Aligning cellular and molecular components in age-dependent tertiary lymphoid tissues of kidney and liver

Abstract

Tertiary lymphoid tissues (TLTs) are ectopic lymphoid structures induced by multiple stimuli, including infection and tissue injuries; however, their clinical relevance in disease progression has remained unclear. We demonstrated previously that TLTs develop in mouse and human kidneys with aging and can be a potential marker of kidney injury and prognosis, and therapeutic targets. In addition, we found that two types of unique lymphocytes that emerge with aging, senescence-associated T cells and age-associated B cells, are essential for TLT formation in the kidney. Although TLTs develop with aging in other organs as well, their cellular and molecular components, and clinical significance remain unclear. In the present study, we found that TLTs developed in the liver with aging, and that their cellular and molecular components were similar to those in the kidneys. Notably, senescence-associated T cells and age-associated B cells were also present in hepatic TLTs. Furthermore, analysis of publicly available data on human liver biopsy transcriptomes revealed that the expression of TLT-related genes was elevated in the liver biopsy samples from hepatitis C virus (HCV)-infected patients compared with those without HCV infection and was associated with liver injury and fibrosis. Therefore, we analyzed liver biopsy samples from 47 HCV patients and found that TLTs were present in 87.2% of cases and that the numbers and stages of TLTs were higher in aged patients and cellular and molecular components of TLTs in humans were similar to those in mice. Our findings suggesting that age-dependent TLT formation is a systemic phenomenon across the tissues and aging is also a predisposing factor for TLT formation across organs.

Fig 1. Superaged mouse develops hepatic TLTs without injury.

(A) Hematoxylin and eosin (H&E) staining. Quantitative analysis of (B) TLT frequencies, (C) TLT numbers, and (D) relative TLT sizes in young and superaged mouse livers (n =  6 per group). Immunofluorescence analysis of (E) lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and smooth muscle actin (α-SMA); (F) CD3ε (a T-cell marker) and B220 (a B-cell marker); (G–I) CD21 (a follicular dendritic cell (FDC) marker) and Ki67; (J) B220 and Ki67; (K, L) CD45 and p75 neurotrophin receptor (p75NTR); (M) p75NTR and CD21; (N, O) Desmin (a hepatic stellate cell marker) and p75NTR; (P) CXCL13 (a homeostatic chemokine) and CD45; (Q) CCL19 (a homeostatic chemokine) and CD45; (R) Desmin and CXCL13; and (S) CD21 and CXCL13. (T) mRNA expression of Cd3e, Cd19, Des (Desmin), and Ngfr (p75ntr) in young and superaged mouse livers (n =  6 per group). (U) Correlations between relative TLT sizes and mRNA levels of Cxcl13 and Ccl19 in young and superaged mouse livers (n =  6 per group). The mRNA expression levels are normalized to those of Gapdh and expressed relative to those of the young mouse. (V) Representative immunofluorescence of three distinct stages (I, II, and III) of TLTs. Immunofluorescence analysis of CD3εand B220; CD21 and Ki67 in each stage are serial sections. Stage I TLTs are the aggregation of lymphocytes with the sign of proliferation, and do not include FDCs or germinal centers; Stage II TLTs include FDCs, but not germinal centers; Stage III TLTs include FDCs and germinal centers. (W) The relative numbers of stages I, II, and III TLTs and the proportions of stages I, II, and III TLTs in superaged mouse livers without injury (n =  6). In (C, D, T, W) data are shown as the median and interquartile range. Mann–Whitney test (C, D, T) was used to analyze the difference. Correlations in (U) are determined by Pearson’s correlation analysis. TLT number is shown as the number per area of section. * p ≤  0.05 and **p ≤  0.01. Arrows indicate TLT localization. Scale bars: 50 µm. Abbreviations: CV, central vein; PT, portal tract.

Fig 2. SAT cells and ABCs expand within hepatic TLTs in superaged mice.

Immunofluorescence analysis of GFP and CD45 in the liver (A, B), kidney (C), and spleen (D) of superaged Spp1-EGFP-KI mice. Immunofluorescence analysis of (E) GFP and CD3ε; (F) GFP and B220; (G) GFP and CD11c (a dendritic cell marker); (H) GFP and CD21; (I) GFP and Desmin; (J) GFP and p75NTR; (K) GFP and Ki67; and (L) GFP and p21 in the livers of superaged Spp1-EGFP-KI mice, and (M) B220 and CD21 in the livers of superaged C57BL6J mice. In situ hybridization images of (N) Cd3e and Tnfsf8 with immunofluorescence co-staining for CD45; and (Ο) Cd19 and Tnfrsf8 with immunofluorescence co-staining for CD45 in the livers of superaged C57BL6J mice. Scale bars in (A-M): 50 µm; in (N-O): 25 µm.

Fig 3. TLT-related gene expression is upregulated in the liver of HCV patients and is associated with liver injury and fibrosis.

(A, C-E) Reanalysis of published RNA-seq data of human liver samples of patients with HCV infection (HCV, n =  6) or without HCV infection (non-HCV, n =  21) and (B) reanalysis of published RNA-seq data of human samples of patients with HBV infection (HBV, n =  4) or without HBV infection (non-HBV, n =  4). In (A, B) data are shown as the median and interquartile range. Mann–Whitney test is used to analyze the difference (A, B). Correlations in (C–E) are determined by Pearson’s correlation analysis. **p ≤  0.01, ***p ≤  0.001. Abbreviation: RPKM, reads per kilobase of exon per million mapped reads; FPKM, fragments per kilobase per million mapped fragments.

Fig 4. Hepatic TLTs in HCV patients.

(A–K) Histological analyses of HCV patients’ liver samples. (A, B) Hematoxylin and eosin (H&E) and (C) silver stain. Immunofluorescence analysis of (D) CD3ε (a T-cell marker) and CD20 (a B-cell marker); (E) Ki67 and CD23 (a follicular dendritic cell marker); (G) Desmin (a satellite cell marker); (H) α-SMA and (I) CXCL13 (a homeostatic chemokine) and CD45. (F) Immunohistochemical analysis of CD21 (a follicular dendritic cell marker). (J) In situ hybridization images of TNFSF8 with immunofluorescence co-staining for CD3ε (K) Representative immunofluorescence analysis of two distinct stages (I and II) of TLTs. Stage I TLTs are the aggregation of lymphocytes with the sign of proliferation, and do not include FDCs or germinal centers; Stage II TLTs include FDCs, but not germinal center. Immunofluorescence analysis of CD3ε and CD20; Ki67 and CD23 in each stage are serial sections. (L–N) Quantification of hepatic TLTs in young and aged patients with HCV infection (n =  22 and 25, respectively). (L) TLT frequencies in young and aged patients are 72.7% and 100%, respectively. (M) The relative numbers of TLTs per portal tract number. The medians of those in young and aged patients are 0.125 and 0.138, respectively. (N) The proportion of patients in each TLT stages. The proportion of patients with stage I TLTs in young and aged patients are 45% and 48%, respectively, while that of patients with stage II TLTs are 27% and 52%, respectively. (O) Percentage of patients in each fibrosis stage according to the new Inuyama classification stratified by hepatic TLT presence (no TLT: n =  6, with TLTs: n =  41). Arrows indicate TLT localization. Scale bars: in (A) 200 µm; (B–K) 50 µm. Abbreviations: CV, central vein; PT, portal tract.