Quantitative Analysis of NKX2-3 Expression in Human Colon: An Immunohistochemical Study.

In mice, Nkx2-3 homeodomain transcription factor defines the vascular specification of secondary and tertiary lymphoid tissues of the intestines. In human studies, polymorphisms in NKX2-3 have been identified as a susceptibility factor in inflammatory bowel diseases, whereas in mice, its absence is associated with protection against experimental colitis and enhanced intestinal epithelial proliferation. Here, we investigated the expression of NKX2-3 in normal, polyp, and adenocarcinoma human colon samples using immunohistochemistry and quantitative morphometry, correlating its expression with endothelial and mesenchymal stromal markers. Our results revealed that the expression of NKX2-3 is regionally confined to the lamina propria and lamina muscularis mucosae, and its production is restricted mostly to endothelial cells and smooth muscle cells with variable co-expression of CD34, alpha smooth muscle antigen (αSMA), and vascular adhesion protein-1 (VAP-1). The frequency of NKX2-3-positive cells and intensity of expression correlated inversely with aging. Furthermore, in most colorectal carcinoma samples, we observed a significant reduction of NKX2-3 expression. These findings indicate that the NKX2-3 transcription factor is produced by both endothelial and non-endothelial tissue constituents in the colon, and its expression changes during aging and in colorectal malignancies. (J Histochem Cytochem XX: XXX-XXX, XXXX).

Absence of Nkx2-3 induces ectopic lymphatic endothelial differentiation associated with impaired extramedullary stress hematopoiesis in the spleen.

The red and white pulps as two main parts of the spleen are arranged around distinct types of vasculature, and perform significantly different functions in both humans and mice. Previous observations indicated a profound alteration of the local vessel specialization in mice lacking Nkx2-3 homeodomain transcription factor, including contradictory results suggesting presence of an ectopic lymphatic vascular structure. Furthermore, how the absence of Nkx2-3 and the consequential changes in endothelial components affect the extramedullary hematopoietic activity restricted to the splenic red pulp is unknown. In this work, we investigated the role of Nkx2-3 homeodomain transcription factor as a major morphogenic determinant for vascular specification, and its effect in the extramedullary hematopoiesis following acute blood loss and pharmacological stimulation of megakaryocyte differentiation after treatment with thrombopoietin-receptor mimetic Romiplostim. We found that, in mice lacking Nkx2-3, Prox1-positive lymphatic capillaries containing gp38/CD31 double positive lymphatic endothelial cells develop, arranged into an extensive meshwork, while the Clever1-positive venous segments of red pulp blood vasculature are absent. This lymphatic endothelial shift is coupled with a severely compromised splenic erythropoiesis and a significantly reduced splenic megakaryocyte colony formation following Romiplostim treatment in mice lacking Nkx2-3. These findings indicate that the shift of microvascular patterning in the absence of Nkx2-3 includes the emergence of ectopic Prox1-positive lymphatic vessels, and that this pivoting towards lymph node-like vascular patterning is associated with an impaired reserve hematopoietic capacity of the splenic red pulp.

An NKX-COUP-TFII morphogenetic code directs mucosal endothelial addressin expression.

Immunoglobulin family and carbohydrate vascular addressins encoded by Madcam1 and St6gal1 control lymphocyte homing into intestinal tissues, regulating immunity and inflammation. The addressins are developmentally programmed to decorate endothelial cells lining gut post-capillary and high endothelial venules (HEV), providing a prototypical example of organ- and segment-specific endothelial specialization. We identify conserved NKX-COUP-TFII composite elements (NCCE) in regulatory regions of Madcam1 and St6gal1 that bind intestinal homeodomain protein NKX2-3 cooperatively with venous nuclear receptor COUP-TFII to activate transcription. The Madcam1 element also integrates repressive signals from arterial/capillary Notch effectors. Pan-endothelial COUP-TFII overexpression induces ectopic addressin expression in NKX2-3+ capillaries, while NKX2-3 deficiency abrogates expression by HEV. Phylogenetically conserved NCCE are enriched in genes involved in neuron migration and morphogenesis of the heart, kidney, pancreas and other organs. Our results define an NKX-COUP-TFII morphogenetic code that targets expression of mucosal vascular addressins.

Role of adipose-associated lymphoid tissues in the immunological homeostasis of the serosal surface.

Although not typical lymphoid organs, analysis of the visceral adipose-associated lymphoid tissues has recently substantially expanded our knowledge about the immunological features of these elusive compartments. Recent data have highlighted their considerable complexity in cellular organization and interactions in several biological processes, including adaptive immune responses, tissue plasticity to accommodate mesenchymal stem cells and progenitors, and providing a suitable microenvironment for serosal tumor propagation. This review aims to present a comprehensive view of the adipose-associated lymphoid tissues in local and systemic immune responsiveness, with particular emphasis on the omental and mesenteric lymphoid tissues in the serosal defense of abdominal organs.

