Tregs protect against invariant NKT cell-mediated autoimmune colitis and hepatitis.

Immunomodulatory T cells play a pivotal role in protection against (auto)immune-mediated diseases that open perspectives for therapeutic modulation. However, how immune regulatory networks operate in vivo is less understood. To this end, we focused on FOXP3+CD4+CD25+ regulatory T cells (Tregs) and invariant natural killer T (iNKT) cells, two lymphocyte populations that independently regulate adaptive and innate immune responses. In vitro, a functional interplay between Tregs and iNKT cells has been described, but whether Tregs modulate the function and phenotype of iNKT cell subsets in vivo and whether this controls iNKT-mediated autoimmunity is unclear. Taking advantage of the conditional depletion of Tregs, we examined the in vivo interplay between iNKT and Treg cells in steady state and in preclinical models of liver and gut autoimmunity. Under non-inflamed conditions, Treg depletion enhanced glycolipid-mediated iNKT cell responses, with a general impact on Type 1, 2 and 17 iNKT subsets. Moreover, in vivo iNKT activation in the absence of Tregs suppressed the induction of iNKT anergy, consistent with a reduction in programmed cell death receptor 1 (PD-1) expression. Importantly, we unveiled a clear role for an in vivo Treg-iNKT crosstalk both in concanavalin A-induced acute hepatitis and oxazolone-induced colitis. Here, the absence of Tregs led to a markedly enhanced liver and gut pathology, which was not observed in iNKT-deficient mice. Taken together, these results provide evidence for a functional interplay between regulatory T cell subsets critical in controlling the onset of autoimmune disease.

Transport by circulating myeloid cells drives liposomal accumulation in inflamed synovium.

The therapeutic potential of liposomes to deliver drugs into inflamed tissue is well documented. Liposomes are believed to largely transport drugs into inflamed joints by selective extravasation through endothelial gaps at the inflammatory sites, known as the enhanced permeation and retention effect. However, the potential of blood-circulating myeloid cells for the uptake and delivery of liposomes has been largely overlooked. Here we show that myeloid cells can transport liposomes to inflammatory sites in a collagen-induced arthritis model. It is shown that the selective depletion of the circulating myeloid cells reduces the accumulation of liposomes up to 50-60%, suggesting that myeloid-cell-mediated transport accounts for more than half of liposomal accumulation in inflamed regions. Although it is widely believed that PEGylation inhibits premature liposome clearance by the mononuclear phagocytic system, our data show that the long blood circulation times of PEGylated liposomes rather favours uptake by myeloid cells. This challenges the prevailing theory that synovial liposomal accumulation is primarily due to the enhanced permeation and retention effect and highlights the potential for other pathways of delivery in inflammatory diseases.

Gut Inflammation in Axial Spondyloarthritis Patients is Characterized by a Marked Type 17 Skewed Mucosal Innate-like T Cell Signature.

OBJECTIVE: Patients with spondyloarthritis (SpA) often present with microscopic signs of gut inflammation, a risk factor for progressive disease. We investigated whether mucosal innate-like T cells are involved in dysregulated interleukin-23 (IL-23)/IL-17 responses in the gut-joint axis in SpA. METHODS: Ileal and colonic intraepithelial lymphocytes (IELs), lamina propria lymphocytes (LPLs), and paired peripheral blood mononuclear cells (PBMCs) were isolated from treatment-naive patients with nonradiographic axial SpA with (n = 11) and without (n = 14) microscopic gut inflammation and healthy controls (n = 15) undergoing ileocolonoscopy. The presence of gut inflammation was assessed histopathologically. Immunophenotyping of innate-like T cells and conventional T cells was performed using intracellular flow cytometry. Unsupervised clustering analysis was done by FlowSOM technology. Serum IL-17A levels were measured via Luminex. RESULTS: Microscopic gut inflammation in nonradiographic axial SpA was characterized by increased ileal intraepithelial γδ-hi T cells, a γδ-T cell subset with elevated γδ-T cell receptor expression. γδ-hi T cells were also increased in PBMCs of patients with nonradiographic axial SpA versus healthy controls and were strongly associated with Ankylosing Spondylitis Disease Activity Score. The abundance of mucosal-associated invariant T cells and invariant natural killer T cells was unaltered. Innate-like T cells in the inflamed gut showed increased RORγt, IL-17A, and IL-22 levels with loss of T-bet, a signature that was less pronounced in conventional T cells. Presence of gut inflammation was associated with higher serum IL-17A levels. In patients treated with tumor necrosis factor blockade, the proportion of γδ-hi cells and RORγt expression in blood was completely restored. CONCLUSION: Intestinal innate-like T cells display marked type 17 skewing in the inflamed gut mucosa of patients with nonradiographic axial SpA. γδ-hi T cells are linked to intestinal inflammation and disease activity in SpA.

