Interleukin-1 receptor accessory protein blockade limits the development of atherosclerosis and reduces plaque inflammation

The interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor required for signalling through the IL-1, IL-33, and IL-36 receptors. Using a novel anti-IL1RAP-blocking antibody, we investigated the role of IL1RAP in atherosclerosis.Single-cell RNA sequencing data from human atherosclerotic plaques revealed the expression of IL1RAP and several IL1RAP-related cytokines and receptors, including IL1B and IL33. Histological analysis showed the presence of IL1RAP in both the plaque and adventitia, and flow cytometry of murine atherosclerotic aortas revealed IL1RAP expression on plaque leucocytes, including neutrophils and macrophages. High-cholesterol diet fed apolipoprotein E-deficient (Apoe-/-) mice were treated with a novel non-depleting IL1RAP-blocking antibody or isotype control for the last 6 weeks of diet. IL1RAP blockade in mice resulted in a 20% reduction in subvalvular plaque size and limited the accumulation of neutrophils and monocytes/macrophages in plaques and of T cells in adventitia, compared with control mice. Indicative of reduced plaque inflammation, the expression of several genes related to leucocyte recruitment, including Cxcl1 and Cxcl2, was reduced in brachiocephalic arteries of anti-IL1RAP-treated mice, and the expression of these chemokines in human plaques was mainly restricted to CD68+ myeloid cells. Furthermore, in vitro studies demonstrated that IL-1, IL-33, and IL-36 induced CXCL1 release from both macrophages and fibroblasts, which could be mitigated by IL1RAP blockade.Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production.Biopharmaceutic

Lymph-borne CD8α+ dendritic cells are uniquely able to cross-prime CD8+ T cells with antigen acquired from intestinal epithelial cells

Cross-presentation of cellular antigens is crucial for priming CD8<sup>+</sup> T cells, and generating immunity to intracellular pathogens—particularly viruses. It is unclear which intestinal phagocytes perform this function in vivo. To address this, we examined dendritic cells (DCs) from the intestinal lymph of IFABP-tOVA 232-4 mice, which express ovalbumin in small intestinal epithelial cells (IECs). Among lymph DCs (LDCs) only CD103<sup>+</sup> CD11b<sup>−</sup> CD8α<sup>+</sup> DCs cross-present IEC-derived ovalbumin to CD8<sup>+</sup> OT-I T cells. Similarly, in the mesenteric lymph nodes (MLNs), cross-presentation of IEC–ovalbumin was limited to the CD11c<sup>+</sup> MHCII<sup>hi</sup> CD8α<sup>+</sup> migratory DCs, but absent from all other subsets, including the resident CD8α<sup>hi</sup> DCs. Crucially, delivery of purified CD8α<sup>+</sup> LDCs, but not other LDC subsets, into the MLN subcapsular lymphatic sinus induced proliferation of ovalbumin-specific, gut-tropic CD8<sup>+</sup> T cells <i>in vivo</i>. Finally, in 232-4 mice treated with R848, CD8α<sup>+</sup> LDCs were uniquely able to cross-prime interferon γ-producing CD8<sup>+</sup>T cells and drive their migration to the intestine. Our results clearly demonstrate that migrating CD8α<sup>+</sup> intestinal DCs are indispensable for cross-presentation of cellular antigens and, in conditions of inflammation, for the initial differentiation of effector CD8<sup>+</sup> T cells. They may therefore represent an important target for the development of antiviral vaccinations

Retinoic acid controls the homeostasis of pre-cDC–derived splenic and intestinal dendritic cells

