Ageing of Plasmodium falciparum malaria sporozoites alters their motility, infectivity and reduces immune activation in vitro.

BACKGROUND: Sporozoites (SPZ), the infective form of Plasmodium falciparum malaria, can be inoculated into the human host skin by Anopheline mosquitoes. These SPZ migrate at approximately 1 µm/s to find a blood vessel and travel to the liver where they infect hepatocytes and multiply. In the skin they are still low in number (50-100 SPZ) and vulnerable to immune attack by antibodies and skin macrophages. This is why whole SPZ and SPZ proteins are used as the basis for most malaria vaccines currently deployed and undergoing late clinical testing. Mosquitoes typically inoculate SPZ into a human host between 14 and 25 days after their previous infective blood meal. However, it is unknown whether residing time within the mosquito affects SPZ condition, infectivity or immunogenicity. This study aimed to unravel how the age of P. falciparum SPZ in salivary glands (14, 17, or 20 days post blood meal) affects their infectivity and the ensuing immune responses. METHODS: SPZ numbers, viability by live/dead staining, motility using dedicated sporozoite motility orienting and organizing tool software (SMOOT), and infectivity of HC-04.j7 liver cells at 14, 17 and 20 days after mosquito feeding have been investigated. In vitro co-culture assays with SPZ stimulated monocyte-derived macrophages (MoMɸ) and CD8+ T-cells, analysed by flow cytometry, were used to investigate immune responses. RESULTS: SPZ age did not result in different SPZ numbers or viability. However, a markedly different motility pattern, whereby motility decreased from 89% at day 14 to 80% at day 17 and 71% at day 20 was observed (p ≤ 0.0001). Similarly, infectivity of day 20 SPZ dropped to ~ 50% compared with day 14 SPZ (p = 0.004). MoMɸ were better able to take up day 14 SPZ than day 20 SPZ (from 7.6% to 4.1%, p = 0.03) and displayed an increased expression of pro-inflammatory CD80, IL-6 (p = 0.005), regulatory markers PDL1 (p = 0.02), IL-10 (p = 0.009) and cytokines upon phagocytosis of younger SPZ. Interestingly, co-culture of these cells with CD8+ T-cells revealed a decreased expression of activation marker CD137 and cytokine IFNγ compared to their day 20 counterparts. These findings suggest that older (day 17-20) P. falciparum SPZ are less infectious and have decreased immune regulatory potential. CONCLUSION: Overall, this data is a first step in enhancing the understanding of how mosquito residing time affects P. falciparum SPZ and could impact the understanding of the P. falciparum infectious reservoir and the potency of whole SPZ vaccines.

IgG Subclasses Shape Cytokine Responses by Human Myeloid Immune Cells through Differential Metabolic Reprogramming.

IgG Abs are crucial for various immune functions, including neutralization, phagocytosis, and Ab-dependent cellular cytotoxicity. In this study, we identified another function of IgG by showing that IgG immune complexes elicit distinct cytokine profiles by human myeloid immune cells, which are dependent on FcγR activation by the different IgG subclasses. Using monoclonal IgG subclasses with identical Ag specificity, our data demonstrate that the production of Th17-inducing cytokines, such as TNF, IL-1ß, and IL-23, is particularly dependent on IgG2, whereas type I IFN responses are controlled by IgG3, and IgG1 is able to regulate both. In addition, we identified that subclass-specific cytokine production is orchestrated at the posttranscriptional level through distinct glycolytic reprogramming of human myeloid immune cells. Combined, these data identify that IgG subclasses provide pathogen- and cell type-specific immunity through differential metabolic reprogramming by FcγRs. These findings may be relevant for future design of Ab-related therapies in the context of infectious diseases, chronic inflammation, and cancer.

Dectin-1/2-induced autocrine PGE2 signaling licenses dendritic cells to prime Th2 responses.

The molecular mechanisms through which dendritic cells (DCs) prime T helper 2 (Th2) responses, including those elicited by parasitic helminths, remain incompletely understood. Here, we report that soluble egg antigen (SEA) from Schistosoma mansoni, which is well known to drive potent Th2 responses, triggers DCs to produce prostaglandin E2 (PGE2), which subsequently-in an autocrine manner-induces OX40 ligand (OX40L) expression to license these DCs to drive Th2 responses. Mechanistically, SEA was found to promote PGE2 synthesis through Dectin-1 and Dectin-2, and via a downstream signaling cascade involving spleen tyrosine kinase (Syk), extracellular signal-regulated kinase (ERK), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase 1 and 2 (COX-1 and COX-2). In addition, this pathway was activated independently of the actions of omega-1 (ω-1), a previously described Th2-priming glycoprotein present in SEA. These findings were supported by in vivo murine data showing that ω-1-independent Th2 priming by SEA was mediated by Dectin-2 and Syk signaling in DCs. Finally, we found that Dectin-2-/-, and to a lesser extent Dectin-1-/- mice, displayed impaired Th2 responses and reduced egg-driven granuloma formation following S. mansoni infection, highlighting the physiological importance of this pathway in Th2 polarization during a helminth infection. In summary, we identified a novel pathway in DCs involving Dectin-1/2-Syk-PGE2-OX40L through which Th2 immune responses are induced.

