MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C.

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability.

Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface.

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of terminal mucin glycans sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.

The power of imaging to understand extracellular vesicle biology in vivo.

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.

MUC1 is a receptor for the Salmonella SiiE adhesin that enables apical invasion into enterocytes.

The cellular invasion machinery of the enteric pathogen Salmonella consists of a type III secretion system (T3SS) with injectable virulence factors that induce uptake by macropinocytosis. Salmonella invasion at the apical surface of intestinal epithelial cells is inefficient, presumably because of a glycosylated barrier formed by transmembrane mucins that prevents T3SS contact with host cells. We observed that Salmonella is capable of apical invasion of intestinal epithelial cells that express the transmembrane mucin MUC1. Knockout of MUC1 in HT29-MTX cells or removal of MUC1 sialic acids by neuraminidase treatment reduced Salmonella apical invasion but did not affect lateral invasion that is not hampered by a defensive barrier. A Salmonella deletion strain lacking the SiiE giant adhesin was unable to invade intestinal epithelial cells through MUC1. SiiE-positive Salmonella closely associated with the MUC1 layer at the apical surface, but invaded Salmonella were negative for the adhesin. Our findings uncover that the transmembrane mucin MUC1 is required for Salmonella SiiE-mediated entry of enterocytes via the apical route.

Lysosome-mediated degradation of a distinct pool of lipid droplets during hepatic stellate cell activation.

Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously provided evidence for the presence of two distinct LD pools, a preexisting and a dynamic LD pool. Here we investigate the mechanisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neutral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation in vitro The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, targeted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa-/- mice. Lalistat partially inhibited the induction of activation marker α-smooth muscle actin (α-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation.

Stearoyl-CoA desaturase activity in bovine cumulus cells protects the oocyte against saturated fatty acid stress.

Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a dose-dependent negative impact on oocyte developmental competence, while monounsaturated free fatty acids appear less harmful. Cumulus cells seem to protect the oocyte against free fatty acids, and the aim of this study was to determine the mechanism behind this protection In particular, the role of the enzyme stearoyl-CoA desaturase (SCD) that converts saturated into monounsaturated fatty acids was investigated. SCD gene and protein were abundantly expressed in cumulus cells, but expression was low in oocytes. The level of SCD protein expression in cumulus cells did not change when COCs were exposed to saturated stearic acid during maturation. SCD inhibition in the presence of stearic acid significantly reduced the developmental competence of oocytes and increased the incidence of apoptosis in cumulus cells. The esterified oleic/stearic acid ratio of the neutral lipid fraction in cumulus cells decreased in the presence of SCD inhibitors when COCs were exposed to saturated free fatty acids during maturation, indicating the SCD-specific conversion of saturated fatty acids under noninhibiting conditions. The observation that cumulus cells can desaturate the potentially toxic stearic acid into oleic acid via SCD activity provides a mechanistic insight into how the cumulus cells protect the oocyte against toxicity by saturated fatty acid.

Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells.

Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. We previously observed that the levels of triacylglycerol (TAG) species containing long polyunsaturated fatty acids (PUFAs) are increased in in vitro activated HSCs. Here we investigated the cause and consequences of the rise in PUFA-TAGs by profiling enzymes involved in PUFA incorporation. We report that acyl CoA synthetase (ACSL) type 4, which has a preference for PUFAs, is the only upregulated ACSL family member in activated HSCs. Inhibition of the activity of ACSL4 by siRNA-mediated knockdown or addition of rosiglitazone specifically inhibited the incorporation of deuterated arachidonic acid (AA-d8) into TAG in HSCs. In agreement with this, ACSL4 was found to be partially localized around lipid droplets (LDs) in HSCs. Inhibition of ACSL4 also prevented the large increase in PUFA-TAGs in HSCs upon activation and to a lesser extent the increase of arachidonate-containing phosphatidylcholine species. Inhibition of ACSL4 by rosiglitazone was associated with an inhibition of HSC activation and prostaglandin secretion. Our combined data show that upregulation of ACSL4 is responsible for the increase in PUFA-TAG species during activation of HSCs, which may serve to protect cells against a shortage of PUFAs required for eicosanoid secretion.

Cholesterol shuttling is important for RNA replication of coxsackievirus B3 and encephalomyocarditis virus.

