Histology Histopathology, 2015 ; 30 (8):931-43

Thymic alterations induced by partial hepatectomy: upregulation of glycoprotein 96, CD91 and TLR2 and generation of regulatory T cells.

Jakovac H, Ćuk M, Trobonjača Z, Mrakovčić-Šutić I, Radošević-Stašić B.

Department of Physiology and Immunology, Medical Faculty, University of Rijeka, Rijeka, Croatia



Glycoprotein 96 (gp96) is an endoplasmic reticulum (ER)-resident heat shock protein. It controls the folding of nascent membrane-spanning and secretory proteins, participates in stress-induced unfolded protein response (UPR) and in pathways leading to proteolysis of damaged proteins through ER-associated degradation pathways and chaperone-mediated autophagy. In addition, gp96 controls the steroid biosynthesis and Ca2+ homeostasis and participates in insulin-IGF/signaling pathways. Besides, owing to its peptide chaperone capacity and ability to interact with antigen-presenting cells, gp96 has been implicated in priming of innate and adaptive immunity.

In an attempt to visualize the intensity of ER-stress in thymus and possible participation of gp96 in generation of auto-reactive T cell clones that were detected in regenerating liver, in this study we investigated the dynamics of gp96 expression in partially hepatectomized (pHx) and sham Hx mice. Simultaneously, we detected the thymic expression of receptors responsible for endocytosis of gp96-chaperoned peptides (CD91) and intracellular activation of ER-stress pathways (TLR2), as well as the expression of TGF-β and the distribution of CD4+CD25+FoxP3+ cells. The data have shown that both pHx and sham Hx induced an accelerated apoptosis and hypoplasia in thymus. Partial Hx induced, however, a higher expression of gp96, the translocation of the CD91, TLR2 and TGF-β immunostaining from medulla to cortex and an appearance of Treg cells.

The data show that pHx triggers in thymus the ER stress and UPR response and suggest that gp96 participates in the generation of natural Treg cells, which might be involved in the control of liver regeneration in the periphery.

Key words: Liver regeneration, Thymus, Glycoprotein 96, CD91, TLR2, TGF-beta, Regulatory T cells DOI: 10.14670/HH-11-597



The thymus is the primary lymphoid organ responsible for T cell development and the establishment of central self-tolerance. The elimination of potentially auto-aggressive clones from the CD4+ and CD8+ T cell repertoires is a result of a multistep process, referred as major histocompatibility complex (MHC) restriction, which is based on complex interaction of developing thymocytes, expressing a T-cell antigen receptor (abTCR)-CD3 complex, with antigen-presenting cells (APC) expressing MHC-self peptides complexes [1-7]. In addition, the intrathymic T-lymphocyte selection is under the tight control of neuro-endocrine system and thymic microenvironments, created by distinct types of stromal cells, which secrete soluble polypeptides, such as thymic hormones and interleukins, and define the matrix through which developing thymocytes migrate toward maturity [2, 8].

According to current models of thymocyte selection the development of T cells depends on the “strength” of the individual TCR/MHC–self peptide interaction. Very strong interactions lead to negative selection and elimination of potentially auto-aggressive cells, whereas the low and intermediate affinity allow the positive selection and maturation of thymocytes to the single-positive CD4+ and CD8+T cells and CD4+CD25+Foxp3+ regulatory T (Treg) cells that are released from the thymus. The later participate in preventing autoimmunity, but many questions about their generation and processes that instruct thymocytes to undergo deletion or survival are still poorly understood [1-7].

In an attempt to visualize the changes in the thymus during disturbance of morphostasis, in this study we used a partial hepatectomy (pHx) as a model for the induction of fast liver growth and activation of innate and adaptive immune system, which during liver regeneration need to prevent the potential development of autoimmune attack and suppress the effects of endogenous- and/or exogenous danger- and pathogen-associated molecular patterns that may be released by tissues undergoing stress, damage or abnormal cell death. Besides, since we previously found that in regenerating liver markedly arose the content of the glycoprotein 96 (gp96)[9], an endoplasmic reticulum (ER) resident, heat shock protein (HSP) with chaperon and immunogenic function [10, 11], in thymus of pHx and sham operated mice we analyzed the expression of gp96 and the relationship of gp96 to CD91 and Toll like receptor 2 (TLR2) that were described as receptors responsible for uptake of gp96-peptide complexes on antigen-presenting cells (APC) [12] and for the antigen-unspecific activation of APC [13]. In addition, in thymi of pHx mice we tested the TGF-b expression, the distribution of Treg cells, the phenotypic profile of T cells and the intensity of apoptosis.

The data are summarized on Fig. 1 which shows the changes induced by pHx in the liver (A) and in the thymus (B) and gives a schematic overview of potential mechanisms by which the gp96 may participate in maintenance of central tolerance (C, D). In the liver we found a high upregulation of gp96 (Fig. 1a) and accumulation of NKT cells, NK and Treg cells [9]. In the thymus we point to  the impressive upregulation of gp96 and high apoptosis induced by pHx (Fig. 1b), to co-localization of gp96 and CD91 (Fig. 1d) and gp96 and TLR2 (Fig. 1e) on thymic stromal cells,  to the expression of gp96 on the surface of thymic nurse cells, containing several apoptotic and Caspase-3+ cells (Fig. 1c),  as well as to the appearance of numerous Treg cells (CD25+FoxP3+ cells) in the thymic cortex (Fig. 1f). In this study we also emphasize that during liver regeneration the TGF-b1 and CD91 expressions were redistributed from medulla to cortical thymic areas.

The data show that pHx triggers in thymus the ER-stress and imply that thymic ER-signaling pathways act as stress sensor that detects various types of cellular insults during liver resection. They also suggest that gp96 by its chaperon-related and immunogenetic properties may participate in re-establishment of thymic homeostasis during stress conditions and in maintenance of hepatic tolerance to self antigens. The mechanisms need to be elucidated, but the data seem to be in agreement with reports pointing to the interconnection of signaling pathways emerging from the endoplasmic reticulum and immune responses during stress, inflammation and infection [14], with the proposal that ER-stress is critically involved in the regulation of immune surveillance [11, 15], with hypothesis that gp96-associated self antigens may be cross-presentation on APC [11-13; 15], as well as with the evidence showing that HSP may directly or indirectly stimulate Tregs, via acetylation, TLR, ligation and induction of changes in thymic cytokine microenvironment [16].


Fig 1

Figure 1. Hepato-thymic circuit activated by partial hepatectomy (pHx)

A) Schematic overview of events occurring in regenerating liver of mice 48h after pHx; B) Thymic structure and stages of T cell development; C and D) Functions of chaperons in unfolded protein response and known effects of gp96 in antigen-presenting cells; a) and b)  Expression of gp96 in regenerating liver and thymus 48h after pHx;  c) Expression of gp96 on the surface of  thymic nurse cells, containing caspase-3+ cells (c); d and e) co-localizations of gp96 with its potential receptors – CD91 and TLR2 in thymic stromal cells; f) Treg cells in the thymus of pHx mice. (Reproduced of from Histology and Histopathology, with permission).



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Acknowledgements:  This study was supported by University of Rijeka, Croatia (research grant

Contact: Biserka Radosevic-Stasic, Ph.D., MD. E-mail: biserkars @medri.uniri.hr



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