Caspase-8 promotes scramblase-mediated phosphatidylserine exposure and fusion of osteoclast precursors.

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Tissue-specific transcriptional programming of macrophages controls the microRNA transcriptome targeting multiple functional pathways.

Recent interest in the biology and function of peritoneal tissue resident macrophages (pMΦ) has led to a better understanding of their cellular origin, programming, and renewal. The programming of pMΦ is dependent on microenvironmental cues and tissue-specific transcription factors, including GATA6. However, the contribution of microRNAs remains poorly defined. We conducted a detailed analysis of the impact of GATA6 deficiency on microRNA expression in mouse pMΦ. Our data suggest that for many of the pMΦ, microRNA composition may be established during tissue specialization and that the effect of GATA6 knockout is largely unable to be rescued in the adult by exogenous GATA6. The data are consistent with GATA6 modulating the expression pattern of specific microRNAs, directly or indirectly, and including miR-146a, miR-223, and miR-203 established by the lineage-determining transcription factor PU.1, to achieve a differentiated pMΦ phenotype. Lastly, we showed a significant dysregulation of miR-708 in pMΦ in the absence of GATA6 during homeostasis and in response to LPS/IFN-γ stimulation. Overexpression of miR-708 in mouse pMΦ in vivo altered 167 mRNA species demonstrating functional downregulation of predicted targets, including cell immune responses and cell cycle regulation. In conclusion, we demonstrate dependence of the microRNA transcriptome on tissue-specific programming of tissue macrophages as exemplified by the role of GATA6 in pMΦ specialization.

3R Blackboard: A platform for animal and organ sharing.

Since the embedding of the principles of the 3Rs (Replacement, Reduction and Refinement) in national and international regulations on the use of animals, scientists have been challenged to find ways to reduce the number of animals in their research. Here, we present a digital platform, called '3R Backboard', linked to a laboratory animal management system, which facilitates sharing of surplus biological materials from animals (e.g. tissues, organs and cells) to other research teams. Based on information provided, such as genotype, age and sex, other animal workers were able to indicate their interest in collecting specific tissues and to communicate with the person providing the animals. A short pilot study of this approach conducted in a limited academic environment presented strong evidence of its effectiveness and resulted in a notable reduction of the number of mice used. In addition, the use of 3R Blackboard led to resource saving, knowledge exchange and even establishment of new collaboration.

Oxylipin metabolism is controlled by mitochondrial ß-oxidation during bacterial inflammation.

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial ß-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin ß-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by ß-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial ß-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease-protective variant PLCγ2 R522.

Variants identified in genome-wide association studies have implicated immune pathways in the development of Alzheimer's disease (AD). Here, we investigated the mechanistic basis for protection from AD associated with PLCγ2 R522, a rare coding variant of the PLCG2 gene. We studied the variant's role in macrophages and microglia of newly generated PLCG2-R522-expressing human induced pluripotent cell lines (hiPSC) and knockin mice, which exhibit normal endogenous PLCG2 expression. In all models, cells expressing the R522 mutation show a consistent non-redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This manifests as enhanced release of cellular calcium ion stores in response to physiologically relevant stimuli like Fc-receptor ligation or exposure to Aß oligomers. Expression of the PLCγ2-R522 variant resulted in increased stimulus-dependent PIP2 depletion and reduced basal PIP2 levels in vivo. Furthermore, it was associated with impaired phagocytosis and enhanced endocytosis. PLCγ2 acts downstream of other AD-related factors, such as TREM2 and CSF1R, and alterations in its activity directly impact cell function. The inherent druggability of enzymes such as PLCγ2 raises the prospect of PLCγ2 manipulation as a future therapeutic approach in AD.

Differential recognition of HIV-stimulated IL-1ß and IL-18 secretion through NLR and NAIP signalling in monocyte-derived macrophages.

Macrophages are important drivers of pathogenesis and progression to AIDS in HIV infection. The virus in the later phases of the infection is often predominantly macrophage-tropic and this tropism contributes to a chronic inflammatory and immune activation state that is observed in HIV patients. Pattern recognition receptors of the innate immune system are the key molecules that recognise HIV and mount the inflammatory responses in macrophages. The innate immune response against HIV-1 is potent and elicits caspase-1-dependent pro-inflammatory cytokine production of IL-1ß and IL-18. Although, NLRP3 has been reported as an inflammasome sensor dictating this response little is known about the pattern recognition receptors that trigger the "priming" signal for inflammasome activation, the NLRs involved or the HIV components that trigger the response. Using a combination of siRNA knockdowns in monocyte derived macrophages (MDMs) of different TLRs and NLRs as well as chemical inhibition, it was demonstrated that HIV Vpu could trigger inflammasome activation via TLR4/NLRP3 leading to IL-1ß/IL-18 secretion. The priming signal is triggered via TLR4, whereas the activation signal is triggered by direct effects on Kv1.3 channels, causing K+ efflux. In contrast, HIV gp41 could trigger IL-18 production via NAIP/NLRC4, independently of priming, as a one-step inflammasome activation. NAIP binds directly to the cytoplasmic tail of HIV envelope protein gp41 and represents the first non-bacterial ligand for the NAIP/NLRC4 inflammasome. These divergent pathways represent novel targets to resolve specific inflammatory pathologies associated with HIV-1 infection in macrophages.

