR4 pathway activation in female, but not male donors. Allopregnanolone completely inhibited TLR7 activation by imiquimod, blocking IL-1-ß, IL-6, pSTAT1 and pIRF7 elevations in females only. 3α,5α-THDOC and SGE-516 partially inhibited TLR7 activation, only in female donors. The results indicate that allopregnanolone inhibits TLR4 and TLR7 activation in cultured human macrophages resulting in diminished cytokine/chemokine production. Allopregnanolone inhibition of TLR4 activation was found in males and females, but inhibition of TLR7 signals exhibited specificity for female donors. 3α,5α-THDOC and SGE-516 inhibited TLR4 and TLR7 pathways only in females. These studies demonstrate anti-inflammatory effects of allopregnanolone in human macrophages for the first time and suggest that inhibition of pro-inflammatory cytokines/chemokines may contribute to its therapeutic actions.

Suppression of HIV-associated Macrophage Activation by a p75 Neurotrophin Receptor Ligand.

Previous studies indicated that nerve growth factor (NGF) and proNGF differentially regulate the phenotype of macrophages and microglia via actions at tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptors (p75NTR), respectively. The ability of HIV gp120 and virions to induce the secretion of factors toxic to neurons was suppressed by NGF and enhanced by proNGF, suggesting the potential for neurotrophin based "anti-inflammatory" interventions. To investigate the "anti-inflammatory" potential of the p75NTR ligand, LM11A-31, we treated cultured macrophages and microglia with HIV gp120 in the presence or absence of the ligand and evaluated the morphological phenotype, intrinsic calcium signaling, neurotoxic activity and proteins in the secretome. LM11A-31 at 10 nM was able to suppress the release of neurotoxic factors from both monocyte-derived macrophages (MDM) and microglia. The protective effects correlated with a shift in morphology and a unique secretory phenotype rich in growth factors that overrode the actions of HIV gp120. The protein pattern was generally consistent with anti-inflammatory, phagocytic and tissue remodeling functions. Although the toxic factor(s) and the source of the neuroprotection were not identified, the data indicated that an increased degradation of NGF induced by HIV gp120 was likely to contribute to neuronal vulnerability. Although substantial work is still needed to reveal the functions of many proteins in the mononuclear phagocyte secretome, such as growth and differentiation factors, the data clearly indicate that the ligand LM11A-31 has excellent therapeutic potential due to its ability to induce a more protective phenotype that restricts activation by HIV.

Neuroinflammation and EIF2 Signaling Persist despite Antiretroviral Treatment in an hiPSC Tri-culture Model of HIV Infection.

HIV-associated neurocognitive disorders (HAND) affect over half of HIV-infected individuals, despite antiretroviral therapy (ART). Therapeutically targetable mechanisms underlying HAND remain elusive, partly due to a lack of a representative model. We developed a human-induced pluripotent stem cell (hiPSC)-based model, independently differentiating hiPSCs into neurons, astrocytes, and microglia, and systematically combining to generate a tri-culture with or without HIV infection and ART. Single-cell RNA sequencing analysis on tri-cultures with HIV-infected microglia revealed inflammatory signatures in the microglia and EIF2 signaling in all three cell types. Treatment with the antiretroviral compound efavirenz (EFZ) mostly resolved these signatures. However, EFZ increased RhoGDI and CD40 signaling in the HIV-infected microglia. This activation was associated with a persistent increase in transforming growth factor α production by microglia. This work establishes a tri-culture that recapitulates key features of HIV infection in the CNS and provides a new model to examine the effects of infection, its treatment, and other co-morbid conditions.

Opposing Effects of NGF and proNGF on HIV Induced Macrophage Activation.

Macrophage and microglial activation by HIV in the central nervous system (CNS) triggers the secretion of soluble factors which damage neurons. Therapeutic approaches designed to restore cognitive function by suppressing this inflammatory activity have not yet been successful. Recent studies have indicated that the phenotype of macrophages is differentially controlled by the mature and pro form of nerve growth factor. These cells therefore may be highly responsive to the imbalance in pro versus mature neurotrophins often associated with neurodegenerative diseases. In this study we evaluated the interactions between neurotrophins and HIV induced macrophage activation. HIV stimulation of macrophages induced a neurotoxic phenotype characterized by the expression of podosomes, suppression of calcium spiking and increased neurotoxin production. The secretome of the activated macrophages revealed a bias toward anti-angiogenic like activity and increased secretion of MMP-9. Co-stimulation with NGF and HIV suppressed neurotoxin secretion, increased calcium spiking, suppressed podosome expression and reversed 86% of the proteins secreted in response to HIV, including MMP-9 and many growth factors. In contrast, co-stimulation of macrophages with proNGF not only failed to reverse the effects of HIV but increased the neurotoxic phenotype. These differential effects of proNGF and NGF on HIV activation provide a potential novel therapeutic avenue for controlling macrophage activation in response to HIV.

Differential regulation of macrophage phenotype by mature and pro-nerve growth factor.

To characterize the role of neurotrophin receptors on macrophages, we investigated the ability of nerve growth factor (NGF) and its precursor, proNGF, to regulate human macrophage phenotype. The p75 neurotrophin receptor (p75(NTR)) and TrkA were concentrated within overlapping domains on membrane ruffles. NGF stimulation of macrophages increased membrane ruffling, calcium spiking, phagocytosis and growth factor secretion. In contrast, proNGF induced podosome formation, increased migration, suppressed calcium spikes and increased neurotoxin secretion. These results demonstrate opposing roles of NGF and proNGF in macrophage regulation providing new avenues for pharmacological intervention during neuroinflammation.

The p75 neurotrophin receptor: at the crossroad of neural repair and death.

The strong repair and pro-survival functions of neurotrophins at their primary receptors, TrkA, TrkB and TrkC, have made them attractive candidates for treatment of nervous system injury and disease. However, difficulties with the clinical implementation of neurotrophin therapies have prompted the search for treatments that are stable, easier to deliver and allow more precise regulation of neurotrophin actions. Recently, the p75 neurotrophin receptor (p75(NTR)) has emerged as a potential target for pharmacological control of neurotrophin activity, supported in part by studies demonstrating 1) regulation of neural plasticity in the mature nervous system, 2) promotion of adult neurogenesis and 3) increased expression in neurons, macrophages, microglia, astrocytes and/or Schwann cells in response to injury and neurodegenerative diseases. Although the receptor has no intrinsic catalytic activity it interacts with and modulates the function of TrkA, TrkB, and TrkC, as well as sortilin and the Nogo receptor. This provides substantial cellular and molecular diversity for regulation of neuron survival, neurogenesis, immune responses and processes that support neural function. Upregulation of the p75(NTR) under pathological conditions places the receptor in a key position to control numerous processes necessary for nervous system recovery. Support for this possibility has come from recent studies showing that small, non-peptide p75(NTR) ligands can selectively modify pro-survival and repair functions. While a great deal remains to be discovered about the wide ranging functions of the p75(NTR), studies summarized in this review highlight the immense potential for development of novel neuroprotective and neurorestorative therapies.