Effect of Leishmania major Infection on the Expression of TGF Beta in Murine

1. Introduction

Leishmaniasis is a disease caused by a protozoan parasite called Leishmania, an obligatory intracellular parasite that resides in the macrophages of the mammalian hosts as round to oval amastigotes phase. Leishmania major is a source of cutaneous leishmaniasis with an infection burden of about 1–1.5 million with mucosal lesions ( 1 ). Leishmania major transmitted by sand flies which causes various lesions of cutaneous bumps, nodules, and gross tissue damage ( 2 ). The immune responses of Leishmania are mostly determined by the expansion of Th1 and Th2 cells of CD4+ T cells. Th1 cells offer IFN-γ, IL-2, and TNF-αprotection which plays a significant role in innate and adaptive immune responses against leishmaniasis in humans and mice ( 3 - 5 ). Th2 responses are determined by the production of IL-4, IL-5, IL-10, TGF-β, and IL-13, which inhibit some macrophage functions ( 4 ). TGF-βwas shown to inhibit releasing of IFN-γ by CD4+ T cells in BALB/c mice infected with visceral leishmaniasis and activating of Th2 cells. Clinical diagnosis of cutaneous lesions and microscopic analysis of Leishmania major is usually performed in endemic countries to detect the infection.

Supported diagnostic techniques allow confirmative identification of limited studies in non-endemic countries of cutaneous leishmaniasis. Similarly, previous studies, to the best of our knowledge, have focused on improving diagnostic experiments to detect the release of TGF-β by IHC ( 6 ). Leishmania antigens were detected by histopathological and immunohistochemical techniques to investigate local immune response in liver development of granulomatous lesions ( 7 ).

Therefore, the present study aimed to detect TGF-β production by independently developing Th2-type cytokines in different organs of mice infected by Leishmaniamajor using immunohistochemistry.

2. Materials and Methods

2.1. Study Samples

A total of 40 samples of paraffin blocks, formalin-fixed paraffin-embedded (FFPE) tissue of different organs (skin, spleen, liver, kidney, and heart) were used in the present study from October 2020 to January 2021. These tissue samples were obtained from the archives of the Faculty of Sciences, University of Kufa. The archival blocks were collected from male and female BALB/c mice (6-8 weeks and weighing 25-30 gm) that had previously been subcutaneously infected with Leishmania major promastigotes at adose of 1×107 promastigotes/mouse.

2.2. Histopathology

Tissues were sectioned by microtome at 4 μm thickness and routinely stained with hematoxylin and eosin (H&E) ( 8 ). After H&E staining, the slides were dehydrated through a series of 70%, 80%, 95%, and twice in 100% ethanol, then twice in xylene for 2 minutes each. Finally, the tissues sections were covered with Permount Mounting Medium (DPX). Tissue slides were examined under magnification of 10× and then 40× of the light microscope to evaluate histopathological changes.

2.3. Immunohistochemistry (IHC)

Unstained glass slides from skin, spleen, liver, kidney, and heart were used to perform IHC. Briefly, the slides were deparaffinized twice for 5 min by xylene and then dehydrated with a series of ethanol concentrations (100 %, 95%, 80%, and 70%) for 5 minutes each, then rinsed with distilled water. Endogenous peroxidase activity was eliminated by incubation with hydrogen peroxide (3%) for 5 min at 37°C, and then the slides were washed with phosphate-buffered saline (PBS) (3 times for 5 min each). The slides were incubated with Blocking Reagent (ab64218) for 20 min and then washed 3 times in PBS to block non-specific binding. After removing the blocking solution, the slides were incubated with diluted primary antibody (anti- TGF beta 1 – BSA) 1:200 for 1 h at 37°C in a humidity chamber, then rinsed with PBS (3 times for 5 min each). Biotinylated- secondary antibody (at assay dependent concentration) was applied to the slides for 30 min at room temperature. Sections were washed with PBS, incubated with a streptavidin-HRP solution for 10 minutes at room temperature, and then washed again with PBS (3 times for 5 min each). Diaminobenzidine hydrochloride (DAB) substrate was added to the glass slide until the desired color was achieved (1-10 min) at room temperature. The tissue sections were counterstained with hematoxylinstain for microscopic examination.

3. Results

Histopathological findings of mice infected with L. major were characterized by variable degrees of inflammatory cells infiltration, mainly mononuclear cells (lymphocytes and macrophages) to micro granulomatous lesions. In response to Leishmaniasis, histopathological changes from skin biopsy of the mice infected with L. major amastigotes indicated epidermal hyperplasia (acanthosis), with diffuse severe lymphohistiocytic inflammatory cells infiltration in the dermis with the presence of the L. major amastigotes within macrophages (Figure 1). Follicular lymphoid hyperplasia (FLH) was observed in the spleen of mice infected with L. major (Figure 2). Scattered polymorphonuclear cells mainly neutrophils accumulated with randomly distributed microgranulomas foci composed of lymphocytes and macrophages accumulations were observed in liver parenchyma around central veins and portal areas with individual necrosis of hepatocytes (Figure 3). Aggregation of perivascular mononuclear cells (lymphocytes and macrophages) was also observed in the renal cortex of the kidney (Figure 4). Mononuclear cells mainly lymphocytes and macrophages were observed in the heart parenchyma especially around blood vessels (Figure 5).