Ameliorated Autoimmune Arthritis and Impaired B Cell Receptor-Mediated Ca2+ Influx in Nkx2-3 Knock-out Mice.

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3-/-) the spleen's histological structure is fundamentally changed; therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3-/- mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3-/- and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca2+ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3-/- mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3-/- mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures; in the sera, we found less anti-CCP-IgG2a, IL-17 and IFNγ, but more IL-1ß, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3-/- mice showed decreased intracellular Ca2+ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation.

Foliate Lymphoid Aggregates as Novel Forms of Serous Lymphocyte Entry Sites of Peritoneal B Cells and High-Grade B Cell Lymphomas.

The cellular homeostasis of lymphoid tissues is determined by the continuous interactions of mobile hematopoietic cells within specialized microenvironments created by sessile stromal cells. In contrast to the lymph nodes and mucosal lymphoid tissues with well-defined entry and exit routes, the movement of leukocytes in the peritoneal cavity is largely unknown. In this study, we report that, in addition to the omental milky spots and fat-associated lymphoid clusters, in mice, the serous surface of the mesenteric adipose streaks contains lymphocyte-rich organoids comprised of a highly compacted leaf-like part connected to the adipose tissue that can also efficiently bind B cells and high-grade B cell lymphoma (diffuse large B cell lymphoma) cells. Denoted as foliate lymphoid aggregates (FLAgs), these structures show incomplete T/B segregation and a partially differentiated stromal architecture. LYVE-1-positive macrophages covering FLAgs efficiently bind i.p. injected normal B cells as well as different types of diffuse large B cell lymphoma cells. Within FLAgs, the lymphocytes compartmentalize according to their chemokine receptor pattern and subsequently migrate toward the mesenteric lymph nodes via the mesenteric lymphatic capillaries. The blood supply of FLAgs includes short vascular segments displaying peripheral lymph node addressin, and the extravasation of lymphocytes to the omental and mesenteric adipose tissues is partly mediated by L-selectin. The appearance of i.p. injected cells in mesenteric lymph nodes suggests that the mesentery-associated lymphatics may also collect leukocytes from the fat-associated lymphoid clusters and FLAgs, thus combining the mucosal and serous exit of mobile leukocytes and increasing the range of drainage sites for the peritoneal expansion of lymphoid malignancies.

Differential Effects of the Absence of Nkx2-3 and MAdCAM-1 on the Distribution of Intestinal Type 3 Innate Lymphoid Cells and Postnatal SILT Formation in Mice.

Seeding of leukocytes to developing lymphoid tissues in embryonic and early postnatal age and to the mucosa throughout adulthood depends on the interaction between endothelial MAdCAM-1 addressin and its cognate ligand α4ß7 integrin. Nkx2-3 as a transcriptional regulator of MAdCAM-1 controls vascular patterning in visceral lymphoid tissues in mice, and has been identified as a susceptibility factor for inflammatory bowel diseases in humans, associated with lymphoid neogenesis in the inflamed intestines. The role of Nkx2-3 in the organogenesis of the solitary intestinal lymphoid tissues (SILTs) involving type 3 innate lymphoid cells (ILC3) is still unknown. Here we investigated the effect of Nkx2-3 on the postnatal distribution of intestinal ILC3s and the development of SILTs, comparing these to mice lacking MAdCAM-1, but preserving Nkx2-3. At 1 week of age small intestines (SI) contained significantly higher number of ILC3s relative to the colon, with a substantial reduction in MAdCAM-1-/- mice compared to C57BL/6 controls. One week later SI ILC3 number decreased in all genotypes, the number of colonic ILC3 of both Nkx2-3-deficient and Nkx2-3-heterozygous mice significantly increased. On the fourth postnatal week a further reduction of SI ILC3s was observed in both Nkx2-3-deficient and Nkx2-3-heterozygous mice, while in the colon the number of ILC3s showed a significant reduction in all genotypes. At 1 week of age only sporadic SILT components were present in all genotypes. By the second week mice deficient for either Nkx2-3 or MAdCAM-1 showed absence of SILT maturation compared to their relevant controls, lacking mature isolated lymphoid follicles (ILF). By the fourth week both Nkx2-3-deficient and Nkx2-3-heterozygous mice showed a similar distribution of ILFs relative to cryptopatches (CP), whereas in MAdCAM-1-/- mice CPs and immature ILFs were present, mature ILFs were scarce. Our data demonstrate that the complete absence of MAdCAM-1 partially impairs intestinal seeding of ILC3s and causes partial blockade of SILT maturation, without affecting peripheral lymph node development. In contrast, the inactivation of Nkx2-3 permits postnatal seeding, and its blocking effect on SILT maturation prevails at later stage, thus other adhesion molecules may compensate for the intestinal homing of ILC3s in the absence of MAdCAM-1.