C-Reactive Protein Promotes Inflammation through FcγR-Induced Glycolytic Reprogramming of Human Macrophages.

C-reactive protein (CRP) is an acute-phase protein produced in high quantities by the liver in response to infection and during chronic inflammatory disorders. Although CRP is known to facilitate the clearance of cell debris and bacteria by phagocytic cells, the role of CRP in additional immunological functions is less clear. This study shows that complexed CRP (phosphocholine [PC]:CRP) (formed by binding of CRP to PC moieties), but not soluble CRP, synergized with specific TLRs to posttranscriptionally amplify TNF, IL-1ß, and IL-23 production by human inflammatory macrophages. We identified FcγRI and IIa as the main receptors responsible for initiating PC:CRP-induced inflammation. In addition, we identified the underlying mechanism, which depended on signaling through kinases Syk, PI3K, and AKT2, as well as glycolytic reprogramming. These data indicate that in humans, CRP is not only a marker but also a driver of inflammation by human macrophages. Therefore, although providing host defense against bacteria, PC:CRP-induced inflammation may also exacerbate pathology in the context of disorders such as atherosclerosis.

Rapamycin and omega-1: mTOR-dependent and -independent Th2 skewing by human dendritic cells.

Recent reports have attributed an immunoregulatory role to the mammalian target of rapamycin (mTOR), a key serine/threonine protein kinase integrating input from growth factors and nutrients to promote cell growth and differentiation. In the present study, we investigated the role of the mTOR pathway in Th2 induction by human monocyte-derived dendritic cells (moDCs). Using a co-culture system of human lipopolysaccharide (LPS)-matured moDCs and allogeneic naive CD4(+) T cells, we show that inhibition of mTOR by the immunosuppressive drug rapamycin reduced moDC maturation and promoted Th2 skewing. Next, we investigated whether antigens from helminth parasites, the strongest natural inducers of Th2 responses, modulate moDCs via the mTOR pathway. In contrast to rapamycin, neither Schistosoma mansoni-soluble egg antigens (SEA) nor its major immunomodulatory component omega-1 affected the phosphorylation of S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), downstream targets of mTORC1. Finally, we found that the effects of rapamycin and SEA/omega-1 on Th2 skewing were additive, suggesting two distinct underlying molecular mechanisms. We conclude that conditioning human moDCs to skew immune responses towards Th2 can be achieved via an mTOR-dependent and -independent pathway triggered by rapamycin and helminth antigens, respectively.

mTORC1 signaling in antigen-presenting cells of the skin restrains CD8+ T cell priming.

How mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of cellular metabolism, affects dendritic cell (DC) metabolism and T cell-priming capacity has primarily been investigated in vitro, but how mTORC1 regulates this in vivo remains poorly defined. Here, using mice deficient for mTORC1 component raptor in DCs, we find that loss of mTORC1 negatively affects glycolytic and fatty acid metabolism and maturation of conventional DCs, particularly cDC1s. Nonetheless, antigen-specific CD8+ T cell responses to infection are not compromised and are even enhanced following skin immunization. This is associated with increased activation of Langerhans cells and a subpopulation of EpCAM-expressing cDC1s, of which the latter show an increased physical interaction with CD8+ T cells in situ. Together, this work reveals that mTORC1 limits CD8+ T cell priming in vivo by differentially orchestrating the metabolism and immunogenicity of distinct antigen-presenting cell subsets, which may have implications for clinical use of mTOR inhibitors.

Treatment with HIV-Protease Inhibitor Nelfinavir Identifies Membrane Lipid Composition and Fluidity as a Therapeutic Target in Advanced Multiple Myeloma.

The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)-refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9-based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI-refractory multiple myeloma. SIGNIFICANCE: Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.

DC-SIGN mediated internalisation of glycosylated extracellular vesicles from Schistosoma mansoni increases activation of monocyte-derived dendritic cells.

Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte-derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC-SIGN (CD209). Blocking DC-SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N-glycans with terminal Galß1-4(Fucα1-3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid-linked glycans, including LewisX, a known ligand for DC-SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD-L1. Furthermore, schistosomula EVs increased expression of IL-12 and IL-10 by moDCs, which was partly dependent on the interaction with DC-SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC-SIGN and EV-associated glycoconjugates in parasite-induced immune modulation.

LKB1 expressed in dendritic cells governs the development and expansion of thymus-derived regulatory T cells.

Liver Kinase B1 (LKB1) plays a key role in cellular metabolism by controlling AMPK activation. However, its function in dendritic cell (DC) biology has not been addressed. Here, we find that LKB1 functions as a critical brake on DC immunogenicity, and when lost, leads to reduced mitochondrial fitness and increased maturation, migration, and T cell priming of peripheral DCs. Concurrently, loss of LKB1 in DCs enhances their capacity to promote output of regulatory T cells (Tregs) from the thymus, which dominates the outcome of peripheral immune responses, as suggested by increased resistance to asthma and higher susceptibility to cancer in CD11cΔLKB1 mice. Mechanistically, we find that loss of LKB1 specifically primes thymic CD11b+ DCs to facilitate thymic Treg development and expansion, which is independent from AMPK signalling, but dependent on mTOR and enhanced phospholipase C ß1-driven CD86 expression. Together, our results identify LKB1 as a critical regulator of DC-driven effector T cell and Treg responses both in the periphery and the thymus.