Dendritic cells (DCs) comprise distinct populations with specialized immune-regulatory functions. However, the environmental factors that determine the differentiation of these subsets remain poorly defined. Here, we report that retinoic acid (RA), a vitamin A derivative, controls the homeostasis of pre-DC (precursor of DC)–derived splenic CD11b(+)CD8α(−)Esam(high) DCs and the developmentally related CD11b(+)CD103(+) subset within the gut. Whereas mice deprived of RA signaling significantly lost both of these populations, neither pre-DC–derived CD11b(−)CD8α(+) and CD11b(−)CD103(+) nor monocyte-derived CD11b(+)CD8α(−)Esam(low) or CD11b(+)CD103(−) DC populations were deficient. In fate-tracking experiments, transfer of pre-DCs into RA-supplemented hosts resulted in near complete conversion of these cells into the CD11b(+)CD8α(−) subset, whereas transfer into vitamin A–deficient (VAD) hosts caused diversion to the CD11b(−)CD8α(+) lineage. As vitamin A is an essential nutrient, we evaluated retinoid levels in mice and humans after radiation-induced mucosal injury and found this conditioning led to an acute VAD state. Consequently, radiation led to a selective loss of both RA-dependent DC subsets and impaired class II–restricted auto and antitumor immunity that could be rescued by supplemental RA. These findings establish a critical role for RA in regulating the homeostasis of pre-DC–derived DC subsets and have implications for the management of patients with immune deficiencies resulting from malnutrition and irradiation

Regionalized Development and Maintenance of the Intestinal Adaptive Immune Landscape

The intestinal immune system has the daunting task of protecting us from pathogenic insults while limiting inflammatory responses against the resident commensal microbiota and providing tolerance to food antigens. This role is particularly impressive when one considers the vast mucosal surface and changing landscape that the intestinal immune system must monitor. In this review, we highlight regional differences in the development and composition of the adaptive immune landscape of the intestine and the impact of local intrinsic and environmental factors that shape this process. To conclude, we review the evidence for a critical window of opportunity for early-life exposures that affect immune development and alter disease susceptibility later in life

Phenotype and function of intestinal CD103+ dendritic cells

Mucosal tissues such as the intestine present an enormous surface area to the outside environment and are continually exposed to foreign antigens in food and microflora. Thus, the intestinal mucosa must must remain tolerant to these innocuous antigens while at same time be able to mount effective immune responses to pathogens. Intestinal dendritic cells (DCs) and macrophages, found throughout the villous lamina propria and intestinal lymphoid tissue in both mice and humans are thought to play key roles in this process. DCs at these sites constantly sample and process both luminal and self-antigens and are key players in presenting these antigens to T cells. Recently, intestinal CD103+ DCs have been shown to promote induction of both gut homing receptors on responding T cells and differentiation of FoxP3+ regulatory T cells via the vitamin A metabolite retinoic acid (RA). Another intestinal DC/macrophage subset, expressing the chemokine receptor CX3CR1+ has been suggested to take up intestinal luminal antigens via transepithelial dendrites. The focus of this thesis has been to phenotypically and functionally characterize intestinal CD103+ DCs and to investigate the mechanisms that underlie their enhanced ability to generate gut homing T cells. In this thesis we demonstrate that CD103+ DCs present in the murine small intestinal mucosa and draining mesenteric lymph nodes (MLN) have an enhanced ability to deliver RA signals and induce gut homing receptors on responding T cells in vitro compared to CD103- DCs and CD103+ DCs outside the intestine. Importantly we show that that CD103+ DCs also are present in human MLN and have a similar ability to induce RA dependent gut tropism on T cells. We demonstrate that CD103+ DCs and CX3CR1+ cells are non- overlapping populations of intestinal cells that differ both in phenotype and in function. Our results suggest that CD103+ DCs are the major migratory DC population in the intestinal mucosa and play a central role in initiating immune responses to luminal antigen in draining MLNs. In contrast we find that CX3CR1+ cells resemble tissue macrophages, are non-migratory and inefficient at activating naïve T cells. We further show that both populations have different growth factor requirements and turnover rates in the intestinal mucosa. We have also examined potential mechanisms underlying the selective ability of intestinal CD103+ DCs to metabolize vitamin A. Results from these studies suggest that the Vitamin A metabolite RA itself induces retinol-metabolizing activity in CD103+ DCs in vivo, and that bile is an important source of these retinoids. Collectively, these studies have contributed to our knowledge of the mechanisms regulating intestinal immune responses and will hopefully lead to novel strategies for treatments for intestinal inflammatory diseases and mucosal vaccine design

The diverse ontogeny and function of murine small intestinal dendritic cell/macrophage subsets.