Distinct immune modulatory roles of regulatory T cells in gut versus joint inflammation in TNF-driven spondyloarthritis.

OBJECTIVES: Gut and joint inflammation commonly co-occur in spondyloarthritis (SpA) which strongly restricts therapeutic modalities. The immunobiology underlying differences between gut and joint immune regulation, however, is poorly understood. We therefore assessed the immunoregulatory role of CD4+FOXP3+ regulatory T (Treg) cells in a model of Crohn's-like ileitis and concomitant arthritis. METHODS: RNA-sequencing and flow cytometry was performed on inflamed gut and joint samples and tissue-derived Tregs from tumour necrosis factor (TNF)∆ARE mice. In situ hybridisation of TNF and its receptors (TNFR) was applied to human SpA gut biopsies. Soluble TNFR (sTNFR) levels were measured in serum of mice and patients with SpA and controls. Treg function was explored by in vitro cocultures and in vivo by conditional Treg depletion. RESULTS: Chronic TNF exposure induced several TNF superfamily (TNFSF) members (4-1BBL, TWEAK and TRAIL) in synovium and ileum in a site-specific manner. Elevated TNFR2 messenger RNA levels were noted in TNF∆ARE/+ mice leading to increased sTNFR2 release. Likewise, sTNFR2 levels were higher in patients with SpA with gut inflammation and distinct from inflammatory and healthy controls. Tregs accumulated at both gut and joints of TNF∆ARE mice, yet their TNFR2 expression and suppressive function was significantly lower in synovium versus ileum. In line herewith, synovial and intestinal Tregs displayed a distinct transcriptional profile with tissue-restricted TNFSF receptor and p38MAPK gene expression. CONCLUSIONS: These data point to profound differences in immune-regulation between Crohn's ileitis and peripheral arthritis. Whereas Tregs control ileitis they fail to dampen joint inflammation. Synovial resident Tregs are particularly maladapted to chronic TNF exposure.

RORγt inhibition ameliorates IL-23 driven experimental psoriatic arthritis by predominantly modulating γδ-T cells.

OBJECTIVE: Divergent therapeutic outcomes on different disease domains have been noted with IL-23 and IL-17A-blockade in PsA. Therefore, elucidating the role of RORγt, the master regulator of type 17 immune responses, is of potential therapeutic interest. To this end, RORγt inhibition was assessed in combined skin, joint and gut inflammation in vivo, using a PsA model. METHODS: We tested the efficacy of a RORγt antagonist in B10.RIII mice challenged with systemic overexpression of IL-23 by hydrodynamic injection of IL-23 enhanced episomal vector (IL-23 EEV). Clinical outcomes were evaluated by histopathology. Bone density and surface erosions were examined using micro-computed tomography. Cytokine production was measured in serum and by intracellular flow cytometry. Gene expression in PsA-related tissues was analysed by qPCR. RESULTS: RORγt-blockade significantly ameliorated psoriasis, peripheral arthritis and colitis development in IL-23 EEV mice (improvement of clinical scores and weight loss respectively by 91.8%, 58.2% and 7.0%, P < 0.001), in line with profound suppression of an enhanced type IL-17 immune signature in PsA-affected tissues. Moreover, inflammation-induced bone loss and bone erosions were reduced (P < 0.05 in calcaneus, P < 0.01 in tibia). Sustained IL-23 overexpression resulted in only mild signs of sacroiliitis. Gamma-delta (γδ)-T cells, the dominant source of T cell-derived IL-17A and IL-22, were expanded during IL-23 overexpression, and together with Th17 cells, clearly countered by RORγt inhibition (P < 0.001). CONCLUSION: RORγt-blockade shows therapeutic efficacy in a preclinical PsA model with protection towards extra-musculoskeletal manifestations, reflected by a clear attenuation of type 17 cytokine responses by γδ-T cells and Th17 cells.

