Arifa Ozir-Fazalalikhan

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.

Schistosome parasites are known to modulate host immune responses, which is achieved in part through the release of excretory/secretory (ES) products, including extracellular vesicles (EVs). During chronic schistosomiasis, increased regulatory responses are found, which include enhanced IL‐10 production by B (Breg) cells. ES products from schistosome eggs are able to induce IL‐10 production by B cells. However, since infection with male worms only (without egg production) also promotes IL‐10 producing B cells, we here studied the stimulatory effects of adult worm ES and EVs on murine and human B cells. Worm ES increased IL‐10 release by mouse splenic B cells; this activity was concentrated in defined size‐separated fractions of adult worm ES. Interestingly, mass spectrometry of the fractions that induced the highest IL‐10 response revealed an enrichment of EV‐associated proteins. Indeed, highly purified adult worm EVs could interact with mouse splenic B cells, visualised by binding of a schistosome‐specific tetraspanin (TSP2) targeting antibody. Furthermore, purified adult worm EVs induced IL‐10 release in both mouse splenic and human peripheral blood B cells, suggesting that adult worm EVs can play a role in immune regulatory processes within their host.

Obesity-associated metabolic inflammation drives the development of insulin resistance and type 2 diabetes, notably through modulating innate and adaptive immune cells in metabolic organs. The nutrient sensor liver kinase B1 (LKB1) has recently been shown to control cellular metabolism and T cell priming functions of DCs. Here, we report that hepatic DCs from high-fat diet–fed (HFD-fed) obese mice display increased LKB1 phosphorylation and that LKB1 deficiency in DCs (CD11c ^ΔLKB1 ) worsened HFD-driven hepatic steatosis and impaired glucose homeostasis. Loss of LKB1 in DCs was associated with increased expression of Th17-polarizing cytokines and accumulation of hepatic IL-17A ⁺ Th cells in HFD-fed mice. Importantly, IL-17A neutralization rescued metabolic perturbations in HFD-fed CD11c ^ΔLKB1 mice. Mechanistically, deficiency of the canonical LKB1 target AMPK in HFD-fed CD11c ^ΔAMPKα1 mice recapitulated neither the hepatic Th17 phenotype nor the disrupted metabolic homeostasis, suggesting the involvement of other and/or additional LKB1 downstream effectors. We indeed provide evidence that the control of Th17 responses by DCs via LKB1 is actually dependent on both AMPKα1 and salt-inducible kinase signaling. Altogether, our data reveal a key role for LKB1 signaling in DCs in protection against obesity-induced metabolic dysfunctions by limiting hepatic Th17 responses.

Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) can induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega-1 (ω1), one of the major S. mansoni immunomodulatory glycoproteins, on metabolic homeostasis. Male C57Bl6/J mice were fed a high-fat diet for 12 weeks followed by bi-weekly injection of SEA, ω1 or vehicle for 4 additional weeks. Whole-body metabolic homeostasis and energy expenditure were assessed by glucose/insulin tolerance tests and indirect calorimetry, respectively. Tissue-specific immune cell phenotypes were determined by flow cytometry. We show that treatment of obese mice with plant-produced recombinant ω1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass and improved systemic insulin sensitivity and glucose tolerance in a time-and dose-dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils and alternatively-activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of ω1 were still observed in obese STAT6-deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that ω1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole-body energy expenditure, an effect also occurring in leptin receptor-deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein ω1 can induce type 2 immunity, it improves whole-body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6-independent mechanism. Author summary: The obesity-induced chronic low-grade inflammation, notably in adipose tissue, contributes to insulin resistance and increased risk of type 2 diabetes. We have previously shown that infection with parasitic helminth worms was associated with protection against obesity-related metabolic dysfunctions both in mice and humans. We have also reported that treatment of obese mice with an extract of Schistosoma mansoni eggs (SEA) improves insulin sensitivity and glucose tolerance, a beneficial effect that was associated with a helminth-specific type 2 immune response in metabolic organs. Here, we studied the effects of omega-1 (ω1), a single immunomodulatory molecule from SEA, on metabolic health in obese mice, and investigated the role of the host immune response elicited. We found that ω1 induced a helminth-characteristic type 2 immune response in adipose tissue and improved both insulin sensitivity and glucose tolerance in obese mice. Yet, in contrast to SEA, ω1’s immunomodulatory properties were dispensable for its metabolic effects. Instead, we show that ω1 inhibited food intake, a feature accounting for most of the improvements in metabolic health. Together, our findings indicate that helminth molecules may improve metabolic health through multiple distinct mechanisms, and further characterization of such molecules could lead to new therapeutic strategies to combat obesity.

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.

Schistosomiasis treatment relies on the use of a single drug, praziquantel, which is insufficient to control transmission in highly endemic areas¹. Novel medicines and vaccines are urgently needed^2,3. An experimental human model for schistosomiasis could accelerate the development of these products. We performed a dose-escalating clinical safety trial in 17 volunteers with male Schistosoma mansoni cercariae, which do not produce eggs (clinicaltrials.gov NCT02755324), at the Leiden University Medical Center, the Netherlands. The primary endpoints were adverse events and infectivity. We found a dose-related increase in adverse events related to acute schistosomiasis syndrome, which occurred in 9 of 17 volunteers. Overall, 5 volunteers (all 3 of the high dose group and 2 of 11 of the medium dose group) reported severe adverse events. Worm-derived circulating anodic antigen, the biomarker of the primary infection endpoint, peaked in 82% of volunteers at 3–10 weeks following exposure. All volunteers showed IgM and IgG1 seroconversion and worm-specific cytokine production by CD4⁺ T cells. All volunteers were cured with praziquantel provided at 12 weeks after exposure. Infection with 20 Schistosoma mansoni cercariae led to severe adverse events in 18% of volunteers and high infection rates. This infection model paves the way for fast-track product development for treatment and prevention of schistosomiasis.

The helminth Schistosoma mansoni (S. mansoni) induces a network of regulatory immune cells, including interleukin (IL)‐10‐producing regulatory B cells (Bregs). However, the signals required for the development and activation of Bregs are not well characterized. Recent reports suggest that helminths induce type I interferons (IFN‐I), and that IFN‐I drive the development of Bregs in humans. We therefore assessed the role of IFN‐I in the induction of Bregs by S. mansoni. Mice chronically infected with S. mansoni or i.v. injected with S. mansoni soluble egg antigen (SEA) developed a systemic IFN‐I signature. Recombinant IFN‐α enhanced IL‐10 production by Bregs stimulated with S. mansoni SEA in vitro, while not activating Bregs by itself. IFN‐I signaling also supported ex vivo IL‐10 production by SEA‐primed Bregs but was dispensable for activation of S. mansoni egg‐induced Bregs in vivo. These data indicate that although IFN‐I can serve as a coactivator for Breg IL‐10 production, they are unlikely to participate in the development of Bregs in response to S. mansoni eggs.

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.