Alwin J. van der Ham

Intestinal dendritic cells (DCs) play a central role in maintaining gut tolerance through priming of peripheral regulatory T cells (pT _reg cells). DC tolerogenicity has been linked to catabolic metabolism, but the role of AMP-activated kinase (AMPK), a key regulator of catabolic metabolism, in regulating intestinal tolerance remains unclear. We found high AMPK activation in intestinal DCs, and loss of AMPKα1 in CD11c-expressing cells (CD11c ^ΔAMPKα1 ) led to reduced frequencies of intestinal RALDH ⁺ CD103 ⁺ cDC2s. This was associated with reduced pT _reg cell induction and consequently increased type 2 immunity in models of intestinal helminth infection. Correspondingly, CD103 ⁺ cDC2s from helminth-infected CD11c ^ΔAMPKα1 mice failed to prime T _reg cells ex vivo. Similarly, T _reg cell induction by AMPK-deficient human retinoic acid (RA)–induced tolerogenic CD103 ⁺ DCs was compromised. Mechanistically, AMPK underpinned RA-driven tolerogenicity by promoting RALDH activity and TGF-β secretion in a FoxO3-dependent manner, independent from metabolic reprogramming. Our findings identify AMPK as a key regulator of intestinal DC-mediated tolerance and as a therapeutic target to counter intestinal inflammatory disease.

Dendritic cell (DC) activation and function are underpinned by profound changes in cellular metabolism. Several studies indicate that the ability of DCs to promote tolerance is dependent on catabolic metabolism. Yet the contribution of AMP-activated kinase (AMPK), a central energy sensor promoting catabolism, to DC tolerogenicity remains unknown. Here, we show that AMPK activation renders human monocyte-derived DCs tolerogenic as evidenced by an enhanced ability to drive differentiation of regulatory T cells, a process dependent on increased RALDH activity. This is accompanied by several metabolic changes, including increased breakdown of glycerophospholipids, enhanced mitochondrial fission–dependent fatty acid oxidation, and upregulated glucose catabolism. This metabolic rewiring is functionally important as we found interference with these metabolic processes to reduce to various degrees AMPK-induced RALDH activity as well as the tolerogenic capacity of moDCs. Altogether, our findings reveal a key role for AMPK signaling in shaping DC tolerogenicity and suggest AMPK as a target to direct DC-driven tolerogenic responses in therapeutic settings.

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-1β, 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.

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 Galβ1‐4(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.

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-1β, 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.

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 CD11c^ΔLKB1 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 β1-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.

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. T helper 2 (Th2) responses, which are initiated by dendritic cells (DCs), can cause allergic diseases, but they can also provide protection against metabolic disorders and parasitic helminth infections. As such, there is great interest in better understanding how their activity is induced and regulated by DCs. Parasitic helminths can potently induce Th2 responses. However, how helminths condition DCs for priming of Th2 responses remains incompletely understood. Here, we find that egg antigens from the parasitic helminth Schistosoma mansoni bind to pattern-recognition receptors (PRRs) Dectin-1 and Dectin-2 on DCs. This binding triggers a signaling cascade in DCs that results in synthesis of eicosanoid prostaglandin E2 (PGE₂). PGE₂ is sensed by the DCs themselves, resulting in expression of OX40 ligand (OX40L), which subsequently enables the DCs to promote Th2 differentiation. We show that this pathway is activated independently of omega-1 (ω-1), which is a glycoprotein secreted by the eggs and previously shown to condition DCs for priming of Th2 responses. Moreover, we demonstrate that this ω-1–independent pathway is crucial for Th2 induction and egg-driven immunopathology following S. mansoni infection in vivo. In summary, we identified a novel pathway in DCs involving Dectin-1/2–induced autocrine PGE₂ signaling through which Th2 responses are induced.

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 (Tregs) 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 has 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 towards IL-10-producing type 1 regulatory T cells (Tr1). This effect was dependent on induction of the retinoic acid-producing enzyme retinaldehyde dehydrogenase 1 (RALDH1) in DCs. The induction of RALDH activity and Tr1 cell differentiation by butyrate was dependent on simultaneous inhibition of histone deacetylases (HDACs) and signaling through G protein-coupled receptor (GPR)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.

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^e96) 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.

There is evidence for an interaction of the commensal flora and development of allergic disease. Epidemiological studies and studies in murine models suggest that infection with H. pylori protects against developing allergic airway disease. Treatment with a lysate of the bacteria was also able to reduce allergic airway disease (Engler et al. PNAS 2014). The effect was mediated by IL-10 producing DC, but in contrast to live infection did not depend on the induction of regulatory T cells (Treg). In our in vitro study the effect of lysate of H. pylori on human monocyte-derived (mo-)DC was evaluated. Immature DC exposed for 48 h to H. pylori lysate in presence or absence of LPS matured and strongly increased the release of the anti-inflammatory cytokine IL-10. DCs exposed to the lysate reduced polarization from naïve T cells to Th1 cells. Instead, T cells differentiated by the lysate-exposed DCs of a subgroup of donors secreted more IL-10 compared to T cells from control DC, suggesting the induction of Treg. These Treg were able to suppress proliferation of alloreactive memory T cells in vitro. Our results suggest that H. pylori lysate has strong IL-10 inducing capacities in human mo-DC, which can induce IL-10 secreting T cells with a suppressive function. Further studies are needed to assess if this approach can be utilized in humans to dampen unwanted immune responses.