Tanja Konijn

Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity

The impact of nutritional status during fetal life on the overall health of adults has been recognized; however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

Identification of Natural RORγ Ligands that Regulate the Development of Lymphoid Cells

Mice deficient in the nuclear hormone receptor RORγt have defective development of thymocytes, lymphoid organs, Th17 cells, and type 3 innate lymphoid cells. RORγt binds to oxysterols derived from cholesterol catabolism, but it is not clear whether these are its natural ligands. Here, we show that sterol lipids are necessary and sufficient to drive RORγt-dependent transcription. We combined overexpression, RNAi, and genetic deletion of metabolic enzymes to study RORγ-dependent transcription. Our results are consistent with the RORγt ligand(s) being a cholesterol biosynthetic intermediate (CBI) downstream of lanosterol and upstream of zymosterol. Analysis of lipids bound to RORγ identified molecules with molecular weights consistent with CBIs. Furthermore, CBIs stabilized the RORγ ligand-binding domain and induced coactivator recruitment. Genetic deletion of metabolic enzymes upstream of the RORγt-ligand(s) affected the development of lymph nodes and Th17 cells. Our data suggest that CBIs play a role in lymphocyte development potentially through regulation of RORγt.

Diet-Derived Short Chain Fatty Acids Stimulate Intestinal Epithelial Cells To Induce Mucosal Tolerogenic Dendritic Cells

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article, we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A–converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro, respectively. Furthermore, our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A–converting enzymes in mesenteric lymph node dendritic cells, along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover, we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion, our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system.

Vitamin A Controls the Presence of RORγ+ Innate Lymphoid Cells and Lymphoid Tissue in the Small Intestine

Changes in diet and microbiota have determining effects on the function of the mucosal immune system. For example, the active metabolite of vitamin A, retinoic acid (RA), has been described to maintain homeostasis in the intestine by its influence on both lymphocytes and myeloid cells. Additionally, innate lymphoid cells (ILCs), important producers of cytokines necessary for intestinal homeostasis, are also influenced by vitamin A in the small intestines. In this study, we show a reduction of both NCR− and NCR+ ILC3 subsets in the small intestine of mice raised on a vitamin A–deficient diet. Additionally, the percentages of IL-22–producing ILCs were reduced in the absence of dietary vitamin A. Conversely, mice receiving additional RA had a specific increase in the NCR− ILC3 subset, which contains the lymphoid tissue inducer cells. The dependence of lymphoid tissue inducer cells on vitamin A was furthermore illustrated by impaired development of enteric lymphoid tissues in vitamin A–deficient mice. These effects were a direct consequence of ILC-intrinsic RA signaling, because retinoic acid–related orphan receptor γt–Cre × RARα-DN mice had reduced numbers of NCR− and NCR+ ILC3 subsets within the small intestine. However, lymphoid tissue inducer cells were not affected in these mice nor was the formation of enteric lymphoid tissue, demonstrating that the onset of RA signaling might take place before retinoic acid–related orphan receptor γt is expressed on lymphoid tissue inducer cells. Taken together, our data show an important role for vitamin A in controlling innate lymphoid cells and, consequently, postnatal formed lymphoid tissues within the small intestines.

A polymorphism in the coding region of Il12b promotes IL-12p70 and IL-23 heterodimer formation.

IL-12 and IL-23 are heterodimeric cytokines involved in the induction of Th1 and Th17 immune responses. Previous work indicated that a region on chromosome 11 encoding the IL-12p40 subunit regulates strain differences in susceptibility to murine trinitrobenzene sulfonic acid-induced colitis. In addition, this region determines strain differences in LPS-induced IL-12 responses. In this study, we investigated how polymorphisms in the coding region of murine Il12b influence IL-12 and IL-23 heterodimer formation. Transfection studies using constructs containing IL-12p35 linked to IL-12p40 from the colitis-resistant C57BL/6 strain or to the polymorphic p40 variant from the colitis-susceptible SJL/J strain demonstrated that SJL/J-derived p40 constructs synthesized significantly more IL-12p70 than did constructs harboring the C57BL/6-p40 variant. This could not be attributed to differences in synthesis rate or secretion, implicating a greater affinity of SJL/J-derived IL-12p40 for its IL-12p35 subunit. This greater affinity is also associated with increased IL-23 synthesis. In addition, C57BL/6 mice transgenic for the SJL/J 40 variant synthesized significantly more IL-12p70 upon LPS challenge and were more prone to develop colonic inflammation than did C57BL/6 mice transgenic for the C57BL/6-p40 variant. The more efficient binding of the polymorphic Il12b variant to p35 and p19 is most likely due to conformational changes following differential glycosylation as a consequence of the polymorphism. The high synthesis rate of the mature cytokines resulting from this efficient binding can lead to rapid proinflammatory skewing of immune responses and distortion of the homeostatic balance underlying the greater susceptibility for colitis.

Nestin-Expressing Precursors Give Rise to Both Endothelial as well as Nonendothelial Lymph Node Stromal Cells.