Picornaviruses are a family of positive-strand RNA viruses that includes important human and animal pathogens. Upon infection, picornaviruses induce an extensive remodelling of host cell membranes into replication organelles (ROs), which is critical for replication. Membrane lipids and lipid remodelling processes are at the base of RO formation, yet their involvement remains largely obscure. Recently, phosphatidylinositol-4-phosphate was the first lipid discovered to be important for the replication of a number of picornaviruses. Here, we investigate the role of the lipid cholesterol in picornavirus replication. We show that two picornaviruses from distinct genera that rely on different host factors for replication, namely the enterovirus coxsackievirus B3 (CVB3) and the cardiovirus encephalomyocarditis virus (EMCV), both recruited cholesterol to their ROs. Although CVB3 and EMCV both required cholesterol for efficient genome replication, the viruses appeared to rely on different cellular cholesterol pools. Treatments that altered the distribution of endosomal cholesterol inhibited replication of both CVB3 and EMCV, showing the importance of endosomal cholesterol shuttling for the replication of these viruses. Summarizing, we here demonstrate the importance of cholesterol homeostasis for efficient replication of CVB3 and EMCV.

FcRγ-chain ITAM signaling is critically required for cross-presentation of soluble antibody-antigen complexes by dendritic cells.

The uptake of Ag-Ab immune complexes (IC) after the ligation of activating FcγR on dendritic cells (DC) leads to 100 times more efficient Ag presentation than the uptake of free Ags. FcγRs were reported to facilitate IC uptake and simultaneously induce cellular activation that drives DC maturation and mediates efficient T cell activation. Activating FcγRs elicit intracellular signaling via the ITAM domain of the associated FcRγ-chain. Studies with FcRγ-chain knockout (FcRγ(-/-)) mice reported FcRγ-chain ITAM signaling to be responsible for enhancing both IC uptake and DC maturation. However, FcRγ-chain is also required for surface expression of activating FcγRs, hampering the dissection of ITAM-dependent and independent FcγR functions in FcRγ(-/-) DCs. In this work, we studied the role of FcRγ-chain ITAM signaling using DCs from NOTAM mice that express normal surface levels of activating FcγR, but lack functional ITAM signaling. IC uptake by bone marrow-derived NOTAM DCs was reduced compared with wild-type DCs, but was not completely absent as in FcRγ(-/-) DCs. In NOTAM DCs, despite the uptake of ICs, both MHC class I and MHC class II Ag presentation was completely abrogated similar to FcRγ(-/-) DCs. Secretion of cytokines, upregulation of costimulatory molecules, and Ag degradation were abrogated in NOTAM DCs in response to FcγR ligation. Cross-presentation using splenic NOTAM DCs and prolonged incubation with OVA-IC was also abrogated. Interestingly, in this setup, proliferation of CD4(+) OT-II cells was induced by NOTAM DCs. We conclude that FcRγ-chain ITAM signaling facilitates IC uptake and is essentially required for cross-presentation, but not for MHC class II Ag presentation.

The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos.

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.

The cumulus cell layer protects the bovine maturing oocyte against fatty acid-induced lipotoxicity.

Mobilization of fatty acids from adipose tissue during metabolic stress increases the amount of free fatty acids in blood and follicular fluid and is associated with impaired female fertility. In a previous report, we described the effects of the three predominant fatty acids in follicular fluid (saturated palmitate and stearate and unsaturated oleate) on oocyte maturation and quality. In the current study, the effects of elevated fatty acid levels on cumulus cells were investigated. In a dose-dependent manner, the three fatty acids induced lipid storage in cumulus cells accompanied by an enhanced immune labeling of perilipin-2, a marker for lipid droplets. Lipidomic analysis confirmed incorporation of the administered fatty acids into triglyceride, resulting in a 3- to 6-fold increase of triglyceride content. In addition, palmitate selectively induced ceramide formation, which has been implicated in apoptosis. Indeed, of the three fatty acids tested, palmitate induced reactive oxygen species formation, caspase 3 activation, and mitochondria deterioration, leading to degeneration of the cumulus cell layers. This effect could be mimicked by addition of the ceramide-C2 analog and could be inhibited by the ceramide synthase inhibitor fumonisin-B1. Interfering with the intactness of the cumulus cell layers, either by mechanical force or by palmitate treatment, resulted in enhanced uptake of lipids in the oocyte and increased radical formation. Our results show that cumulus cells act as a barrier, protecting oocytes from in vitro induced lipotoxic effects. We suggest that this protective function of the cumulus cell layers is important for the developmental competence of the oocyte. The relevance of our findings for assisted reproduction technologies is discussed.

Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture.

Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV.

Endosomal sorting of MHC class II determines antigen presentation by dendritic cells.

