Lymphoid neogenesis and immune infiltration in aged liver

Abstract

Immune dysregulation and inflammation play a major role in the pathology of age-related disorders. In an earlier study, the microarray data from our laboratory indicated an increase in inflammation-related gene expression in the liver with age. We further investigated immune-related changes in the aged liver and found that the levels of inflammatory cytokines, chemokines, and inflammatory genes were higher in aged animals. Immunohistochemical studies showed that immune cells formed clusters or foci in the livers of old mice, preferentially near the perivascular regions. Further analysis revealed an enrichment of macrophages, T cells, B cells, natural killer cells, and neutrophils in old liver. Characterization of the immune clusters showed the presence of shared markers of tertiary lymphoid neogenesis. Levels of lymph node homing cytokines were elevated. Expression of immunoglobulin and recombinase gene transcripts was also higher, indicating the presence of ectopic lymphoid structures in the aged liver.

Conclusion: Aged liver exhibits a marked inflammatory status accompanied by increased immune cell infiltration. Inflammation and ectopic lymphoid structures have previously been shown to be associated with carcinogenesis, a condition that becomes more prevalent with age. Thus, further study of inflammation-related changes in the microenvironment of the aged liver could provide insights into these disorders.

Fig. 1

Changes in the immune cell population in liver with age. (A, B) Liver tissue from young (3-month-old) and old (24-month-old) mice was stained with H&E to ascertain tissue morphology and cell distribution. The immune clusters have been outlined and their respective size per square micrometer reported. (C, D) Frozen liver sections were stained for pan leukocytic marker CD45 (green) along with nuclear 4′,6-diamidino-2-phenylindole (DAPI)-stained (blue) nuclei. Hepatocytes do not show any staining with CD45, whereas the smaller nuclei in the clusters show strong peripheral CD45 staining, indicating the presence of immune cells. (magnification ×20).

Fig. 2

Distribution of the immune clusters between the perivascular (PV) and the lobular (LB) regions of the liver in young (3-month-old) and aged (24-month-old) mice in different size brackets. Six fields per individual animal at magnification ×10 (total area corresponding to 3.63 mm²) were analyzed for each sample (n = 3 young and 3 old; *P < 0.05.

Fig. 3

Frozen sections from old liver tissue (24 months) were paraformaldehyde-fixed and stained with (A) nuclear DAPI stain (blue) and the following immune cell markers: (B) CD45 for leukocytes, (C) B220 for B cells, (D) F4/80 for macrophages, (E) GR1 for granulocytes (neutrophils), and (F) CD3 for T cells (total). Hepatocytes can be visualized with DAPI as larger, round, isolated nuclei, whereas the immune cell cluster can be seen as a region of denser concentration of DAPI staining smaller nuclei (magnification ×40).

Fig. 4

Bar graph compares the mRNA levels of NK cell-specific genes in old and young mice. Gene expression was assessed by real-time PCR using cDNA from young (3-month-old) and old (24-month-old) mice (*P value < 0.05).

Fig. 5

Distribution of various immune markers in the hepatic tissue of old and young mice. (A) Number of immune clusters staining positive for a given marker per square millimeter of the tissue as well as the number of single isolated immune cells present per square millimeter of the tissue in the old and young mice (*All comparisons between young and old mice showed a significant difference [P < 0.05} except granulocytes, which showed no significant difference in single-cell distribution with age. (B) Resident macrophages of liver were visualized by immunohistochemistry for the F4/80 antigen. F4/80 staining was seen in the immune foci observed in the old mice, indicating the presence of macrophages in these foci. In both young and old liver samples, Kupffer cells were present in the sinusoids. Nuclei have been counterstained with hematoxylin (mgnification ×20).

Fig. 6

Frozen sections from liver tissue of old mice were paraformaldehyde- fixed and stained with the following markers associated with lymphoid neogenesis and nuclear DAPI stain (blue): (A) proliferation marker PCNA (magnification ×20); (B) dendritic cell marker CD11c (magnification ×40); and (C) germinal center marker peanut agglutinin (magnification ×20).