Deletion of Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 in Mouse T Cells Protects Against Development of Autoimmune Arthritis but Leads to Spontaneous Osteoporosis.

OBJECTIVE: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) plays a crucial role in innate and adaptive immune signaling by modulating the threshold for activation of immune cells, including Treg cells. Therefore, MALT-1 is regarded to be an interesting therapeutic target in several immune-mediated diseases. The goal of this study was to examine the role of MALT-1 in experimental animal models of rheumatoid arthritis (RA). METHODS: MALT-1 activation was assessed by measuring cleavage of the deubiquitinase CYLD in lymphocytes from mice with collagen-induced arthritis (CIA). Furthermore, the impact of MALT-1 deficiency on arthritis was evaluated in Malt1KO mice with CIA or with collagen antibody-induced arthritis (CAIA). T cell-specific MALT-1 deficiency was measured in mice with deletion of T cell-specific MALT-1 (Malt1Tcell KO ), and the time-dependent effects of MALT-1 deficiency were assessed in mice with deletion of tamoxifen-inducible T cell-specific MALT-1 (Malt1iTcell KO ). Bone density was determined in MALT-1-deficient mice using micro-computed tomography and femur-bending tests. Reconstitution of Treg cells was performed using adoptive transfer experiments. RESULTS: MALT-1 activation was observed in the lymphocytes of mice with CIA. T cell-specific MALT-1 deletion in the induction phase of arthritis (incidence of arthritis, 25% in control mice versus 0% in Malt1iTcell KO mice; P < 0.05), but not in the effector phase of arthritis, completely protected mice against the development of CIA. Consistent with this finding, MALT-1 deficiency had no impact on CAIA, an effector phase model of RA. Finally, mice with MALT-1 deficiency showed a spontaneous decrease in bone density (mean ± SEM trabecular thickness, 46.3 ± 0.7 µm in control mice versus 40 ± 1.1 µm in Malt1KO mice; P < 0.001), which was linked to the loss of Treg cells in these mice. CONCLUSION: Overall, these data in murine models of RA highlight MALT-1 as a master regulator of T cell activation, which is relevant to the pathogenesis of autoimmune arthritis. Furthermore, these findings show that MALT-1 deficiency can lead to spontaneous osteoporosis, which is associated with impaired Treg cell numbers.

RORγt inhibition selectively targets IL-17 producing iNKT and γδ-T cells enriched in Spondyloarthritis patients.

Dysregulated IL-23/IL-17 responses have been linked to psoriatic arthritis and other forms of spondyloarthritides (SpA). RORγt, the key Thelper17 (Th17) cell transcriptional regulator, is also expressed by subsets of innate-like T cells, including invariant natural killer T (iNKT) and γδ-T cells, but their contribution to SpA is still unclear. Here we describe the presence of particular RORγt+T-betloPLZF- iNKT and γδ-hi T cell subsets in healthy peripheral blood. RORγt+ iNKT and γδ-hi T cells show IL-23 mediated Th17-like immune responses and were clearly enriched within inflamed joints of SpA patients where they act as major IL-17 secretors. SpA derived iNKT and γδ-T cells showed unique and Th17-skewed phenotype and gene expression profiles. Strikingly, RORγt inhibition blocked γδ17 and iNKT17 cell function while selectively sparing IL-22+ subsets. Overall, our findings highlight a unique diversity of human RORγt+ T cells and underscore the potential of RORγt antagonism to modulate aberrant type 17 responses.

