Histopathological changes following immunization with recombinant leukemia inhibitory factor

doi:10.21203/rs.3.rs-3892018/v1

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Histopathological changes following immunization with recombinant leukemia inhibitory factor

https://doi.org/10.21203/rs.3.rs-3892018/v1

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Background

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein of the interleukin-6 superfamily that regulates many physiological processes as well as pathological conditions. LIF plays an important role in the initiation and progression of solid tumors as well as its role in leukemia suppression. The purpose of this study is to determine whether exogenous LIF has any side effects in liver and kidney tissues that have receptors for this cytokine in immunized mice compared with controls.

Methods

LIF and complete Freund's adjuvant were injected subcutaneously for the test group in the initial injection. Freund's incomplete adjuvant was injected intraperitoneal for the last injection. Phosphate-buffered saline was used with the adjuvant for the control group.

Results

The liver structure in the control group was normal. However, the LIF immunized group exhibited irregular sinusoidal plates and hyperemia in central and portal veins compared to the control group. The kidney tissue in the control group was also normal, with preserved tubule shapes and no cell destruction or degeneration. In the LIF injected group, hyperemia and an increase in the urinary space were observed, but the overall kidney tissue condition was similar to the control group, with no tissue destruction or infiltration of mononuclear cells, and the renal corpuscle and glomerular coil remained normal.

Conclusion

The results indicated that while the injection of exogenous LIF into the mouse body caused changes and destructive effects on the liver tissue, including the development of an inflammatory condition in the liver, it had no discernible effect on the kidney tissue.

leukemia inhibitory factor

liver

kidney

pathology

mouse

antibody

ELISA

As a differentiation and proliferation inducer of a myeloid leukemia cell line (M1), leukemia inhibitory factor (LIF) was first introduced [1]. The precursor of glycoprotein LIF is 202 amino acids, which are processed post-translationally into a 20 kDa form by removing 22 amino acids at the N-terminus [2]. In the interleukin-6 family, LIF is a pleiotropic cytokine, meaning it acts on a wide variety of cell types [3–5]. It has a very broad range of effects on virtually all organ systems, including hemopoietic, bone remodeling, neural, muscular, endocrine, and reproductive functions. Among the cells that express LIF receptors are neurons (macrophages, hepatocytes, osteoblasts, megakaryocytes, myoblasts, adipocytes) as well as tumor cell lines taken from these tissues [6–8].

Furthermore, LIF is reported to regulate multiple signaling pathways, including the mTOR, TGF-β, VEGF/HIF-1, Toll/NF-κB and etc pathways. A growing number of studies suggest that LIF plays a role not only in suppressing leukemia, but also in solid tumor initiation and progression [9]. As well as suppressing embryonic stem-cell differentiation, it inhibits lipoprotein lipase expression and stimulates liver acute-phase protein production [10–13]. LIF increases bone resorption and is associated with pathological conditions such as cachexia, tissue calcification, pancreatitis, and gonadal abnormalities [14, 15].

A polyclonal anti-LIF antibody is produced in a laboratory animal through active immunization with LIF protein. By preventing the implantation of mouse embryos, anti-LIF polyclonal antibodies may have contraceptive effects. Furthermore, anti-LIF antibody treatments significantly reduced the length of the placenta, the ossification of the exoccipital bone, and the width of the vertebral space in comparison with controls [16].

Considering that targeting LIF with vaccination is done for various purposes today, in this study, we intend to investigate the side effects of the antibody against it on liver and kidney tissues that have receptors for this cytokine in immunized mice compared to the control group from a histopathological point of view.

Female BALB/c mice 6-8 weeks old were obtained from the Pasteur Institute (Karaj, Iran) and randomly divided into two groups. A protocol of animal handling and treatment for the animals has been developed by the Institutional Ethical Committee of Shahid Sadoughi University of Medical Sciences (IR.SSU.SPH.REC.1399.211). Mice were kept with unrestricted access to water and food in cages at room temperature (22–24°C) in ventilated rooms.

Immunization

For the test group in the first injection, LIF was injected subcutaneously (SC) along with Freund's complete adjuvant (FCA). Injections one to four were done SC and the last one was done intraperitoneally (IP) with Freund's incomplete adjuvant (FIA). For the control group, the adjuvant was injected with Phosphate-buffered saline (PBS) (Fig. 1).

Antibody determination

Two weeks after the last injection, blood samples were taken from the mice. The serum was separated by centrifugation for 10 minutes at 2800 g and kept at -70°C until the complete collection of the samples. The titer of antibodies in serum was measured using ELISA (enzyme-linked immunosorbent assay). In this way, a certain amount of LIF protein was added to the bottom of the 96-well plates and the desired samples to determine the antibody titer. Then HRP anti-mouse antibody solution was added to the plates and after washing the substrate chromogen solution and then stop solution was added and the absorbance of the wells was read with an ELISA reader at a wavelength of 450 nm.

Histology

The liver and kidney tissues were fixed in formalin and placed in a tissue processor for tissue preparation and then molded with melted paraffin. Five micrometers (μm) sections of tissues were prepared from the tissue molds by microtome and examined from the tissue point of view.

Possible pathological changes in tissue, liver, and kidney were examined by hematoxylin-eosin (H&E) staining. Under a light microscope the picture of each field was captured from several sections to find out changes in histopathology.