Dendritic cells (DCs) initiate primary immune responses by presenting pathogen-derived antigens in association with major histocompatibility Class II molecules (MHC II) to T cells. In DCs, MHC II is constitutively synthesized and loaded at endosomes with peptides from hydrolyzed endogenous proteins or exogenously acquired antigens. Whether peptide loaded MHC II (MHC II-p) is subsequently recruited to and stably expressed at the plasma membrane or degraded in lysosomes is determined by the status of the DC. In immature DCs, MHC II-p is ubiquitinated after peptide loading, driving its sorting to the luminal vesicles of multivesicular bodies. These luminal vesicles, and the MHC II-p they carry, are delivered to lysosomes for degradation. MHC II-p is inefficiently ubiquitinated in DCs that are activated by pathogens or inflammatory stimuli, thus allowing its transfer to and stable expression at the plasma membrane.

Spermatozoa recruit prostasomes in response to capacitation induction.

Seminal plasma contains various types of extracellular vesicles, including 'prostasomes'. Prostasomes are small vesicles secreted by prostatic epithelial cells that can be recruited by and fuse with sperm cells in response of progesterone that is released by oocyte surrounding cumulus cells. This delivers Ca(2+) signaling tools that allow the sperm cell to gain hypermotility and undergo the acrosome reaction. Conditions for binding of prostasomes to sperm cells are however unclear. We found that classically used prostasome markers are in fact heterogeneously expressed on distinct populations of small and large vesicles in seminal plasma. To study interactions between prostasomes and spermatozoa we used the stallion as a model organism. A homogeneous population of ~60nm prostasomes was first separated from larger vesicles and labeled with biotin. Binding of biotinylated prostasomes to individual live spermatozoa was then monitored by flow cytometry. Contrary to assumptions in the literature, we found that such highly purified prostasomes bound to live sperm only after capacitation had been initiated, and specifically at pH ≥7.5. Using fluorescence microscopy, we observed that prostasomes bound primarily to the head of live sperm. We propose that in vivo, prostasomes may bind to sperm cells in the uterus, to be carried in association with sperm cells into oviduct and to fuse with the sperm cell only during the final approach of the oocyte. This article is part of a Special Issue entitled: An Updated Secretome.

The paracrine feedback loop between vitamin D3 (1,25(OH)2D3) and PTHrP in prehypertrophic chondrocytes.

The endocrine feedback loop between vitamin D3(1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10(-8) M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration.

Fungicidal mechanisms of cathelicidins LL-37 and CATH-2 revealed by live-cell imaging.

Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptides kill C. albicans rapidly. CATH-2 is the most active peptide and kills C. albicans within 5 min. Both cathelicidins induce cell membrane permeabilization and simultaneous vacuolar expansion. Minimal fungicidal concentrations (MFC) are in the same order of magnitude for all three peptides, but the mechanisms of antifungal activity are very different. The activity of cathelicidins is independent of the energy status of the fungal cell, unlike Hst5 activity. Live-cell imaging using fluorescently labeled peptides showed that both CATH-2 and LL-37 quickly localize to the C. albicans cell membrane, while Hst5 was mainly directed to the fungal vacuole. Small amounts of cathelicidins internalize at sub-MFCs, suggesting that intracellular activities of the peptide could contribute to the antifungal activity. Analysis by flow cytometry indicated that CATH-2 significantly decreases C. albicans cell size. Finally, electron microscopy showed that CATH-2 affects the integrity of the cell membrane and nuclear envelope. It is concluded that the general mechanisms of action of both cathelicidins are partially similar (but very different from that of Hst5). CATH-2 has unique features and possesses antifungal potential superior to that of LL-37.

Extracellular vesicles from seminal plasma interact with T cells in vitro and drive their differentiation into regulatory T-cells.

Seminal plasma induces immune tolerance towards paternal allogenic antigens within the female reproductive tract and during foetal development. Recent evidence suggests a role for extracellular vesicles in seminal plasma (spEVs). We isolated spEVs from seminal plasma that was donated by vasectomized men, thereby excluding any contributions from the testis or epididymis. Previous analysis demonstrated that such isolated spEVs originate mainly from the prostate. Here we observed that when isolated fluorescently labelled spEVs were mixed with peripheral blood mononuclear cells, they were endocytosed predominantly by monocytes, and to a lesser extent also by T-cells. In a mixed lymphocyte reaction, T-cell proliferation was inhibited by spEVs. A direct effect of spEVs on T-cells was demonstrated when isolated T cells were activated by anti-CD3/CD28 coated beads. Again, spEVs interfered with T cell proliferation, as well as with the expression of CD25 and the release of IFN-γ, TNF, and IL-2. Moreover, spEVs stimulated the expression of Foxp3 and IL-10 by CD4+CD25+CD127- T cells, indicating differentiation into regulatory T-cells (Tregs). Prior treatment of spEVs with proteinase K revoked their effects on T-cells, indicating a requirement for surface-exposed spEV proteins. The adenosine A2A receptor-specific antagonist CPI-444 also reduced effects of spEVs on T-cells, consistent with the notion that the development of Tregs and their immune suppressive functions are under the influence of adenosine-A2A receptor signalling. We found that adenosine is highly enriched in spEVs and propose that spEVs are targeted to and endocytosed by T-cells, after which they may release their adenosine content into the lumen of endosomes, thus allowing endosome-localized A2A receptor signalling in spEVs targeted T-cells. Collectively, these data support the idea that spEVs can prime T cells directly for differentiation into Tregs.