Figure 1. Histopathology of the skin in mice infected with L. major

Figure 2. Histopathology of the spleen in mice infected with L. major

Figure 3. Histopathology of the liver in mice infected with L. major

Figure 4. Histopathology of the kidney in mice infected with L. major

Figure 5. Histopathology of the heart in mice infected with L. major

Representative images of skin (Figure 1) from BALB/c mice inoculated with PBS (upper left panel) and L. major amastigotes (1 × 10⁷ amastigotes /mouse) (upper right panel). The images below are magnifications of the upper sections. Animals infected with L. major amastigotes showed epidermal hyperplasia (acanthosis), with diffuse severe lymphohistiocytic inflammatory cells infiltration in the dermis with the presence of the L. major amastigotes in macrophages. None of the BALB/c mice inoculated with PBS indicated remarkable lesions.

Representative images of spleen (Figure 2) from BALB/c mice inoculated with PBS (upper left panel) and L. major amastigotes (1 × 10⁷amastigotes /mouse) (upper right panel). The images below are magnifications of the upper sections. Animals infected with L. major amastigotes showed hyperplasia of the lymphoid follicles. None of the BALB/c mice inoculated with PBS indicated remarkable lesions.

Representative images of liver (Figure 3) from BALB/c mice inoculated with PBS (upper left panel) and L. major amastigotes (1 × 10⁷amastigotes /mouse) (upper right panel). The images below are magnifications of the upper sections. Animals infected with L. major amastigotes showed scattered polymorphonuclear cells, mainly neutrophil masses, with a random distribution of microgranulomas foci composed of lymphocytes and macrophages accumulations within liver parenchyma around central veins and portal areas. None of the BALB/c mice inoculated with PBS showed remarkable lesions.

Representative images of kidney (Figure 4) from BALB/C mice inoculated with PBS (upper left panel) and L. major amastigotes (1 × 10⁷ amastigotes /mouse) (upper right panel). The images below are magnifications of the upper sections. Animals infected with L. major amastigotes showed perivascular mononuclear cells (lymphocytes and macrophages) aggregation in the renal cortex. While the BALB/C mice inoculated with PBS failed to show remarkable lesions.

Representative images of the heart (Figure 5) from BALB/c mice inoculated with PBS (upper left panel) and L. major amastigotes (1 × 10⁷ amastigotes /mouse) (upper right panel). The images below are magnifications of the upper sections. Animals infected with L. major amastigotes showed mononuclear cells mainly lymphocytes and macrophages aggregation within the heart parenchyma, especially around blood vessels. Whereas, the BALB/c mice inoculated with PBS failed to show remarkable lesions.

3.1. Immunohistochemistry Detection

Tissue expression of TGF-β1 was directly accompanied with L. major in mice. Immunohistochemistry (IHC) was performed to evaluate the TGF-β1 expression in the skin, spleen, liver, heart, and kidney. The result of skin activity against the TGF-β1 biomarker was a very strong signal as shown in table 1 and figure 6. IHC has demonstrated that the spleen and the liver showed moderate to strong immunoreactivity, followed by moderate to weak positive TGF-β1 signalling in the heart and kidney. Table 1 represents the expression level of TGF-β1 signalling depending on immunoreactivity and intensity scores in five tissue organs of mice infected with L. major amastigotes, and the brown signal was measured according to Rezaee Movassaghi ( 9 ).

Figure 6. Immunohistochemistry of the skin in mice infected with L. major. TGF-β expression of BALB/c mice injected with L. major amastigotes (1 X 10⁷amastigotes /mouse).

Mice skin tissues indicated strong brown signal staining of positive cells to TGF-β biomarker of BALB/c mice (n=4), 40× magnification.

Mice spleen tissues indicated strong brown signal staining of positive cells to TGF-β biomarker of BALB/c mice (n=4), 10× magnification (Figure 7).

Figure 7. Immunohistochemistry of the spleen in mice infected with L. major. TGF-β expression of BALB/c mice injected with L. major amastigotes (1 × 10⁷ amastigotes /mouse).

Mice liver tissues indicated strong brown signal staining of positive cells to TGF-β biomarker of BALB/c mice (n=4), 40× magnification (Figure 8).

Figure 8. Immunohistochemistry of the liver in mice infected with L. major. TGF-β expression of BALB/c mice injected with L. major amastigotes (1 × 10⁷ amastigotes /mouse).

Mice heart tissues detected moderate brown signal staining of positive cells to TGF-β biomarker of BALB/c mice (n=4), 40× magnification (Figure 9).

