Murine Liver-draining Lymph Nodes And Their Functions

Liver is the largest solid organ with critical functions in metabolism in our body. It also works as an immunological organ with predominant innate immunity and propensity for tolerance. On the one hand, innate immune cells are abundant in the liver and respond rapidly to clear harmful exogenous substances, such as microorganism-derived toxins and blood-borne pathogens. On the other hand, due to abundant inhibitory signals and the lack of co-stimulatory signals, immune responses initiated in the liver always exhibit a propensity for tolerance. The liver has well-developed lymphatic networks and produces large amounts of lymphatic fluid that drains into downstream liver-draining lymph nodes (LNs). However, although liver-draining LNs are important parts of the liver immune system, their roles in fulfilling liver immune functions are still unclear. Besides, basic knowledge of liver-draining LNs is still lacking.In this study, we identified portal LN and celiac LN in the hepatic hilar region as liver-draining LNs in mice and studied their basic structure, cellular composition,organogenesis and immune functions. The results were divided into four major aspects:1. The anatomical location of liver-draining LNsLymph drainage of liver was visullized by injecting Even's blue dye into the liver parenchyma. Two LNs in the hepatic lilar region were identified as liver-draining LNs:one LN was stably attached to the portal vein and named the portal LN, the other one which lied in the hepatoduodenal ligament and was not amccompanied with portal vein,was much deeper in the peritoneal cavity and named the celiac LN. Portal LN and celiac LN drain liver lymph through different lymphatic vessels, indicating that they are independent liver-draining LNs. Beside. the anatomical location of portal LN and celiac LN were conserved in mouse, rat and treeshrew.2. The structure and cellular compositions of liver-draining LNsAs LN is composed of highly organized cells, we observed the structure of these two liver-draining LNs. Both portal LN and celiac LN showed typical LN structures.The expression patterns of vascular addressins were also examined. Noteworthily, both peripheral node addressin and mucosal addressin cell adhesion molecule-1(MAdCAM-1) were expressed in liver-draining LNs. We further analyzed the immune cell cormpositions of portal LN and celiac LN. Although the proportions of several major immune cells were similar among different LNs. obvious differences were observed in particular cell subsets. For example, more NKT cells, DX5-CXCR3+ NK cells and CD 103+ DCs were observed in liver-draining LNs and mesenteric LNs compared to peripheral LNs. Conversely, more ICOS+ Tregs, CD27+CD44hi ??T and CD11b+ NK cells were observed in peripheral LNs compared to liver-draining LNs and mesenteric LNs. Moreover. substantial differences were detected between portal LN and celiac LN: NKT and ??T cells were more abundant in celiac LN; germinal center B cells were present in portal LN but rare in celiac LN: CD103+ DCs was a major population of DCs in portal LN but minor in celiac LN.3. The organogenesis of liver-draining LNsWe studied the timeline for the organogenesis of portal LN and celiac LN by treating pregnant mice with LT?R-Ig-Fc, which blocks the LT(3R signaling and thus abolished the iniation of LN organogenesis, at sequential gestational days. We identified that the organogenesis of portal LN was initiated before gestational day 15.5 and partially dependent on LT?R signaling, while the organogenesis of celiac LN initiated between gestational day 16.5 and 18.5 and completely dependent on LT?R signaling. Interestingly, the organogenesis of celiac LN was abnormal in Nfil3-/- mice,which showed decreased number and size of celiac LN, while the organogenesis of portal LN was not impaired by NFIL3 deficiency. Overall, the developmental differences in the signaling pathways involved, time of formation, and controlling transcription factor characterize portal LN and celiac LN as two different LNs.4. The functions of liver-draining LNsSince portal LN and celiac LN differ in their immune cell compositions and organogenesis processes, we examined whether they have distinct functions. As clearing blood-borne pathogens and inducing systemic tolerance to harmless molecules are two major immune function of the liver, we studied the functions of portal LN and celiac LN by establishing mouse models of blood-borne viral infections and oral tolerance. Utilizing hepatotropic adenovirus infection model, hepatitis B virus infection model, acute and chronic lymphocytic choriomeningitis virus infection models, we revealed that celiac LN was the predominant site for liver antiviral immune responses,while portal LN only induced modest immune responses. Utilizing a common model to study oral tolerance by observing antigen-specific Treg induction after oral administration of ovabulmin, we found that portal LN was the site for in situ induction of antigen-specific Treg cells. Althought antigen recognition of oral antigen also happened in celiac LN, no antigen-specific Treg cells were in situ induced.Conclusion: Portal LN and celiac LN are two independent liver-draining LNs in mice.Although portal LN and celiac LN show typical LN structures and expressed both peripheral node addressin and mucosal addressin, they exhibit differences in immune cell compositions and organogenesis. Portal LN and celiac LN also manifest distinct functional priorities in fulfilling the major immune functions of the liver. The current study highlights the celiac LN as the predominant site for liver antiviral immune responses and the portal LN as the site for the induction of antigen-specific Tregs following oral antigen administration. Further insights into the organogenesis and functions of liver-draining LNs will lead to a better understanding of immune homeostasis in the liver.