Retinoic acid modulates IL-4, IL-10 and MCP-1 pathways in immune mediated hepatitis and interrupts CD4+ T cells infiltration

https://doi.org/10.1016/j.intimp.2019.105808Get rights and content

Highlights

  • Retinoic acid can protect against immune mediated hepatitis.

  • Retinoic acid interrupts infiltration of neutrophils, monocytes and CD4+ T cells.

  • Retinoic acid modulates IL-4 level and production of TNF-α and NF-κb activation.

  • Retinoic acid mitigates second inflammatory responses by increasing IL-10.

Abstract

Aims

Immune mediated liver injury includes activation of different immune pathways that requires various modalities to control their consequences. The current study involves evaluation of retinoic acid (RA) modulatory effects on immune responses induced in concanavalin A (ConA) model of acute hepatitis.

Main methods

Mice were divided as follows: Control group; RA group: received 35 mg/kg RA; ConA group: received 15 mg/kg ConA; ConA + RA group: received ConA and RA as described. Liver function biomarkers were measured in addition to malondialdehyde as lipid peroxidation biomarker. Liver tissue sections were scored for necro-inflammation, neutrophils infiltration, CD4+ T cells infiltration and NF-κb positive cells. Effect on hepatic levels of TNF-α, IL-4, IL-10 and MCP-1 was evaluated as well.

Key findings

Injection of RA before ConA significantly (p < 0.001) decreased ALT, AST and LDH levels compared to their levels in ConA group. Hepatic infiltration of neutrophils and CD4+ T cells was markedly (p < 0.001) reduced by RA. Hepatic injury, necrosis and expression of NF-κb were significantly decreased by RA when injected before ConA challenge. A significant decrease in the measured cytokines TNF-α and IL-4 was observed in ConA + RA group in addition to a decrease in MCP-1 level. On the other hand, IL-10 was significantly increased in the latter group compared to ConA group.

Significance

RA can protect against ConA-induced hepatitis through: interrupting early inflammatory response as neutrophils, monocytes and CD4+ T cells infiltration, modulating IL-4 level and subsequent production of TNF-α and NF-κb activation, mitigating second inflammatory responses through increasing IL-10 liver production.

Introduction

Immune-mediated liver injuries such as autoimmune hepatitis (AIH) and viral hepatitis are executed by many players that include T cells, macrophages (Kupffer cells), hepatic stellate cells (HSCs), liver resident lymphocytes such as natural killer (NK), natural killer T (NKT) cells, and dendritic cells [1,2]. Among these players the most important are the resident Kupffer cells that represent the first responder through release of the pro-inflammatory cytokines tumor-necrosis factor alpha (TNF-α), IL-1β, and subsequently the anti-inflammatory cytokines such as IL-10.

Activated HSCs play a pivotal role in orchestrating hepatic immune responses. They produce a number of chemokines inducing recruitment and infiltration of CD8+ and CD4+ T cells that amplify the inflammatory response and also interact directly with NK cells. This path may help clearing activated HSCs and limit the overwhelmed immune response that would damage the whole organ [[2], [3], [4]].

Concanavalin A (ConA) is a lectin derived from the seeds of jack beans Canavalia ensiformis. Acute hepatitis induced by ConA is considered a typical and well established model for investigating T-cell and macrophage dependent liver injury in mice. It is regarded as the best experimental model for AIH research that mimics immune reactions observed in human so far [5,6]. Besides AIH, ConA animal model involves activation of many inflammatory responses and release of various cytokines commonly involved in different liver diseases as viral hepatitis acute liver failure and alcoholic steatohepatitis in which T cell infiltration is observed [7].