A Human Dectin-2 Deficiency Associated With Invasive Aspergillosis.

Immunocompromised patients are highly susceptible to invasive aspergillosis. Herein, we identified a homozygous deletion mutation (507 del C) resulting in a frameshift (N170I) and early stop codon in the fungal binding Dectin-2 receptor, in an immunocompromised patient. The mutated form of Dectin-2 was weakly expressed, did not form clusters at/near the cell surface and was functionally defective. Peripheral blood mononuclear cells from this patient were unable to mount a cytokine (tumor necrosis factor, interleukin 6) response to Aspergillus fumigatus, and this first identified Dectin-2-deficient patient died of complications of invasive aspergillosis.

Erratum: Effective In Vivo Gene Modification in Mouse Tissue-Resident Peritoneal Macrophages by Intraperitoneal Delivery of Lentiviral Vectors.

[This corrects the article DOI: 10.1016/j.omtm.2019.10.004.].

Tissue-resident macrophages actively suppress IL-1beta release via a reactive prostanoid/IL-10 pathway.

The alarm cytokine interleukin-1ß (IL-1ß) is a potent activator of the inflammatory cascade following pathogen recognition. IL-1ß production typically requires two signals: first, priming by recognition of pathogen-associated molecular patterns leads to the production of immature pro-IL-1ß; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of IL-1ß from its pro-form. However, despite the important role of IL-1ß in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissue-resident macrophages use an active inhibitory pathway, to suppress IL-1ß processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of IL-10. In the absence of secondary signal, IL-10 potently inhibits IL-1ß processing, providing a previously unrecognized control of IL-1ß in tissue-resident macrophages.

Effective In Vivo Gene Modification in Mouse Tissue-Resident Peritoneal Macrophages by Intraperitoneal Delivery of Lentiviral Vectors.

Tissue-resident macrophages exhibit specialized phenotypes dependent on their in vivo physiological niche. Investigation of their function often relies upon complex whole mouse transgenic studies. While some appropriate lineage-associated promoters exist, there are no options for tissue-specific targeting of macrophages. We have developed full protocols for in vivo productive infection (defined by stable transgene expression) of tissue-resident macrophages with lentiviral vectors, enabling RNA and protein overexpression, including expression of small RNA species such as shRNA, to knock down and modulate gene expression. These approaches allow robust infection of peritoneal tissue-resident macrophages without significant infection of other cell populations. They permit rapid functional study of macrophages in homeostatic and inflammatory settings, such as thioglycolate-induced peritonitis, while maintaining the cells in their physiological context. Here we provide detailed protocols for the whole workflow: viral production, purification, and quality control; safety considerations for administration of the virus to mice; and assessment of in vivo transduction efficiency and the low background levels of inflammation induced by the virus. In summary, we present a quick and accessible protocol for the rapid assessment of gene function in peritoneal tissue-resident macrophages in vivo.

Dendritic Cells Promote the Spread of Human T-Cell Leukemia Virus Type 1 via Bidirectional Interactions with CD4+ T Cells.

Human T-cell leukemia virus type 1 (HTLV-1) propagates within and between individuals via cell-to-cell transmission, and primary infection typically occurs across juxtaposed mucosal surfaces during breastfeeding or sexual intercourse. It is therefore likely that dendritic cells (DCs) are among the first potential targets for HTLV-1. However, it remains unclear how DCs contribute to virus transmission and dissemination in the early stages of infection. We show that an HTLV-1-infected cell line (MT-2) and naturally infected CD4+ T cells transfer p19+ viral particles to the surface of allogeneic DCs via cell-to-cell contacts. Similarly organized cell-to-cell contacts also facilitate DC-mediated transfer of HTLV-1 to autologous CD4+ T cells. These findings shed light on the cellular structures involved in anterograde and retrograde transmission and suggest a key role for DCs in the natural history and pathogenesis of HTLV-1 infection.

TGFß Induces a SAMHD1-Independent Post-Entry Restriction to HIV-1 Infection of Human Epithelial Langerhans Cells.

