Dicky J. Lindenbergh-Kortleve

Temporal and spatial interplay of microbiota and intestinal mucosa drive establishment of immune homeostasis in conventionalized mice

During colonization of germfree mice with the total fecal microbial community of their conventionally born and raised siblings (conventionalization), the intestinal mucosal immune system initiates and maintains a balanced immune response. However, the genetic regulation of these balanced, appropriate responses to the microbiota is obscure. Here, combined analysis of germfree and conventionalized mice revealed that the major molecular responses could be detected initiating at day 4 post conventionalization, with a strong induction of innate immune functions followed by stimulation of adaptive immune responses and development and expansion of adaptive immune cells at later stages of conventionalization. This study provides a comprehensive overview of mouse developmental and immune-related cellular pathways and processes that were co-mediated by the commensal microbiota and suggests which mechanisms were involved in this reprogramming. The dynamic, region-dependent mucosal responses to the colonizing microbiota revealed potential transcriptional signatures for the control of intestinal homeostasis in healthy mice, which may help to decipher the genetic basis of pathway dysregulation in human intestinal inflammatory diseases.

Gene expression of the insulin-like growth factor system during mouse kidney development

Expression of the insulin-like growth factor (IGF) system was investigated in mouse renal development and physiology, using non radioactive in situ hybridization and quantitative RT-PCR. IGF-I mRNA levels increased after birth and were confined to distal tubules and peritubular capillaries in the outer medulla. IGF-II mRNA levels were high in developing kidneys and peaked after birth. The type I receptor mRNA expression pattern mostly parallelled those of IGF-I and IGF-II. The IGF binding proteins (IGFBPs) showed weak mRNA expression for IGFBP-1 and -6. High fetal mRNA levels were measured for IGFBP-2, showing a similar profile in time as observed for IGF-II. Low fetal IGFBP-3 and -5 mRNA levels increased after birth. IGFBP-2, -4 and -5 mRNA expression was localized to differentiating cells. In the mature kidney predominant expression was confined to proximal tubules (IGFBP-4), thin limbs of Henles Loop (IGFBP-2), glomerular mesangial cells (IGFBP-5) and peritubular capillaries of the medulla (IGFBP-5). IGFBP-3 mRNA was exclusively expressed in endothelial cells of the renal capillary system. Distinct mRNA expression for each member of the IGF system may point to specific roles in development and physiology of the mouse kidney.

Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage

Type 3 lymphoid cells (ILC3s) are required for epithelial activation and proliferation in response to small intestinal tissue damage induced by chemotherapeutics. Multiple ILC3 subsets are activated after intestinal damage, and the absence of ILC3s or their signature cytokine IL-22 results in severely impaired maintenance of intestinal stem cells.

The IGF system during fetal-placental development of the mouse

Insulin-like growth factors (IGF-I and -II) promote cellular mitosis and differentiation and have been implicated in fetal and placental growth. Together with the IGF receptors and IGF binding proteins (IGFBPs) they form a complex network, with tissue specific activity. This review will discuss the data generated to elucidate the functions of the IGF system during mouse development.

mRNA expression patterns of the IGF system during mouse limb bud development, determined by whole mount in situ hybridization.

During limb development the primary limb bud requires various signals to differentiate. Insulin-like growth factor (IGF)-I and IGF-II serve as ubiquitous cellular growth promoters and are modulated by their binding proteins (IGFBPs), which inhibit or augment IGF bioavailability. This is the first study to give a complete overview of the mRNA expression patterns of Igf-1, Igf-2, type 1 Igf receptor (Igf1r) and six Igf binding proteins (IGFBP-1-6) in embryonic mouse limbs, at various stages of development, by whole mount in situ hybridization (ISH). Our results show that all the members of the Igf system, except Igfbp-1 and -6, have specific spatio-temporal mRNA expression patterns. IGFBP-2 and -5 are found in the apical ectodermal ridge (AER), and IGF-I and IGFBP-4 in the region of the zone of polarizing activity (ZPA). IGF-II and IGF1R are found in regions of pre-cartilage formation. At 13.5 days post coitus (dpc) the IGF system colocalizes with apoptosis areas; IGFBP-2, -4 and -5 are found in the interdigital zone, while IGFBP-3 and IGF-I border this region. Furthermore, IGFBP-3, -4 and -5 are found in the phalangeal joint areas, at an early stage of joint formation. This supports the hypothesis that the IGF system may be involved in chondrogenic differentiation of mesenchyme and the regulation of apoptosis in the developing limb.

