HSP90 function is required for T2R bitter taste receptor nitric oxide production and innate immune responses in human airway epithelial cells and macrophages

Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two airway innate immune cell types are activated by bitter products, including those secreted by common airway pathogens like Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO production leads to enhanced mucociliary clearance and direct antibacterial effects by ciliated epithelial cells as well as increased phagocytosis by macrophages. Using biochemistry and live cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). We used immunofluorescence to show that H441 cells express eNOS and certain T2Rs and that the bitterant denatonium benzoate activates NO production in an HSP90-dependent manner in cells grown either as submerged cultures and at air liquid interface. In primary sinonasal epithelial cells, we determined that HSP-90 inhibition reduces T2R-stimulated NO production and ciliary beating which are crucial for pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves an innate immune role by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream calcium signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases like chronic rhinosinusitis, asthma, or cystic fibrosis.

ACSL1 Inhibits ALV-J Replication by IFN-Ⅰ Signaling and PI3K/Akt Pathway

J subgroup avian leukosis virus (ALV-J) infection causes serious immunosuppression problems, leading to hematopoietic malignancy tumors in chicken. It has been demonstrated that interferon-stimulated genes (ISGs) could limit ALV-J replication; nevertheless, the underlying mechanisms remain obscure. Here, we demonstrate that Long-chain Acyl-CoA synthetase 1 (ACSL1) is an interferon (IFN)-stimulated gene that specifically restricts the replication of ALV-J due to the higher IFN-I production. More importantly, ACSL1 induces primary monocyte-derived macrophages (MDMs) to pro-inflammatory phenotypic states during ALV-J infection, and ACSL1 mediates apoptosis through the PI3K/Akt signaling pathway in ALV-J-infected primary monocyte-derived macrophages (MDMs). Overall, these results provide evidence that ACSL1 contributes to the antiviral response against ALV-J.

Biodegradation of graphdiyne oxide in classically activated (M1) macrophages modulates cytokine production

Graphdiyne oxide (GDYO) is a carbon‐based nanomaterial possessing sp2 and sp-hybridized carbon atoms with many promising applications. However, its biocompatibility and potential biodegradability remain poorly understood. Using human primary monocyte-derived...

Saturated Fatty Acids Promote GDF15 Expression in Human Macrophages through the PERK/eIF2/CHOP Signaling Pathway

Growth differentiation factor-15 (GDF-15) and its receptor GFRAL are both involved in the development of obesity and insulin resistance. Plasmatic GDF-15 level increases with obesity and is positively associated with disease progression. Despite macrophages have been recently suggested as a key source of GDF-15 in obesity, little is known about the regulation of GDF-15 in these cells. In the present work, we sought for potential pathophysiological activators of GDF15 expression in human macrophages and identified saturated fatty acids (SFAs) as strong inducers of GDF15 expression and secretion. SFAs increase GDF15 expression through the induction of an ER stress and the activation of the PERK/eIF2/CHOP signaling pathway in both PMA-differentiated THP-1 cells and in primary monocyte-derived macrophages. The transcription factor CHOP directly binds to the GDF15 promoter region and regulates GDF15 expression. Unlike SFAs, unsaturated fatty acids do not promote GDF15 expression and rather inhibit both SFA-induced GDF15 expression and ER stress. These results suggest that free fatty acids may be involved in the control of GDF-15 and provide new molecular insights about how diet and lipid metabolism may regulate the development of obesity and T2D.

620 Tumor cell-intrinsic STING pathway is activated in the presence of cues from immune cells and contributes to the anti-tumor activity of tumor cell-targeted STING agonist antibody-drug conjugates

BackgroundSTING pathway agonism has emerged as a potential therapeutic mechanism to stimulate an innate anti-tumor immune response. While in principle systemic administration of a STING agonist would have many therapeutic benefits, including the delivery of STING to all tumor lesions, such an approach may be limited by toxicity. Antibody-drug conjugates (ADCs) constitute a proven therapeutic modality that is ideally suited to allow systemic administration while stimulating the innate immunity in a targeted manner. We have previously demonstrated that targeted delivery of a STING agonist with an ADC induces robust anti-tumor immune responses.MethodsHerein we investigated the mechanism of action of tumor cell-targeted STING agonist ADCs. We evaluated STING pathway activation and anti-tumor activity elicited by ADCs harboring either wild type (wt) or mutant Fc deficient in Fcγ receptor (FcγR) binding in wt or STING knockout (ko) cancer cell mono-cultures, immune cell co-cultures, and in in vivo tumor models.ResultsConsistent with previous reports, the majority of cancer cell lines tested failed to induce STING pathway following STING agonist payload treatment in mono-cultures. In cancer cell:THP1 monocytic cell co-cultures, tumor-targeted STING agonist ADCs with wt Fc exhibited robust STING activation, whereas Fc-mutant ADCs or non-targeted control ADCs had minimal activity. Similar results were obtained when THP1 cells were treated in plates coated with target antigen without cancer cells, demonstrating STING activation in THP1 cells following FcγR-mediated uptake of antigen-bound ADCs. Tumor-targeted Fc-wt ADCs led to marked induction of STING pathway and cancer cell-killing in cancer cell:PBMC or primary monocyte co-cultures, and complete tumor regressions in in vivo tumors. Surprisingly, while at reduced levels relative to the Fc-wt ADCs, Fc-mutant ADCs exhibited significant activity in these in vitro and in vivo models, suggesting that tumor cell-intrinsic STING pathway may be activated in the presence of cues from immune cells. Consistently, STING agonist payload treatment in the presence of conditioned media from PBMC and primary monocyte but not from THP1 cultures, led to STING activation in cancer cell mono-cultures. Moreover, Fc-mutant ADCs had diminished activity in STING ko cancer cell:PBMC or primary monocyte co-cultures, demonstrating the contribution of tumor cell-intrinsic STING activation to the anti-tumor activity elicited by tumor cell-targeted STING agonist ADCs.ConclusionsIn conclusion, we demonstrated that tumor cell-targeted STING agonist ADCs induce robust anti-tumor activity through mechanisms involving both FcγR and tumor antigen-mediated ADC internalization and subsequent induction of STING pathway in immune cells and tumor cells.