All-trans-retinoic acid (RA) is considered the most active metabolite of vitamin A and it is essential in the regulation of both innate and adaptive immunity [8]. It is used primarily for treating dermatological disorders as acne vulgaris and psoriasis through anti-inflammatory effects. It has been also used to treat several types of human cancer as acute promyelocytic leukemia [9]. Retinoic acid was suggested to differentially regulate NKT cell-mediated hepatitis and production of cytokines as IL-4 [10]. However, the direct in-vivo effects of RA on anti-inflammatory cytokines and various immune cells as CD+4 T cells is still not fully investigated. The current study was conducted to track potential mechanisms involved in the hepato-protective effect of RA using a ConA induced acute hepatitis model in mice that mimics autoimmune and viral hepatitis in human.

Section snippets

Animals

Sixty adult healthy albino male BALB/c mice weighing 25–30 g (VACSERA Egypt) were allowed free access to water and food and were kept for 2 accommodation weeks before starting the study. Animal care and experimental protocol carried out in this study was reviewed and approved from the Faculty of Pharmacy Scientific Research Ethics Committee (Mansoura University) with code number:

Chemicals

All-trans retinoic acid (RA) and concanavalin A (ConA) were purchased from Alfa Aesar, Thermo Fisher Scientific

Experimental design

Mice were randomly divided into four groups as follows: Control group (n = 15, received 10 ml/kg RA vehicle, i.p, 19 h before sacrifice in addition to 7.7 ml/kg ConA vehicle once i.v via the retro-orbital venous plexus under light ether anesthesia 3 h before sacrifice); ConA group (n = 15, received 15 mg/kg ConA, 0.5% w/v in normal saline, 7.7 ml/kg, once i.v via the retro-orbital venous plexus under light ether anesthesia in addition to vehicle 10 ml/kg i.p, 16 h before ConA challenge); RA

Methods

Three hours after ConA injection, mice were anesthetized by 70 mg/kg thiopental (i.p). Serum samples were separated (at 3000 rpm at 4 °C) from blood collected via cardiac puncture. Samples were then divided into aliquots and stored at −80 °C for serum biomarkers analysis. Liver sections for histopathological procedures were isolated and fixed in 8% v/v neutral buffered formalin solutions. Liver tissue homogenate (10% w/v) was prepared using phosphate buffer saline (pH 7.4–7.5).

Statistical analysis

Data (mean ± S.E, n = 10) were statistically evaluated using ANOVA test followed by Tukey–Kramer multiple comparison test as post-hoc test. Histopathological and immunohistochemistry scores were compared using Kruskal Wallis test by rank followed by Dunn's multiple comparison test. Statistical tests and figures were carried out using GraphPad Prism V5.01 (GraphPad Software Inc., CA).

Effect on liver function biomarkers

Levels of ALT and AST were found to be sharply increased (p < 0.001) when they were measured in serum 3 h after ConA injection. Their levels reached about 15 fold and 6 fold the levels in control group respectively [Table 1]. Level of LDH was also increased significantly (p < 0.001) following ConA injection. Furthermore, total serum protein level was significantly decreased (p < 0.001) by ConA compared with its level in control group.

Injection of RA 16 h prior to ConA injection resulted in

Discussion

Immune-mediated hepatitis such as AIH and acute viral hepatitis is characterized by progressive necro-inflammation and destruction of the hepatic parenchyma triggered by different immune-mediated processes [14]. Immunosuppressants and liver transplantation represent the main current treatment for AIH and the specific immune targets as therapeutic options still a major clinical challenge [15].

The present study investigated effects of RA administration before ConA challenge on subsequent immune

Conclusion

Collectively, results obtained from the current study propose a hepatoprotective effect of RA against Con A-induced hepatitis. Retinoic acid may interrupt early inflammatory response including infiltration of neutrophils, monocytes and CD4+ T cells. It can modulate IL-4 level and subsequent production of TNF-α and NF-κb activation in turn. Besides, RA can interfere with second inflammatory responses through increasing hepatic production of IL-10. Further studies are needed to evaluate effect of

Declaration of competing interest

The authors declare that they have no conflict of interest.

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