Generation of antisera to mouse insulin-like growth factor binding proteins (IGFBP)-1 to -6: comparison of IGFBP protein and messenger ribonucleic acid localization in the mouse embryo.

The insulin-like growth factor (IGF) system is an important regulator of fetal growth and differentiation. IGF bioavailability is modulated by IGF binding proteins (IGFBPs). We have generated six different antisera, directed to synthetic peptide fragments of mouse IGFBP-1 through -6. The specificity of the produced antisera was demonstrated by enzyme-linked immunosorbent assay, Western blotting, and by immunohistochemistry on sections of mouse embryos of 13.5 days post coitum. Specificity for the IGFBP-2 through -6 antisera also was confirmed immunohistochemically in liver and lung of corresponding gene deletion (knock-out) mutant mice and wild-type litter mates. Immunohistochemistry and messenger RNA (mRNA) in situ hybridization on sections of mouse embryos of 13.5 days post coitum revealed tissue-specific expression patterns for the six IGFBPs. The only site of IGFBP-1 protein and mRNA production was the liver. IGFBP-2, -4, and -5 protein and mRNA were detected in various organs and tissues. IGFBP-3 and -6 protein and mRNA levels were low. In several tissues, such as lung, liver, kidney, and tongue, more than one IGFBP (protein and mRNA) could be detected. Differences between mRNA and protein localization were extensive for IGFBP-3, -5, and -6, suggesting that these IGFBPs are secreted and transported. These results confirm the different spatial localization of the IGFBPs, on the mRNA and protein level. The overlapping mRNA and protein localization for IGFBP-2 and -4, on the other hand, may indicate that these IGFBPs also function in an auto- or paracrine manner. (Endocrinology 140: 5944 ‐5952, 1999)

T‐cell regulation of neutrophil infiltrate at the early stages of a murine colitis model

Background: T‐cells are a main target for antiinflammatory drugs in inflammatory bowel disease. As the innate immune system is also implicated in the pathogenesis of these diseases, T‐cell suppressors may not only inhibit T‐cell‐dependent production of proinflammatory mediators but also affect innate immune cell function. Specifically, these drugs may impair innate immune cell recruitment and activation through inhibition of T‐cells or act independent of T‐cell modulation. We explored the extent of immune modulation by the T‐cell inhibitor tacrolimus in a murine colitis model. Methods: We assessed the effects of tacrolimus on trinitro‐benzene sulphonic acid (TNBS) colitis in wildtype and Rag2‐deficient mice. The severity of colitis was assessed by means of histological scores and weight loss. We further characterized the inflammation using immunohistochemistry and by analysis of isolated intestinal leukocytes at various stages of disease. Results: Tacrolimus‐treated wildtype mice were less sensitive to colitis and had fewer activated T‐cells. Inhibition of T‐cell function was associated with strongly diminished recruitment of infiltrating neutrophils in the colon at the early stages of this model. In agreement, immunohistochemistry demonstrated that tacrolimus inhibited production of the neutrophil chemoattractants CXCL1 and CXCL2. Rag2‐deficient mice displayed an enhanced baseline level of lamina propria neutrophils that was moderately increased in TNBS colitis and remained unaffected by tacrolimus. Conclusions: Both the innate and the adaptive mucosal immune system contribute to TNBS colitis. Tacrolimus suppresses colitis directly through inhibition of T‐cell activation and by suppression of T‐cell‐mediated recruitment of neutrophils. (Inflamm Bowel Dis 2010)

Increased production of interleukin-21, but not interleukin-17A, in the small intestine characterizes pediatric celiac disease.

