TNFα-dependent development of lymphoid tissue in the absence of RORγt⁺ lymphoid tissue inducer cells

Abstract

Lymphoid tissue often forms within sites of chronic inflammation. Here we report that expression of the proinflammatory cytokine tumor necrosis factor α (TNFα) drives development of lymphoid tissue in the intestine. Formation of this ectopic lymphoid tissue was not dependent on the presence of canonical RORgt(+) lymphoid tissue-inducer (LTi) cells, because animals expressing increased levels of TNFα but lacking RORgt(+) LTi cells (TNF/Rorc(gt)(-/-) mice) developed lymphoid tissue in inflamed areas. Unexpectedly, such animals developed several lymph nodes (LNs) that were structurally and functionally similar to those of wild-type animals. TNFα production by F4/80(+) myeloid cells present within the anlagen was important for the activation of stromal cells during the late stages of embryogenesis and for the activation of an organogenic program that allowed the development of LNs. Our results show that lymphoid tissue organogenesis can occur in the absence of LTi cells and suggest that interactions between TNFα-expressing myeloid cells and stromal cells have an important role in secondary lymphoid organ formation.

Figure 1. TLO are formed in the ileum TNF/Rorc(γt)^−/− mice

(a) Representative H&E sections of the Ileum of WT, Rorc(γt)^−/−, TNF/Rorc(γt)^+/+ and TNF/Rorc(γt)^−/− mice at 16 wks. Notice the presence of inflammatory infiltrates in the ileum of TNF/Rorc(γt)^+/+ and TNF/Rorc(γt)^−/− mice. (b) Ileum sections of indicated mice were stained with anti-B220 antibody to visualize B cell aggregates and DAPI for nuclear staining. Small B cell clusters were found in the ileum of WT but were absent in the ileum of Rorc(γt) ^−/− mice. (c) Overexpression of TNF induced the formation of large B cell clusters with few T cells in the ileum of TNF/Rorc(γt)^+/+ and TNF/Rorc(γt)^−/− mice. Scale bars = 250 μm, n=4/group.

Figure 2. Increased expression of TNF induces development SLO in the absence of RORγt⁺ LTi cells

(a) Photograph of the mesentery of WT, Rorc(γt)^−/−, TNF/Rorc(γt)^+/+, and TNF/Rorc(γt)^−/− mice. Photograph of axilary (b) and cervical (c) lymph nodes of WT, TNF/Rorc(γt)^+/+ and TNF/Rorc(γt)^−/− mice. (d) Incidence of mesenteric (Mes), axillary (Axi), cervical (Cer), brachial (Bra), inguinal (Ing), para aortic (PA), peripancreatic (Pan), popliteal (Pop), mediastinal (Med) lymph nodes, and Peyer’s patches (PP) formed in TNF/Rorc(γt)^−/− mice (n = 80). (e) Lymph nodes from TNF/Rorc(γt)^+/+ and TNF/Rorc(γt)^−/− mice at 6 wk of age were analyzed by immunostaining. Note segregation of T and B cell areas, presence of PNAd⁺ HEV and lymphatic vessels, normal distribution of ER-TR7⁺ meshwork and CD35^bright FDC in mesenteric (mLN) and axillary (aLN) lymph nodes of TNF/Rorc(γt)^−/− mice. These features were similar to the ones observed in the mLn of TNF/Rorc(γt)^+/+ mice (n = 5 mice/group). Scale bars = 250μm. (f) OVA-specific IgG and IgA measured in the serum of TNF/Rorc(γt)^+/+ (n = 5) and TNF/Rorc(γt)^−/− (n = 4) obtained after 5 rounds of immunization. (g) IFN-γ and (h) IL-17 levels in supernatants of cultured MLN cells 7 weeks after OVA immunization.

Figure 3. Phenotype of the cells present in the mLN anlagen of Rorc(γt)^−/− and TNF/Rorc(γt)^−/− mice

Cell suspensions from the mLN region of RORγt^−/− and TNF/Roc(γt)^−/− mice at P0.5-P1 stage were analyzed by flow cytometry for the expression of the indicated markers. Cells were gated on PI⁻CD45⁺. Representative plots of 3 independent experiments (n=2–3/group).