Butyrate Conditions Human Dendritic Cells to Prime Type 1 Regulatory T Cells via both Histone Deacetylase Inhibition and G Protein-Coupled Receptor 109A Signaling.

Recently, it has become clear that short-chain fatty acids (SCFAs), and in particular butyrate, have anti-inflammatory properties. Murine studies have shown that butyrate can promote regulatory T cells via the induction of tolerogenic dendritic cells (DCs). However, the effects of SCFAs on human DCs and how they affect their capacity to prime and polarize T-cell responses have not been addressed. Here, we report that butyrate suppresses LPS-induced maturation and metabolic reprogramming of human monocyte-derived DCs (moDCs) and conditions them to polarize naive CD4+ T cells toward IL-10-producing type 1 regulatory T cells (Tr1). This effect was dependent on induction of the retinoic acid-producing enzyme retinaldehyde dehydrogenase 1 in DCs. The induction of retinaldehyde dehydrogenase activity and Tr1 cell differentiation by butyrate was dependent on simultaneous inhibition of histone deacetylases and signaling through G protein-coupled receptor 109A. Taken together, we reveal that butyrate is a potent inducer of tolerogenic human DCs, thereby shedding new light on the cellular and molecular mechanisms through which SCFAs can exert their immunomodulatory effects in humans.

Analysis of TLR-Induced Metabolic Changes in Dendritic Cells Using the Seahorse XF(e)96 Extracellular Flux Analyzer.

Engagement of Toll-like receptors (TLRs) on dendritic cells (DCs) triggers the expression of a large set of genes involved in DC activation and maturation, which allow them to act efficiently as antigen-presenting cells. Recently, it has become clear that TLR signalling in DCs also results in dramatic metabolic changes that are integral to their changed biology. Here, we describe a detailed protocol on how DC metabolism can be studied after TLR stimulation using the 96-well format Extracellular Flux (XF(e)96) Analyzer from Seahorse Bioscience, a machine that allows one to simultaneously assess rates of oxidative phosphorylation and glycolysis in real-time, in live cells and in a high-throughput manner.

Differences in innate cytokine responses between European and African children.

Although differences in immunological responses between populations have been found in terms of vaccine efficacy, immune responses to infections and prevalence of chronic inflammatory diseases, the mechanisms responsible for these differences are not well understood. Therefore, innate cytokine responses mediated by various classes of pattern-recognition receptors including Toll-like receptors (TLR), C-type lectin receptors (CLRs) and nucleotide-binding oligomerisation domain-like receptors (NLRs) were compared between Dutch (European), semi-urban and rural Gabonese (African) children. Whole blood was stimulated for 24 hours and the pro-inflammatory tumor necrosis factor (TNF) and the anti-inflammatory/regulatory interleukin-10 (IL-10) cytokines in culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Gabonese children had a lower pro-inflammatory response to poly(I:C) (TLR3 ligand), but a higher pro-inflammatory response to FSL-1 (TLR2/6 ligand), Pam3 (TLR2/1 ligand) and LPS (TLR4 ligand) compared to Dutch children. Anti-inflammatory responses to Pam3 were also higher in Gabonese children. Non-TLR ligands did not induce substantial cytokine production on their own. Interaction between various TLR and non-TLR receptors was further assessed, but no differences were found between the three populations. In conclusion, using a field applicable assay, significant differences were observed in cytokine responses between European and African children to TLR ligands, but not to non-TLR ligands.

Schistosome-derived omega-1 drives Th2 polarization by suppressing protein synthesis following internalization by the mannose receptor.

Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming.

Omega-1, a glycoprotein secreted by Schistosoma mansoni eggs, drives Th2 responses.

Soluble egg antigens of the parasitic helminth Schistosoma mansoni (S. mansoni egg antigen [SEA]) induce strong Th2 responses both in vitro and in vivo. However, the specific molecules that prime the development of Th2 responses have not been identified. We report that omega-1, a glycoprotein which is secreted from S. mansoni eggs and present in SEA, is capable of conditioning human monocyte-derived dendritic cells in vitro to drive T helper 2 (Th2) polarization with similar characteristics as whole SEA. Furthermore, using IL-4 dual reporter mice, we show that both natural and recombinant omega-1 alone are sufficient to generate Th2 responses in vivo, even in the absence of IL-4R signaling. Finally, omega-1-depleted SEA displays an impaired capacity for Th2 priming in vitro, but not in vivo, suggesting the existence of additional factors within SEA that can compensate for the omega-1-mediated effects. Collectively, we identify omega-1, a single component of SEA, as a potent inducer of Th2 responses.