HIV-1 replication complexes accumulate in nuclear speckles and integrate into speckle-associated genomic domains

The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.

Massive reorganization of the genome during primary monocyte differentiation into macrophage

Monocyte-to-macrophage trans-differentiation has long been studied to better understand this immunological response and aspects of developmental processes more generally. A key question is the nature of the corresponding changes in chromatin conformation and its relationship to the transcriptome during this process. This question is especially intriguing since this trans-differentiation is not associated with progression through mitosis, often considered a necessary step for gross changes in chromosomal structure. Here, we characterized the transcriptional and genomic structural changes during macrophage development of primary human monocytes using RNA-seq and in situ Hi-C. We found that, during this transition, the genome architecture undergoes a massive remodeling to a degree not observed before between structured genomes, with changes in ~90% of the topologically associating domains (TADs). These changes in the TADs are associated with changed expression of immunological genes. These structural changes, however, differ extensively from those described recently in a study of the leukemia cell line, THP-1. Furthermore, up-regulation of the AP-1 family of genes that effected functionally important changes in the genomic structure during the differentiation of the THP-1 cells was not corroborated with the primary cells. Taken together, our results provide a comprehensive characterization of the changes in genomic structure during the monocyte-to-macrophage transition, establish a framework for the elucidation of processes underlying differentiation without proliferation, and demonstrate the importance of verifying with primary cells the mechanisms discovered with cultured cells.

The Nef Protein of the Macrophage Tropic HIV-1 Strain AD8 Counteracts Human BST-2/Tetherin

Bone Marrow Stromal Cell Antigen 2 (BST-2)/tetherin inhibits the release of numerous enveloped viruses by physically tethering nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency virus (HIV), and pandemic main (M) group HIV type 1s (HIV-1s) are thought to rely exclusively on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express Vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 Nef protein can compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous cell surface tetherin levels, physically separating it from the site of viral budding, thus preventing HIV retention. Mechanistically, AD8 Nef enhances internalisation of the long isoform of human tetherin, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, we show that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.

Proliferation of HIV-infected renal epithelial cells following virus acquisition from infected macrophages

ABSTRACTObjectivesHIV-1 can infect and persist in different organs and tissues, resulting in the generation of multiple viral compartments and reservoirs. Increasing evidence supports the kidney as such a reservoir. Previous work demonstrated that HIV-1 infected CD4+ T-cells transfer virus to renal tubule epithelial (RTE) cells through cell-to-cell contact. In addition to CD4+ T-cells, macrophages represent the other major target of HIV-1. Renal macrophages induce and regulate inflammatory responses and are critical to homeostatic regulation of the kidney environment. Combined with their ability to harbor virus, macrophages may also play an important role in the spread of HIV-1 infection in the kidney.Design and MethodsMultiparametric histochemistry analysis was performed on kidney biopsies from individuals with HIV-1 associated nephropathy (HIVAN). Primary monocyte-derived macrophages were infected with a (GFP)-expressing replication competent HIV-1. HIV-1 transfer from macrophages to RTE cells was carried out in a co-culture system and evaluated by fluorescence-microscopy and flow-cytometry. Live imaging was performed to assess the fate of HIV-1 infected RTE cells over time.ResultsWe show that macrophages are abundantly present in the renal inflammatory infiltrate of individuals with HIVAN. We observed contact-dependent HIV-1 transfer from infected macrophages to both primary and immortalized renal cells. Live imaging of HIV-1 infected RTE cells revealed four different fates: proliferation, hypertrophy, latency and cell death.ConclusionsOur study suggests that macrophages may play a role in the dissemination of HIV-1 in the kidney and that proliferation of infected renal cells may contribute to HIV-1 persistence in this compartment.

The Nef protein of the macrophage tropic HIV-1 strain AD8 counteracts human Bst-2/tetherin

Bst-2/tetherin inhibits the release of numerous enveloped viruses by physically attaching nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency viruses (HIV), and pandemic main (M) group HIV-1s are thought to exclusively rely on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 was able to use its Nef protein to compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous tetherin levels from the cell surface, physically separating it from the site of viral budding and thus preventing HIV retention. Mechanistically, AD8 Nef enhances l-tetherin internalisation, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, this is the first report showing that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.