Celiac disease (CD) is caused by inflammatory CD4+ T-cell responses to dietary gluten. It is unclear whether interleukin (IL)-21 and IL-17A contribute to CD onset and lesion severity; therefore, we investigated IL-21 and IL-17A expression in biopsies from pediatric CD patients with different histopathological scores. High numbers of IL-21-producing cells were observed in pediatric CD lesions, even Marsh 1–2 lesions, whereas increased numbers of IL-17 secreting cells were not observed. Intraepithelial lymphocytes, CD4+ T cells and also neutrophils secreted IL-21. Flow cytometry of lamina propria cells revealed a large population of IL-21- and interferon-γ (IFN-γ)-secreting CD3+ T cells that did not secrete IL-17A. Adult CD patient biopsies also contained high numbers of IL-21-positive cells; however, enhanced numbers of IL-17-positive cells were observed in a small subgroup of patients with severe lesions. As duodenal tissue damage increases contact with microbe-associated molecular patterns, we hypothesized that microbial sensing by Toll-like receptors (TLRs) modulates T cell–derived cytokine secretion. Costimulation with TLR3 ligands during polyclonal T-cell activation significantly increased IL-21 secretion, whereas TLR2 ligands selectively enhanced IL-17A. These results demonstrate that an IL-17A-independent increase in IL-21 production by CD4+ T cells is characteristic of pediatric CD. We hypothesize that incidental IL-17 secretion is caused by tissue damage rather than gluten-specific responses.

Methotrexate-induced mucositis in mucin 2-deficient mice

The mucin Muc2 or Mycin2 (Muc2), which is the main structural component of the protective mucus layer, has shown to be upregulated during chemotherapy‐induced mucositis. As Muc2 has shown to have protective capacities, upregulation of Muc2 may be a counter reaction of the intestine protecting against mucositis. Therefore, increasing Muc2 protein levels could be a therapeutic target in mucositis prevention or reduction. Our aim was to determine the role of Muc2 in chemotherapy‐induced mucositis. Mucositis was induced in Muc2 knockout (Muc2−/−) and wild type (Muc2+/+) mice by injecting methotrexate (MTX). Animals were weighed and sacrificed on Days 2–6 after MTX treatment and jejunal segments were analyzed. Before MTX treatment, the small intestine of Muc2+/+ and Muc2−/− mice were similar with respect to epithelial morphology and proliferation. Moreover, sucrase‐isomaltase and trefoil factor‐3 protein expression levels were comparable between Muc2+/+ and Muc2−/− mice. Up to Day 3 after MTX treatment, percentages of weight‐loss did not differ. Thereafter, Muc2+/+ mice showed a trend towards regaining weight, whereas Muc2−/− mice continued to lose weight. Surprisingly, MTX‐induced intestinal damage of Muc2−/− and Muc2+/+ mice was comparable. Prior to MTX‐injection, tumor necrosis factor‐α and interleukin‐10 mRNAs were upregulated in Muc2−/− mice, probably due to continuous exposure of the intestine to luminal antigens. Muc2 deficiency does not lead to an increase in chemotherapy‐induced mucositis. A possible explanation is the mechanism by which Muc2 deficiency may trigger the immune system to release interleukin‐10, an anti‐inflammatory cytokine before MTX‐treatment. J. Cell. Physiol. 210: 144–152, 2007.

Insulin-like growth factor binding protein-2, 28 kDa and 24 kDa insulin-like growth factor binding protein levels are decreased in fluid of dominant follicles, obtained from normal and polycystic ovaries

In order to investigate potential changes in insulin-like growth factor binding proteins (IGFBPs) during human follicle maturation, we examined the IGFBP profiles in follicular fluid from follicles in different stages of maturation. Samples were obtained from ovaries of women with regular menstrual cycles and of subjects with cycle abnormalities and polycystic ovaries (diagnosed as polycystic ovary syndrome (PCOS)) and analyzed by Western ligand blotting. IGFBPs of 43 kDa, 37 kDa, 31 kDa, a doublet around 28 kDa and a minor band of 24 kDa were detected in follicle fluid of normal non-dominant (size < 10 mm) and atretic (androstenedione/estradiol ratio > 4) follicles of both regularly menstruating women and PCOS patients. The 43 and 37 kDa IGFBPs could be identified as IGFBP-3 and the 31 kDa IGFBP as IGFBP-2, whereas the 28 kDa IGFBP could not be identified as IGFBP-1, all by immunoblotting techniques. A dramatic decrease in IGFBP-2, the 28 kDa and 24 kDa IGFBPs was observed in follicular fluid of dominant follicles (size > 10 mm) of both regular menstruating individuals and one PCOS patient as compared with follicular fluid of normal non-dominant or atretic follicles. These observations indicate that the PCOS follicle may not be different from normal with respect to IGFBP profiles. Furthermore, these results suggest that at least one of these IGFBPs might be involved in human folliculogenesis.