4"-O-Alkylated α-Galactosylceramide Analogues as iNKT-Cell Antigens: Synthetic, Biological, and Structural Studies.

Invariant natural killer T-cells (iNKT) are a glycolipid-responsive subset of T-lymphocytes that fulfill a pivotal role in the immune system. The archetypical synthetic glycolipid, α-galactosylceramide (α-GalCer), whose molecular framework is inspired by a group of amphiphilic natural products, remains the most studied antigen for iNKT-cells. Nonetheless, the potential of α-GalCer as an immunostimulating agent is compromised by the fact that this glycolipid elicits simultaneous secretion of Th1- and Th2-cytokines. This has incited medicinal chemistry efforts to identify analogues that are able to perturb the Th1/Th2 balance. In this work, we present the synthesis of an extensive set of 4"-O-alkylated α-GalCer analogues, which were evaluated in vivo for their cytokine induction. We have found that conversion of the 4"-OH group to ether moieties decreases the immunogenic potential in mice relative to α-GalCer. Yet, the benzyl-modified glycolipids are able to produce a distinct pro-inflammatory immune response. The crystal structures suggest an extra hydrophobic interaction between the benzyl moiety and the α2-helix of CD1d.

Efficient Divergent Synthesis of New Immunostimulant 4″-Modified α-Galactosylceramide Analogues.

A synthesis strategy for the swift generation of 4″-modified α-galactosylceramide (α-GalCer) analogues is described, establishing a chemical platform to comprehensively investigate the structure-activity relationships (SAR) of this understudied glycolipid part. The strategy relies on a late-stage reductive ring-opening of a p-methoxybenzylidene (PMP) acetal to regioselectively liberate the 4″-OH position. The expediency of this methodology is demonstrated by the synthesis of a small yet diverse set of analogues, which were tested for their ability to stimulate invariant natural killer T-cells (iNKT) in vitro and in vivo. The introduction of a p-chlorobenzyl ether yielded an analogue with promising immunostimulating properties, paving the way for further SAR studies.

Galactosylsphingamides: new α-GalCer analogues to probe the F'-pocket of CD1d.

Invariant Natural Killer T-cells (iNKT-cells) are an attractive target for immune response modulation, as upon CD1d-mediated stimulation with KRN7000, a synthetic α-galactosylceramide, they produce a vast amount of cytokines. Here we present a synthesis that allows swift modification of the phytosphingosine side chain by amidation of an advanced methyl ester precursor. The resulting KRN7000 derivatives, termed α-galactosylsphingamides, were evaluated for their capacity to stimulate iNKT-cells. While introduction of the amide-motif in the phytosphingosine chain is tolerated for CD1d binding and TCR recognition, the studied α-galactosylsphingamides showed compromised antigenic properties.

Synthesis of C6''-modified α-C-GalCer analogues as mouse and human iNKT cell agonists.

α-GalCer analogues that combine known Th1 polarizing C6''-modifications with a C-glycosidic linkage were synthesized. We employed a protecting group strategy that allowed the preparation of both saturated and unsaturated derivatives with variable C6''-substituents. Selected analogues demonstrate promising activity in mice. Interestingly, the introduction of a 6''-O-pyridinylcarbamoyl substituent to α-C-GalCer restores its antigenicity in human iNKT cells.

A bidirectional crosstalk between iNKT cells and adipocytes mediated by leptin modulates susceptibility for T cell mediated hepatitis.