Sterile alpha motif (SAM) and histidine-aspartic (HD) domains protein 1 (SAMHD1) was previously identified as a critical post-entry restriction factor to HIV-1 infection in myeloid dendritic cells. Here we show that SAMHD1 is also expressed in epidermis-isolated Langerhans cells (LC), but degradation of SAMHD1 does not rescue HIV-1 or vesicular stomatitis virus G-pseudotyped lentivectors infection in LC. Strikingly, using Langerhans cells model systems (mutz-3-derived LC, monocyte-derived LC [MDLC], and freshly isolated epidermal LC), we characterize previously unreported post-entry restriction activity to HIV-1 in these cells, which acts at HIV-1 reverse transcription, but remains independent of restriction factors SAMHD1 and myxovirus resistance 2 (MX2). We demonstrate that transforming growth factor-ß signaling confers this potent HIV-1 restriction in MDLC during their differentiation and blocking of mothers against decapentaplegic homolog 2 (SMAD2) signaling in MDLC restores cells' infectivity. Interestingly, maturation of MDLC with a toll-like receptor 2 agonist or transforming growth factor-α significantly increases cells' susceptibility to HIV-1 infection, which may explain why HIV-1 acquisition is increased during coinfection with sexually transmitted infections. In conclusion, we report a SAMHD1-independent post-entry restriction in MDLC and LC isolated from epidermis, which inhibits HIV-1 replication. A better understanding of HIV-1 restriction and propagation from LC to CD4(+) T cells may help in the development of new microbicides or vaccines to curb HIV-1 infection at its earliest stages during mucosal transmission.

TLR-4 engagement of dendritic cells confers a BST-2/tetherin-mediated restriction of HIV-1 infection to CD4+ T cells across the virological synapse.

BACKGROUND: Dendritic cells and their subsets, located at mucosal surfaces, are among the first immune cells to encounter disseminating pathogens. The cellular restriction factor BST-2/tetherin (also known as CD317 or HM1.24) potently restricts HIV-1 release by retaining viral particles at the cell surface in many cell types, including primary cells such as macrophages. However, BST-2/tetherin does not efficiently restrict HIV-1 infection in immature dendritic cells. RESULTS: We now report that BST-2/tetherin expression in myeloid (myDC) and monocyte-derived dendritic cells (DC) can be significantly up-regulated by IFN-α treatment and TLR-4 engagement with LPS. In contrast to HeLa or 293T cells, infectious HIV-1 release in immature DC and IFN-α-matured DC was only modestly affected in the absence of Vpu compared to wild-type viruses. Strikingly, immunofluorescence analysis revealed that BST-2/tetherin was excluded from HIV containing tetraspanin-enriched microdomains (TEMs) in both immature DC and IFN-α-matured DC. In contrast, in LPS-mediated mature DC, BST-2/tetherin exerted a significant restriction in transfer of HIV-1 infection to CD4+ T cells. Additionally, LPS, but not IFN-α stimulation of immature DC, leads to a dramatic redistribution of cellular restriction factors to the TEM as well as at the virological synapse between DC and CD4+ T cells. CONCLUSIONS: In conclusion, we demonstrate that TLR-4 engagement in immature DC significantly up-regulates the intrinsic antiviral activity of BST-2/tetherin, during cis-infection of CD4+ T cells across the DC/T cell virological synapse. Manipulating the function and potency of cellular restriction factors such as BST-2/tetherin to HIV-1 infection, has implications in the design of antiviral therapeutic strategies.

HIV impairment of immune responses in dendritic cells.

Dendritic cells and their subsets are diverse populations of immune cells in the skin and mucous membranes that possess the ability to sense the presence of microbes and orchestrate an efficient and adapted immune response. Dendritic cells (DC) have the unique ability to act as a bridge between the innate and adaptive immune responses. These cells are composed of a number of subsets behaving with preferential and specific features depending on their location and surrounding environment. Langerhans cells (LC) or dermal DC (dDC) are readily present in mucosal areas. Other DC subsets such as plasmacytoid DC (pDC), myeloid DC (myDC), or monocyte-derived DC (MDDC) are thought to be recruited or differentiated in sites of pathogenic challenge. Upon HIV infection, DC and their subsets are likely among the very first immune cells to encounter incoming pathogens and initiate innate and adaptive immune responses. However, as evidenced during HIV infection, some pathogens have evolved subtle strategies to hijack key cellular machineries essential to generate efficient antiviral responses and subvert immune responses for spread and survival.In this chapter, we review recent research aimed at investigating the involvement of DC subtypes in HIV transmission at mucosal sites, concentrating on HIV impact on cellular signalling and trafficking pathways in DC leading to DC-mediated immune response alterations and viral immune evasion. We also address some aspects of DC functions during the chronic immune pathogenesis and conclude with an overview of the current and novel therapeutic and prophylactic strategies aimed at improving DC-mediated immune responses, thus to potentially tackle the early events of mucosal HIV infection and spread.