The activation of the adaptor protein STING depends on its interactions with the phospholipid PI4P.

Activation of the endoplasmic reticulum (ER)-resident adaptor protein STING, a component of a cytosolic DNA-sensing pathway, induces the transcription of genes encoding type I interferons (IFNs) and other proinflammatory factors. Because STING is activated at the Golgi apparatus, control of the localization and activation of STING is important in stimulating antiviral and antitumor immune responses. Through a genome-wide CRISPR interference screen, we found that STING activation required the Golgi-resident protein ACBD3, which promotes the generation of phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network, as well as other PI4P-associated proteins. Appropriate localization and activation of STING at the Golgi apparatus required ACBD3 and the PI4P-generating kinase PI4KB. In contrast, STING activation was enhanced when the lipid-shuttling protein OSBP, which removes PI4P from the Golgi apparatus, was inhibited by the US Food and Drug Administration-approved antifungal itraconazole. The increase in the abundance of STING-activating phospholipids at the trans-Golgi network resulted in the increased production of IFN-ß and other cytokines in THP-1 cells. Furthermore, a mutant STING that could not bind to PI4P failed to traffic from the ER to the Golgi apparatus in response to a STING agonist, whereas forced relocalization of STING to PI4P-enriched areas elicited STING activation in the absence of stimulation with a STING agonist. Thus, PI4P is critical for STING activation, and manipulating PI4P abundance may therapeutically modulate STING-dependent immune responses.

Endosomally stored MHC class II does not contribute to antigen presentation by dendritic cells at inflammatory conditions.

Major histocompatibility complex (MHC) class II (MHCII) is constitutively expressed by immature dendritic cells (DC), but has a short half-life as a consequence of its transport to and degradation in lysosomes. For its transfer to lysosomes, MHCII is actively sorted to the intraluminal vesicles (ILV) of multivesicular bodies (MVB), a process driven by its ubiquitination. ILV have, besides their role as an intermediate compartment in lysosomal transfer, also been proposed to function as a site for MHCII antigen loading and temporal storage. In that scenario, DC would recruit antigen-loaded MHCII to the cell surface in response to a maturation stimulus by allowing ILV to fuse back with the MVB delimiting membrane. Other studies, however, explained the increase in cell surface expression during DC maturation by transient upregulation of MHCII synthesis and reduced sorting of newly synthesized MHCII to lysosomes. Here, we have characterized the relative contributions from the biosynthetic and endocytic pathways and found that the vast majority of antigen-loaded MHCII that is stably expressed at the plasma membrane by mature DC is synthesized after exposure to inflammatory stimuli. Pre-existing endosomal MHCII contributed only when it was not yet sorted to ILV at the moment of DC activation. Together with previous records, our current data are consistent with a model in which passage of MHCII through ILV is not required for antigen loading in maturing DC and in which sorting to ILV in immature DC provides a one-way ticket for lysosomal degradation.

Trafficking of MHC class II in dendritic cells is dependent on but not regulated by degradation of its associated invariant chain.

In dendritic cells (DC), newly synthesized MHCII is directed to endosomes by its associated invariant chain (Ii). Here, Ii is degraded after which MHCII is loaded with peptides. In immature DC, ubiquitination of peptide-loaded MHCII drives its sorting to lysosomes for degradation. Ubiquitination of MHCII is strongly reduced in response to inflammatory stimuli, resulting in increased expression of MHCII at the plasma membrane. Whether surface exposure of MHCII is also regulated during DC maturation by changing the rate of Ii degradation remained unresolved by conflicting results in the literature. We here pinpoint experimental problems that have contributed to these controversies and demonstrate that immature and mature DC degrade Ii equally efficient at proper culture conditions. Only when DC were cultured in glutamine containing media, endosome acidification and Ii degradation were restricted in immature DC and enhanced in response to lipopolysaccharide (LPS). These effects are caused by ammonia, a glutamine decomposition product. This artificial behavior could be prevented by culturing DC in media containing a stable dipeptide as glutamine source. We conclude that Ii degradation is a prerequisite for but not a rate limiting step in MHCII processing.