During embryogenesis, lymph nodes form through intimate interaction between lymphoid tissue inducer and lymphoid tissue organizer (LTo) cells. Shortly after birth in mice, specialized stromal cell subsets arise that organize microenvironments within the lymph nodes; however, their direct precursors have not yet been identified. In the bone marrow, mesenchymal stem cells are labeled with GFP in nestin-GFP mice, and we show that during all stages of development, nestin+ cells are present within lymph nodes of these mice. At day of birth, both mesenchymal CD31− and endothelial CD31+ LTo cells were GFP+, and only the population of CD31− LTo cells contained mesenchymal precursors. These CD31−nestin+ cells are found in the T and B cell zones or in close association with high endothelial venules in adult lymph nodes. Fate mapping of nestin+ cells unambiguously revealed the contribution of nestin+ precursor cells to the mesenchymal as well as the endothelial stromal populations within lymph nodes. However, postnatal tamoxifen induced targeting of nestin+ cells in nes-creER mice showed that most endothelial cells and only a minority of the nonendothelial cells were labeled. Overall our data show that nestin+ cells contribute to all subsets of the complex stromal populations that can be found in lymph nodes.

The production of secretory leukocyte protease inhibitor by dendritic cells

Secretory leukocyte protease inhibitor (SLPI) is produced at mucosal sites where it plays an important role in the homeostatic control of local inflammation. In addition to its anti-protease activity SLPI is able to reduce LPS activity by interfering with the transfer of LPS to CD14. In addition SLPI can be taken up by cells where it can prevent signaling via the NF-κB route. It is preferentially expressed in dendritic cells from mucosal sites, suggesting a role in the maintenance of a tolerogenic environment, but it is unclear how this differential expression is regulated. Here we analyzed the regulation of SLPI expression in dendritic cells and found that activation by TLR ligands but not via antiCD40 leads to its expression, which is predominantly dependent on p38 activation. This induced expression is late compared to the induction of cytokines and co-stimulatory molecules, is not dependent on factors that are secreted by the cell itself and may be related to cellular feedback mechanisms involved in inflammation and immunity. In correlation with the differential expression by TLR ligands and antiCD40, SLPI deficient mice show enhanced specific immunity when antigen is co-injected with LPS, but not with antiCD40. The results underscore the importance of SLPI as a modulator of specific immunity that can also function at peripheral sites under pathogenic pressure.

Vitamin A metabolism and mucosal immune function are distinct between BALB/c and C57BL/6 mice

The vitamin A metabolite retinoic acid (RA) has been reported to suppress Th1 responses and enhance Th2 responses. Here, we investigated whether differences in vitamin A metabolism could underlie the differences between C57BL/6 and BALB/c mice, which are reportedly seen as Th1 and Th2 responders, respectively. BALB/c mice were shown to have higher intestinal epithelial expression of RALDH1 (where RALDH is retinaldehyde dehydrogenase), and, consequently, higher RALDH activity in MLN‐DCs, leading to an increased ability to induce IgA class switching in B cells. Furthermore, within BALB/c mice, induction of IgA secretion as well as increased accumulation of regulatory T cells (Treg) in the intestinal lamina propria was observed. Additionally, as BALB/c mice are more resistant to dextran sulphate sodium (DSS) induced colitis, mice that lacked vitamin A in their diet had a more severe form of DSS‐induced colitis compared to control mice. Therefore, the level of RA production and consequently the degree of RA‐mediated signaling is crucial for the efficiency of the mucosal immune system.

Involvement of neurons and retinoic acid in lymphatic development: new insights in increased nuchal translucency

Increased nuchal translucency originates from disturbed lymphatic development. Abnormal neural crest cell (NCC) migration may be involved in lymphatic development. Because both neuronal and lymphatic development share retinoic acid (RA) as a common factor, this study investigated the involvement of NCCs and RA in specific steps in lymphatic endothelial cell (LEC) differentiation and nuchal edema, which is the morphological equivalent of increased nuchal translucency.

Dendritic Cell Migration to Skin-Draining Lymph Nodes Is Controlled by Dermatan Sulfate and Determines Adaptive Immunity Magnitude

For full activation of naïve adaptive lymphocytes in skin-draining lymph nodes (LNs), presentation of peptide:MHC complexes by LN-resident and skin-derived dendritic cells (DCs) that encountered antigens (Ags) is an absolute prerequisite. To get to the nearest draining LN upon intradermal immunization, DCs need to migrate from the infection site to the afferent lymphatics, which can only be reached by traversing a collagen-dense network located in the dermis of the skin through the activity of proteolytic enzymes. Here, we show that mice with altered collagen fibrillogenesis resulting in thicker collagen fibers in the skin display a reduced DC migration to the draining LN upon immune challenge. Consequently, the initiation of the cellular and humoral immune response was diminished. Ag-specific CD8+ and CD4+ T cells as well as Ag-specific germinal center B cells and serum immunoglobulin levels were significantly decreased. Hence, we postulate that alterations to the production of extracellular matrix, as seen in various connective tissue disorders, may in the end affect the qualitative outcome of adaptive immunity.