Ethics Approval and Consent to Participate

This study was approved by the Animal Guidelines Ethics Committee of Shahid Sadoughi University of Medical Sciences in Yazd, Iran (Ethics Code No: IR.SSU.SPH.REC.1399.211).

The results of the antibody titer showed that there was a significant increase in the serum anti-LIF antibody titer in all immunized mice against control group (Fig. 2).

In the control group, the structure of the liver parenchyma was preserved normally, and there was no sign of confusion and disorder in the sinusoidal plates around the central vein, and there was no hyperemia and pressure increase in the portal vein, central vein, and sinusoidal spaces. Hepatocytes and Kupffer cells have normal size and condition, and infiltration of mononuclear cells, steatosis and inclusion accumulation in the liver parenchyma were not observed.

The results of histological investigations of the group immunized with LIF showed that the sinusoidal plates became irregular and lost their radial position compared to the control group, and some degree of hyperemia was seen in the central vein and portal vein. In some lobules, the central vein loses its spherical shape and becomes oval. In the parenchyma of the liver, seepage and infiltration of mononuclear inflammatory cells can be clearly seen. Canalicular cholestasis was seen in some bile canaliculi. There was no significant difference in the amount and number of microvesicular steatosis and councilman body compared to the control group. Overall, the findings show an inflammatory condition in the liver (Fig. 3).

Examination of the kidney tissue showed that in the control group, the state of the kidney parenchyma was normal and the shape of the proximal convoluted tubule (PCT), distal convoluted tubule (DCT) and urine collecting tubes were preserved normally, and there was no sign of cell destruction and degeneration or the presence of nuclei in the lumen and cytoplasm vacuolization. The condition of glomeruli and urinary space is normal and there is no trace of hyperemia and tissue inflammation, as well as infiltration of immune mononuclear cells. In the LIF injected group, there is some hyperemia and an increase in the urinary space, but the general condition of the kidney tissue is similar to that of the control group, and there is no effect of tissue destruction and infiltration of mononuclear cells, and the renal corpuscle and glomerular coil have maintained their normal condition. Also, destruction is not seen in the PCT, DCT and urine collecting tubes (Fig. 4).

LIF is a multifunctional glycoprotein that regulates many physiological processes as well as pathological conditions such as leukemia proliferation and differentiation, tissue/organ regeneration, neurogenesis, neural regeneration, maternal reproduction, immune response, and cancer development [9,17]. Patients with pancreatic cancer and nasopharyngeal cancer had significantly elevated circulating LIF levels and high levels of LIF expressed in tumor tissues [18,19]. The metabolic regulation of LIF has also been suggested by recent studies [20]. Considering the distribution of LIF receptors in different organs of the body, in the present study, we aimed to investigate the effect of exogenous LIF in two organs, the liver and kidney.

In the present study, the examination of the liver tissue of the group of mice immunized with LIF comparing the sinusoidal plates to the control group showed that the plates became irregular and lost their radial position. The central vein and portal vein also had some degree of hyperemia. It is clearly visible that mononuclear inflammatory cells infiltrate into and infiltrate the liver parenchyma. While Metcalf's study showed that the liver had no evidence of hematopoietic cell infiltration, an increase in resident Kupffer cells, and calcification [21].

Yuan et al.'s study in non-alcoholic fatty liver patients showed that circulating LIF levels were associated with the severity of liver steatosis, while the animal phase of their study showed that LIF derived from white adipose tissue can improve liver steatosis by activating hepatic LIF receptor signaling pathways [17].

Among LIF functions, it plays a key role in nephrogenesis and extracellular matrix repair. LIF excretion is also enhanced during local inflammatory reactions, such as rejection of kidney grafts or infections of the urinary tract [22]. It has been reported that LIF reduces the death of myocardium cells and fibrosis of the heart. In Yu et al.'s study, LIF was found to play a role in renal anti-fibrosis by competitively activating Stat3, which regulates microRNA-29c to suppress collagen expression [23]. However, the results of the present study showed that the general condition of the kidney tissue was similar to the control group, and no effect of tissue destruction and infiltration of mononuclear cells can be seen.

According to the mentioned issues, the different effects of LIF in the studies can be based on its type (exogenous or endogenous), the different expression of its tissue receptor, depending on its dose, and even humans or laboratory animals.

The findings showed that the injection of exogenous LIF into the mouse body didn't have a significant effect on the kidney tissue, but it has destructive effects on the liver tissue and causes changes in this tissue, including creating an inflammatory condition in the liver.

Acknowledgments

The authors thank the staff in the Department of Immunology and the Reproductive Immunology Research Center and Shahid Sadoughi Hospital at the Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Authors’ Contributions

F.Z contributed to the conception and design of this study. SM.S, F.Ph, and A.H ‎performed material preparation. F.F and F.Z conducted data collection and analysis. The first draft of the manuscript was ‎written by H.A and all authors commented on the previous versions of the ‎manuscript. All authors have read and approved the final manuscript. ‎

Availability of data and materials

No data sets were produced or examined during the present study.

Competing interests

The authors declare that they have no conflicts of interest.

Funding Information

Financial assistance was provided through a grant from Shahid Sadoughi University of Medical Sciences, Yazd, Iran (grant number 9127).

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No competing interests reported.

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Histopathological changes following immunization with recombinant leukemia inhibitory factor

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Abstract

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Background

Materials and methods

Results

Discussion

Conclusion

Declarations

References

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