Figure 4. F4/80⁺/CD11b⁺ cells produce increased levels of TNF in the mLN of TNF/RORγt^−/− mice

(a) Flow cytometric analysis of TNF production by CD45⁻, CD45⁺F4/80⁻ and CD45⁺F4/80⁺ cells isolated from the mLN region of RORγt^−/− and TNF/Roc(γt)^−/− mice at P0.5-P1 stage. (b) Analysis of TNF production by CD45⁺F4/80^hiCD11b^low and CD45⁺F4/80^lowCD11b^high cells. Representative plot of two independent experiments, (n=4–5 animals/group).

Figure 5. TNF/Id2^−/− mice lack F4/80⁺ cells in the mLN anlagen and do not develop SLO

mLN region of TNF/Id2^+/− and TNF/Id2^−/− at P0 stained with CD45, F4/80, LYVE-1 antibodies. Notice the absence of F4/80⁺ cells in the MLN region of Id2^−/− and TNF/Id2^−/− mice (dashed lines). Representative staining (n = 3/group). Scale bars; 250μm.

Figure 6. Increased expression of genes involved in lymph node organogenesis in the mLN of TNF/Rorc(γt)^−/−mice

(a–b) Transcriptional profiles of mLN anlagen of Rorc(γt)^−/− and TNF/Rorc(γt)^−/−mice at P1. Samples (each column corresponds to a pool of 2–3 anlagen) were compared using MouseRef-8 v2.0 Expression BeadChip. Quantile-normalized expression values were filtered for p < 0.01 and log fold change (logFC) > 1.25 (= fold 2.38). (a) Heatmap analysis sorted by logFC of the 193 resulting probe sets were Z-score normalized and subjected to hierarchical clustering; increased (red) decreased (green) expression in TNF/Rorc(γt)^−/− compared to Rorc(γt)^−/− mice. (b) Fold-change of selected up-regulated genes. (c) qPCR analysis of selected genes in the MLN region of Rorc(γt)^−/− and TNF/Rorc(γt)^−/−mice at different stages (n=3/group).

Figure 7. TNF induces stromal cell activation

(a) FACS analysis of single-cell suspensions from the mLN region of Rorc(γt)^−/− and TNF/Rorc(γt)^−/− mice at E17.5, P0, P1, and P3 showing the increased expression of ICAM-1/VCAM-1 in the CD45 negative stromal cell population. (b) Expression of ICAM-1/VCAM-1 in stromal cell population in the mLN of WT and TNF^ΔARE/+ mice at P1. (c) Relative number of ICAM-1^hiVCAM-1^hi cells in the CD45 negative stromal cells in the mLN region of Rorc(γt)^−/− and TNF/Rorc(γt)^−/− at E17.5, E18.5, and P0 (n = 4 mice/group), P1 (n = 7 mice/group), and P3 (n= 6 mice/group). (d) Expression of CXCL13, CXCL19 and CCL21 in the stromal (CD45 negative) cell population sorted from the mLN region of Rorc(γt)^−/− and TNF/Rorc(γt)^−/− mice at P0.5-P1 (n = 9–10 anlagen/group).

Figure 8. TNF-driven formation of most SLO is dependent on LTβR signaling

(a) Mesentery of LTβR^−/− and TNF/LTβR^−/− mice. (b) With the exception of mLN, SLO were absent in TNF/LTβR^−/− mice (n = 16). (c) Abnormal organogenesis of mLN in TNF/LTβR^−/− mice. mLN of TNF/LTβR^+/+ and TNF/LTβR^−/− mice were stained with the indicated antibodies. Notice the lack of distinct T and B cell areas, absence of of PNAd+ HEVs and reduced lymphatic vasculature, lack of CD35⁺ FDC, and disorganized ER-TR7 stroma in the MLN of TNF/LTβR^−/− mice. Representative staining (n = 5/group). Scale bars = 250μm.

Figure 9. Hematopoietic cell influx into the mLN anlagen

Sections of the mLN region of WT, Rorc(γt)^−/− and TNF/Rorc(γt)^−/− (P0 and P5) mice were stained with anti-CD45, -F4/80 and -LYVE-1 antibodies. Representative staining (n = 3/group). Scale bars = 250μm.

Figure 10. mLN anlagen from WT and TNF/Rorc(γt)^−/− mice promote development of LN-like structures when grafted under the kidney capsule

(a) Schematic representation of the transplantation experiment. (b) mLN anlagen isolated from WT (n = 3) and TNF/Rorc(γt)^−/− (n = 14) newborn mice were grafted under the kidney capsule of Rorc(γt)^−/− mice. Notice normal segregation of T and B cells and development of PNAd⁺ HEV from WT and TNF/Rorc(γt)^−/− grafts. Scale bars = 250μm.