Macrophages serve as vital defenders, protecting the body by exhibiting remarkable cellular adaptability in response to invading pathogens and various stimuli. These cells express nicotinic acetylcholine receptors, with the α7-nAChR being extensively studied due to its involvement in activating the cholinergic anti-inflammatory pathway. Activation of this pathway plays a crucial role in suppressing macrophages’ production of proinflammatory cytokines, thus mitigating excessive inflammation and maintaining host homeostasis. Macrophage polarization, which occurs in response to specific pathogens or insults, is a process that has received limited attention concerning the activation of the cholinergic anti-inflammatory pathway and the contributions of the α7-nAChR in this context. This review aims to present evidence highlighting how the cholinergic constituents in macrophages, led by the α7-nAChR, facilitate the polarization of macrophages towards anti-inflammatory phenotypes. Additionally, we explore the influence of viral infections on macrophage inflammatory phenotypes, taking into account cholinergic mechanisms. We also review the current understanding of macrophage polarization in response to these infections. Finally, we provide insights into the relatively unexplored partial duplication of the α7-nAChR, known as dup α7, which is emerging as a significant factor in macrophage polarization and inflammation scenarios.

Since their discovery, nicotinic acetylcholine receptors (nAChRs) have been extensively studied to understand their function, as well as the consequence of alterations leading to disease states. Importantly, these receptors represent pharmacological targets to treat a number of neurological and neurodegenerative disorders. Nevertheless, their therapeutic value has been limited by the absence of high-resolution structures that allow for the design of more specific and effective drugs. This article offers a comprehensive review of five decades of research pursuing high-resolution structures of nAChRs. We provide a historical perspective, from initial structural studies to the most recent X-ray and cryogenic electron microscopy (Cryo-EM) nAChR structures. We also discuss the most relevant structural features that emerged from these studies, as well as perspectives in the field.

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145, which is currently in Phase I clinical trials, produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5–5.5 and 6.0–7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.

Despite current pharmacological intervention strategies, patients with HIV still suffer from chronic inflammation. The nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the nervous and immune systems. In macrophages, activation of alpha7-nAChR (α7-nAChR) controls inflammatory processes through the cholinergic anti-inflammatory response (CAR). Given that this innate immune response controls inflammation and α7-nAChR plays a critical role in the regulation of systemic inflammation, we investigated the effects of an R5-tropic HIV soluble component, gp120JRFL, on the CAR functioning. We previously demonstrated that X4-tropic HIV-1 gp120IIIB disrupts the CAR as well as inducing upregulation of the α7-nAChR in vitro in monocyte-derived macrophages (MDMs) which correlates with the upregulation observed in monocytes, T-lymphocytes, and MDMs recovered from HIV-infected people. We demonstrate here using imaging and molecular assays that the R5-tropic HIV-1 glycoprotein gp120JRFL upregulates the α7-nAChR in MDMs dependent on CD4 and/or CCR5 activation. This upregulation was also dependent on MEK1 since its inhibition attenuates the upregulation of α7-nAChR induced by gp120JRFL and was concomitant with an increase in basal calcium levels which did not result in apoptosis. Moreover, the CAR was determined to be disrupted, since α7-nAChR activation in MDMs did not reduce the production of the proinflammatory cytokines IL-6, GRO-α, or I-309. Furthermore, a partial antagonist of α7-nAChR, bupropion, rescued IL-6 but not GRO-α or I-309 production. Together, these results demonstrate that gp120JRFL disrupts the CAR in MDMs. Other medications targeting the α7-nAChR need to be tested to reactivate the CAR to ameliorate inflammation in HIV-infected subjects.

We have developed a pipeline to express, purify, and characterize HIV envelope protein (Env) gp145 from Chinese hamster ovary cells, to accelerate the production of a promising vaccine candidate. First in shake flasks, then in bioreactors, we optimized the growth conditions. By adjusting the pH to 6.8, we increased expression levels to 101 mg/L in a 50 L bioreactor, nearly twice the previously reported titer value. A battery of analytical methods was developed in accordance with current good manufacturing practices to ensure a quality biopharmaceutical. Imaged capillary isoelectric focusing verified proper glycosylation of gp145; dynamic light scattering confirmed the trimeric arrangement; and bio-layer interferometry and circular dichroism analysis demonstrated native-like properties (i.e., antibody binding and secondary structure). MALDI-TOF mass spectrometry was used as a multi-attribute platform for accurate mass determination, glycans analysis, and protein identification. Our robust analysis demonstrates that our gp145 product is very similar to a reference standard and emphasizes the importance of accurate characterization of a highly heterogeneous immunogen for the development of an effective vaccine. Finally, we present a novel guanosine microparticle with gp145 encapsulated and displayed on its surface. The unique properties of our gp145 microparticle make it amenable to use in future preclinical and clinical trials.

Macrophages serve as vital defenders, protecting the body by exhibiting remarkable cellular adaptability in response to invading pathogens and various stimuli. These cells express nicotinic acetylcholine receptors, with the α7-nAChR being extensively studied due to its involvement in activating the cholinergic anti-inflammatory pathway. Activation of this pathway plays a crucial role in suppressing macrophages' production of proinflammatory cytokines, thus mitigating excessive inflammation and maintaining host homeostasis. Macrophage polarization, which occurs in response to specific pathogens or insults, is a process that has received limited attention concerning the activation of the cholinergic anti-inflammatory pathway and the contributions of the α7-nAChR in this context. This review aims to present evidence highlighting how the cholinergic machinery in macrophages, led by the α7-nAChR, facilitates the polarization of macrophages towards anti-inflammatory phenotypes. Additionally, we explore the influence of viral infections on macrophage inflammatory phenotypes, taking into account cholinergic mechanisms. We also review the current understanding of macrophage polarization in response to these infections. Finally, we provide insights into the relatively unexplored partial duplication of the α7-nAChR, known as dup α7, which is emerging as a significant factor in macrophage polarization and inflammation scenarios.