Histopathological and immunohistochemical characterisation of hepatic granulomas in Leishmania donovani-infected BALB/c mice: a time-course study

Abstract

Background: Visceral leishmaniasis (VL) is a neglected tropical disease (NTD), caused by the intracellular protozoan parasites Leishmania donovani and Leishmania infantum. Symptomatic VL is considered fatal when left untreated. At present, there is no effective vaccine licensed for human use and available chemotherapies have limitations. Understanding the local immune mechanisms required for the control of infection is a key factor for developing effective vaccines and therapeutics.

Methods: We have investigated the development of the typical granulomatous lesions in the liver in experimental VL over time, together with the local immune responses. BALB/c mice were infected intravenously with a dose of 2 × 10⁷ L. donovani amastigotes (MHOM/ET/67/HU3) and sacrificed at 15, 35 and 63 days post-infection (dpi). Histopathology and immunohistochemical techniques were used for the detection of Leishmania antigen, selected cell types including B and T lymphocytes, macrophages and neutrophils (CD45R-B220+, CD3+, F4/80+ and Ly-6G+) and iNOS.

Results: Granulomatous lesions were identified as early as 15 dpi in the livers of all infected animals. Three categories were used to classify liver granulomas (immature, mature and clear). Clear granulomas were exclusively detected from 35 dpi onwards. Kupffer cells (F4/80+) were predominant in immature granulomas, regardless of the dpi. Nonetheless, the highest expression was found 63 dpi. Positive staining for iNOS was mainly observed in the cytoplasm of fused Kupffer cells and the highest expression observed at 35 dpi. T cells (CD3+) and B cells (CD45R-B220+) were predominant in more advanced granuloma stages, probably related to the establishment of acquired immunity. Neutrophils (Ly-6G+) were predominantly observed in mature granulomas with the highest expression at 15 dpi. Neutrophils were lower in numbers compared to other cell types, particularly at later time points.

Conclusions: Our results reflect the role of macrophages during the early stage of infection and the establishment of a lymphocytic response to control the infection in more advanced stages.

Keywords: Granuloma; Histopathology; Immunohistochemistry; Leishmania donovani; Macrophage; Visceral leishmaniasis.

Fig. 1

a H&E-stained liver section 15 dpi (400×). Kupffer cell harbouring a high amount of amastigotes (arrowhead). Immature granulomas formed by fused macrophages harbouring few amastigotes (arrows). Well-formed mature granuloma with a complete cellular infiltrate (*). b H&E-stained liver section 35 dpi (400×). Mature functional granuloma with few fused macrophages harbouring very few amastigotes (*). Clear granuloma without amastigotes and with presence of collagen deposition (arrow). c H&E-stained liver section 63 dpi (400×). Clear granuloma composed mainly of lymphocytes (arrow). d Immunohistochemical detection of Leishmania donovani antigens (400×). Immature granulomas harbouring a high amount of amastigotes (arrows) and mature granulomas with fewer number of amastigotes (*). Scale-bars: 100 μm

Fig. 2

Distribution (total numbers and percentages) of hepatic granulomas induced by Leishmania donovani divided into category 1 (immature), category 2 (mature) and category 3 (clear) at three different time points (15 dpi, 35 dpi and 63 dpi)

Fig. 3

Immunohistochemical detection of macrophages (F4/80+) within hepatic granulomas at 15 dpi (a), 35 dpi (b) and 63 dpi (c). d Percentage of immunostained area for F4/80 at 15, 35 and 63 dpi within immature (I), mature (M) and clear (C) granulomas. Immunohistochemical detection of iNOS+ cells at 15 dpi (e), 35 dpi (f) and 63 dpi (g). h Percentage of immunostained area for iNOS at 15, 35 and 63 dpi within immature (I), mature (M) and clear (C) granulomas. *P < 0.05. Scale-bars: 50 μm

Fig. 4

Immunohistochemical detection of CD3+ T cells within hepatic granulomas at 15 dpi (a), 35 dpi (b) and 63 (c) dpi. d Percentage of immunostained area for CD3 at 15, 35 and 63 dpi within immature (I), mature (M) and clear (C) granulomas. Immunohistochemical detection of CD45R/B220+ cells (B cells) at 15 dpi (e), 35 dpi (f) and 63 dpi (g). h Percentage of immunostained area for CD45R/B220 at 15, 35 and 63 dpi within immature (I), mature (M) and clear (C) granulomas. Immunohistochemical detection of Ly6G+ cells (neutrophils) at 15 dpi (i), 35 dpi (j) and 63 dpi (k). l Percentage of immunostained area for Ly6G at 15, 35 and 63 dpi within immature (I), mature (M) and clear (C) granulomas. *P < 0.05. Scale-bars: 50 μm