BACKGROUND & AIMS: Immunometabolism is an emerging field of clinical investigation due to the obesity epidemic worldwide. A reciprocal involvement of immune mediators in the body energy metabolism has been recognized for years, but is only partially understood. We hypothesized that the adipokine leptin could provide an important modulator of iNKT cells. METHODS: The expression of leptin receptor (LR) on resting and activated iNKT cells was measured by flow cytometry. FACS-sorted hepatic iNKT cells were stimulated with anti-CD3/CD28Ab coated beads in the absence or presence of a neutralizing anti-leptin Ab. Furthermore, we evaluated the outcome of LR blocking nanobody treatment in ConA induced hepatitis and towards metabolic parameters in WT and iNKT cell deficient mice. RESULTS: The LR is expressed on iNKT cells and leptin suppresses iNKT cell proliferation and cytokine production in vitro. LR deficient iNKT cells are hyper-responsive further enforcing the role of leptin as an important inhibitor of iNKT cell function. Consistently, in vivo blockade of LR signaling exacerbated ConA hepatitis in wild-type but not in iNKT cell deficient mice, through both Janus kinase (JAK)2 and mitogen-activated protein kinase (MAPK) dependent mechanisms. Moreover, LR inhibition altered fat pad features and was accompanied by insulin resistance, only in wild-type mice. Curiously, this interaction was strictly dependent on MAPK mediated LR signaling in iNKT cells and uncoupled from the more central effects of leptin. CONCLUSIONS: Our data support a new concept of immune regulation by which leptin protects towards T cell mediated hepatitis via modulation of iNKT cells.

Bacterial CD1d-restricted glycolipids induce IL-10 production by human regulatory T cells upon cross-talk with invariant NKT cells.

Invariant NKT (iNKT) cells and CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) are important immune regulatory T cells with Ag reactivity to glycolipids and peptides, respectively. However, the functional interplay between these cells in humans is poorly understood. We show that Tregs suppress iNKT cell proliferation induced by CD1d-restricted glycolipids, including bacterial-derived diacylglycerols, as well as by innate-like activation. Inhibition was related to the potency of iNKT agonists, making diacylglycerol iNKT responses very prone to suppression. Cytokine production by iNKT cells was differentially modulated by Tregs because IL-4 production was reduced more profoundly compared with IFN-γ. A compelling observation was the significant production of IL-10 by Tregs after cell contact with iNKT cells, in particular in the presence of bacterial diacylglycerols. These iNKT-primed Tregs showed increased FOXP3 expression and superior suppressive function. Suppression of iNKT cell responses, but not conventional T cell responses, was IL-10 dependent, suggesting that there is a clear difference in mechanism between the Treg-mediated inhibition of these cell types. Our data highlight a physiologically relevant interaction between human iNKT and Tregs upon pathogen-derived glycolipid recognition that has a significant impact on the design of iNKT cell-based therapeutics.

Selection of non-competitive leptin antagonists using a random nanobody-based approach.

The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. Accumulating evidence suggests that leptin plays a role in human pathologies, such as autoimmune diseases and cancer, thus providing a rationale for the development of leptin antagonists. In the present study, we generated and evaluated a panel of neutralizing nanobodies targeting the LR (leptin receptor). A nanobody comprises the variable domain of the naturally occurring single-chain antibodies found in members of the Camelidae family. We identified three classes of neutralizing nanobodies targeting different LR subdomains: i.e. the CRH2 (cytokine receptor homology 2), Ig-like and FNIII (fibronectin type III) domains. Only nanobodies directed against the CRH2 domain inhibited leptin binding. We could show that a nanobody that targets the Ig-like domain potently interfered with leptin-dependent regulation of hypothalamic NPY (neuropeptide Y) expression. As a consequence, daily intraperitoneal injection increased body weight, body fat content, food intake, liver size and serum insulin levels. All of these characteristics resemble the phenotype of leptin and LR-deficient animals. The results of the present study support proposed models of the activated LR complex, and demonstrate that it is possible to block LR signalling without affecting ligand binding. These nanobodies form new tools to study the mechanisms of BBB (blood-brain barrier) leptin transport and the effect of LR inhibition in disease models.

Galactose-modified iNKT cell agonists stabilized by an induced fit of CD1d prevent tumour metastasis.