Figure 9. Immunohistochemistry of the heart in mice infected with L. major. TGF-β expression of BALB/c mice injected with L. major amastigotes (1 × 10⁷ amastigotes /mouse).

Mice kidney tissues indicated weak brown signal staining of positive cells to TGF-β biomarker of BALB/c mice (n=4), 40× magnification (Figure 10)

Figure 10. Immunohistochemistry of the kidney in mice infected with L. major. TGF-β expression of BALB/c mice injected with L. major amastigotes (1 × 10⁷amastigotes /mouse).

4. Discussion

Cutaneous Leishmaniasis caused by L. major manifested clinical lesions in patients ranged from weeks to months ( 10 ). In the present study, histopathological and immunohistochemical changes of different tissue sections of mice infected with L. major were reported to better understand the tissue damage of different sections during infection.

In response to Leishmaniasis, hyperkeratosis and parakeratosis of the dermis were observed from the skin biopsies of mice infected with L. major. Variable hallmarks were observed in skin lesions of patients with cutaneous Leishmaniasis ( 11 ) and in mice ( 12 ) to discover the structural component of the tissue against L. major.

Obvious granuloma lesions were shown in liver and spleen biopsies with aggregation of lymphocytes, plasma cells, and macrophages. These results are usually present in the chronic inflammatory response and are associated with the development of a Th1 response initiated by the IL-12 response to control the disease ( 13 , 14 ). Previous studies have reported that macrophages activated polarised Th1 cells to eradicate L. major inIL-4-/- BALB/c mice ( 15 , 16 ). The granulomatous lesions were less common in the kidney and heart as these organs were not similar to lymphatic tissues and less important in the immune response of the mice ( 17 ).

Five organs (skin, spleen, liver, heart, and kidney) were selected and bioactivity scoring was investigated to screen and quantify the expression of TGF-β intensity and their effects. The immunohistochemical staining method was described as one of the methods for measuring TGF-β activation and quantitation of TGF-β synthesis levels in different experimental situations ( 18 ). Also, this method has previously been used for measuring the growth of cancer cells and tumorigenesis where there are substantial changes in TGF-β synthesis, secretion, or activation ( 19 ). In the present study, samples stained with anti-TGF-β biomarkers would provide information on the presence of active TGF-β that elicit intracellular signalling and develop an immune response in BLAB/c mice ( 20 , 21 ).

In mice, successful primary immunity against L. major includes IL-12 dependent IFN- ˠ production from CD4+ and CD8+ T cells (Th1 response) which mediates macrophage killing mechanism ADDIN CSL_CITATION {"citationItems":,"container-title":"Nature Reviews Immunology","id":"ITEM-1","issue":"11","issued":{"date-parts":]},"page":"845-858","title":"The immunology of susceptibility and resistance to Leishmania major in mice","type":"article-journal","volume":"2"},"uris":}],"mendeley":{"formattedCitation":"( 20 )","plainTextFormattedCitation":"( 20 )","previouslyFormattedCitation":"(Sacks and Noben-Trauth, 2002)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}( 20 ). In addition, the dominance of an IL-4 induces a strongTh2 response in BALB/c that subcutaneously inoculated with a high dose of promastigotes of L. major resulted in rapidly evolving cutaneous lesions ( 22 ). A significant increase was detected in the expression of TGIF-β in skin tissues as a result of the cutaneous infection effect. BALB/c mice are vulnerable to L. major infection and fail to develop cellular mediated immunity to produce self-healing lesions compared toother breeds of mice (such as the C3H, C57BL/6, and B10.D2) ( 14 ). Subsequently, a strong signal (brown staining) was observed in the spleen and liver as a consequence of immune infiltration and the binding of active TGF-β to its cell surface receptors ( 19 ).

The elucidation of TGF-β from the matrix in the heart and kidney which is slightly activated in the tissues that displayed the ability of L. major parasites might display mildvisceralization incomparable to the cutaneous manifestation ( 23 ).

Leishmania major is a parasitic disease that can cause cutaneous Leishmaniasis. It appears with a wide range of clinical, histological, and inflammatory manifestations. In tissue sections indicating histopathological infiltration of a granulomatous reaction that is included in the diagnosis of the disease. Immunohistochemical detection of Leishmania major depends on identifying the expression level of TGF-beta in lesion samples.

Authors' Contribution

Study concept and design: D. A. K.

Acquisition of data: F. A. A.

Analysis and interpretation of data: B. M. K.

Drafting of the manuscript: D. A. K.

Critical revision of the manuscript for important intellectual content: D. A. K.

Statistical analysis: F. A. A.

Administrative, technical, and material support: D. A. K.

Ethics

All the procedures were approved by the Ethics Committee at the University of Baghdad, Baghdad, Iraq.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgment

The authors would like to express their sincere appreciation to Dr. Omar H. Khalaf, Department of Veterinary Pathology & Poultry Diseases, Faculty of Veterinary Medicine, University of Baghdad, Baghdad, Iraq to assist in reading histopathological results.

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