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. 2019 Mar 5:10:366.
doi: 10.3389/fimmu.2019.00366. eCollection 2019.

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

Dóra Vojkovics  1   2 , Zoltán Kellermayer  1   2 , Fanni Gábris  1   2 , Angela Schippers  3 , Norbert Wagner  3 , Gergely Berta  4 , Kornélia Farkas  5 , Péter Balogh  1   2
Affiliations

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

Dóra Vojkovics et al. Front Immunol. .

Abstract

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 α4β7 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.

Keywords: ILC3; MAdCAM-1; NKX2-3; cryptopatch; isolated lymphoid follicle.

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Figures

Figure 1
Figure 1
Different distribution of ILC3 in the postnatal gut in Nkx2-3−/− and MAdCAM-1−/− mice. (A) Gating strategy for the identification of ILC3s defined on the basis the lymphoid gate (left: FSC/SSC ellipse) and CD45+/non-T/B lineage (middle: rectangle) combined with CD90/RORγt (right, rectangle; representative example from a small intestine of a 4 weeks old C57BL/6 mouse). (B) Kinetics of the absolute number of ILC3s in Nkx2-3−/− and Nkx2-3+/− mice (top) and MAdCAM-1−/− and wild-type C57BL/6 (bottom) intestine (small intestine: left, colon: right) at various ages as indicated (n = 3–7, mean ± SEM, *p < 0.05). (C) Representative example of the appearance of ILC3s in the small intestine and colon of Nkx2-3−/−, Nkx2-3+/−, MAdCAM-1−/−, and wild-type C57BL/6 mice at 4 weeks of age.
Figure 2
Figure 2
Composition of postnatal colonic SILT spectrum. (A) Representative samples for cryptopatches (CP), immature (imILF), and mature (matILF) isolated lymphoid follicles of a 4 weeks old Nkx2-3+/− mouse using CD45/Thy-1/B220 combined immunofluorescence staining with the markers as indicated (scale bar = 100 μm). (B) Composition of colonic SILT spectrum in Nkx2-3+/− (black filled) and Nkx2-3−/− (gray filled) mice at 1, 2, and 4 weeks of age as indicated; n = 4; mean ± SEM, *p < 0.05). (C) Composition of SILT spectrum in the colon of wild-type C57BL/6 (black filled) and MAdCAM-1−/− (gray filled) mice at 1, 2, and 4 weeks of age as indicated (n = 4; mean ± SEM *p < 0.05).
Figure 3
Figure 3
Comparison of expression of mRNA expression for peripheral lymph node addressin core proteins and glycosylation enzymes in Peyer's patches between MAdCAM-1−/− and wild-type mice and the expression of MECA-79 PNAd epitope. (A) qPCR analyses were performed for the various core proteins (GlyCAM, endomucin, podocalyxin-like protein, CD34, and nepmucin) and glycosylation enzymes, expressed as a mRNA level relative to β-actin (n = 6; mean ± SEM, in a duplicate measurement; *p < 0.05). (B) Immunofluorescence detection of MECA-79 epitope (green) in wild-type (left) and MAdCAM-1−/− Peyer's patches with Hoechst nuclear counterstaining (blue) (representative example of a cohort of 3 mice; scale bar = 100 μm).
Figure 4
Figure 4
Quantification of the expression of endothelial addressins in the postnatal period. (A) Relative pixel intensities (mean gray values, y-axis) of MAdCAM-1 labeling of small intestine (left) and colon (right) in Nkx2-3+/− (black filled) and Nkx2-3−/− (gray filled) at the periods indicated at the x-axis (n = 5, mean ± SEM, in a duplicate measurement; *p < 0.05). (B) Relative pixel intensities (mean gray values, y-axis) of PNAd labeling of small intestine (left) and colon (right) in C57BL/6 (black filled) and MAdCAM-1−/− (gray filled) at the periods indicated at the x-axis (n = 5, mean ± SEM, in a duplicate measurement; *p < 0.05).
Figure 5
Figure 5
Preserved structure of pLN in the absence of MAdCAM-1. Cryostat sections from inguinal lymph nodes of 6 weeks old Nkx2-3−/−, Nkx2-3+/−, MAdCAM-1−/−, and C57BL/6 mice were stained for B cells (B220/turquoise), T cells (Thy-1/red), and follicular dendritic cells (CD21/35/green) as indicated (scale bar: 100 μm. Representative image, the staining was repeated twice).
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