Invariant natural killer T (iNKT) cells are known to have marked immunomodulatory capacity due to their ability to produce copious amounts of effector cytokines. Here, we report the structure and function of a novel class of aromatic α-galactosylceramide structurally related glycolipids with marked Th1 bias in both mice and men, leading to superior tumour protection in vivo. The strength of the Th1 response correlates well with enhanced lipid binding to CD1d as a result of an induced fit mechanism that binds the aromatic substitution as a third anchor, in addition to the two lipid chains. This induced fit is in contrast to another Th1-biasing glycolipid, α-C-GalCer, whose CD1d binding follows a conventional key-lock principle. These findings highlight the previously unexploited flexibility of CD1d in accommodating galactose-modified glycolipids and broaden the range of glycolipids that can stimulate iNKT cells. We speculate that glycolipids can be designed that induce a similar fit, thereby leading to superior and more sustained iNKT cell responses in vivo.

A multiparameter approach to monitor disease activity in collagen-induced arthritis.

INTRODUCTION: Disease severity in collagen-induced arthritis (CIA) is commonly assessed by clinical scoring of paw swelling and histological examination of joints. Although this is an accurate approach, it is also labour-intensive and the application of less invasive and less time-consuming methods is of great interest. However, it is still unclear which of these methods represents the most discriminating measure of disease activity. METHODS: We undertook a comparative analysis in which different measurements of inflammation and tissue damage in CIA were studied on an individual mouse level. We compared the current gold standard methods - clinical scoring and histological examination - with alternative methods based on scoring of X-ray or micro-computed tomography (CT) images and investigated the significance of systemically expressed proteins, involved in CIA pathogenesis, that have potential as biomarkers. RESULTS: Linear regression analysis revealed a marked association of serum matrix metalloproteinase (MMP)-3 levels with all features of CIA including inflammation, cartilage destruction and bone erosions. This association was improved by combined detection of MMP-3 and anti-collagen IgG2a antibody concentrations. In addition, combined analysis of both X-ray and micro-CT images was found to be predictive for cartilage and bone damage. Most remarkably, validation analysis using an independent data set proved that variations in disease severity, induced by different therapies, could be accurately represented by predicted values based on the proposed parameters. CONCLUSIONS: Our analyses revealed that clinical scoring, combined with serum MMP-3, anti-collagen IgG2a measurement and scoring of X-ray and micro-CT images, yields a comprehensive insight into the different aspects of disease activity in CIA.

The synthesis and in vivo evaluation of 2',2'-difluoro KRN7000.

The synthesis of 2',2'-difluoro KRN7000 is described. In vivo evaluation demonstrates that this fluorinated glycolipid induces CD1d-dependent TCR activation of NKT cells, with a bias towards Th2 cytokine production.

Synthesis and in vivo evaluation of 4-deoxy-4,4-difluoro-KRN7000.

The synthesis of 4-deoxy-4,4-difluoro-KRN7000 starting from phytosphingosine is described. Key steps include a regioselective benzylation of azidophytosphingosine and a deoxofluor-mediated fluorination of the corresponding 4-ketone. This fluorination failed completely when the adjacent 3-OH was protected as benzyl ether but proceeded well when a benzoyl group was used. The biological evaluation reveals a bias toward Th1 cytokine induction upon Natural Killer T cell activation.

Synthesis and in vitro evaluation of alpha-GalCer epimers.

alpha-GalCer (also known as KRN7000) is an immunomodulatory glycolipid that is known to potently activate invariant natural killer T (NKT) cells upon CD1d-mediated stimulation. Because Th1 and Th2 cytokines, which are released after alpha-GalCer presentation, antagonize each other's effects, alpha-GalCer analogues that induce a biased Th1/Th2 response are highly awaited. In this context, we report the synthesis and in vitro evaluation of alpha-Gal-D-xylo-Cer and two alpha-Gal-L-lyxo-Cer analogues, one with the natural acyl chain, the other with a truncated chain.