Intestinal dendritic cell and macrophage subsets are believed to play key roles in maintaining intestinal homeostasis in the steady state and in driving protective immune responses in the setting of intestinal infection. This mini-review focuses on recent progress regarding the ontogeny and function of small intestinal lamina propria dendritic cell/macrophage subsets. In particular we discuss recent findings suggesting that small intestinal CD103(+) dendritic cells and Cx3cr1(+) cells derive from distinct precursor populations and that CD103(+) dendritic cells represent the major migratory population of cells with a key role in initiating adaptive immune responses in the draining mesenteric lymph node. In contrast, Cx3cr1(+) cells appear to represent a tissue resident population, phenotypically indistinguishable from tissue resident macrophages. These latter observations suggest an important division of labour between dendritic cell/macrophage subsets in the regulation of intestinal immune responses in the steady state

Bile retinoids imprint intestinal CD103(+) dendritic cells with the ability to generate gut-tropic T cells.

Small intestinal lamina propria (SI-LP) CD103(+) dendritic cells (DCs) are imprinted with an ability to metabolize vitamin A (retinol), a property underlying their enhanced capacity to induce the gut-homing receptors CC chemokine receptor-9 and α4β7 on responding T cells. In this study, we demonstrate that imprinting of CD103(+) DCs is itself critically dependent on vitamin A and occurs locally within the small intestine (SI). The major vitamin A metabolite retinoic acid (RA) induced retinol-metabolizing activity in DCs both in vitro and in vivo, suggesting a direct role for RA in this process. Consistent with this, SI-LP CD103(+) DCs constitutively received RA signals in vivo at significantly higher levels than did colonic CD103(+) DCs. Remarkably, SI CD103(+) DCs remained imprinted in mice depleted of dietary but not of systemic retinol. We found that bile contained high levels of retinol, induced RA receptor-dependent retinol-metabolizing activity in bone marrow-derived DCs, and imprinted these cells with the ability to generate gut-tropic T cells. Taken together, these results suggest a novel and unexpected role for bile in SI-LP CD103(+) DC imprinting.Mucosal Immunology advance online publication 2 February 2011. doi:10.1038/mi.2010.91

Cellular Barcoding Links B-1a B Cell Potential to a Fetal Hematopoietic Stem Cell State at the Single-Cell Level

Hematopoietic stem cells (HSCs) undergo a functional switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Here, we investigated whether the developmental attenuation of B-1a cell output is a consequence of a shift in stem cell state during ontogeny. Using cellular barcoding for in vivo single-cell fate analyses, we found that fetal liver definitive HSCs gave rise to both B-1a and B-2 cells. Whereas B-1a potential diminished in all HSCs with time, B-2 output was maintained. B-1a and B-2 plasticity could be reinitiated in a subset of adult HSCs by ectopic expression of the RNA binding protein LIN28B, a key regulator of fetal hematopoiesis, and this coincided with the clonal reversal to fetal-like elevated self-renewal and repopulation potential. These results anchor the attenuation of B-1a cell output to fetal HSC behavior and demonstrate that the developmental decline in regenerative potential represents a reversible HSC state

Retinoic acid receptor signaling levels and antigen dose regulate gut homing receptor expression on CD8(+) T cells

Recent studies have highlighted a central role for intestinal dendritic cells (DCs) and vitamin A metabolite retinoic acid (RA) in the generation of alpha 4 beta 7(+) CCR9(+) "gut tropic" effector T cells. Here, using RA-responsive element reporter mice, we demonstrate that both splenic and mesenteric lymph node (MLN) DCs enhanced retinoic acid receptor (RAR) signaling in CD8(+) T cells; however, only a subset of MLN DCs, expressing the integrin alpha-chain CD103, induced an early RAR signal that is required for efficient CCR9 induction. MLN-primed CD8(+) T cells also received enhanced RAR-dependent signals compared with splenic-primed CD8(+) T cells in vivo. Further DC-mediated induction of gut homing receptors was inhibited at a high antigen dose without influencing RAR signaling events, and resulted in less efficient CD8(+) T-cell entry into the small intestinal mucosa. These results highlight a complex interplay between antigen dose and DC subset-induced RAR signaling events in the generation of tissue tropic effector T-cell subsets