Mast-Cell Response to Leishmania mexicana and Sand-Fly Salivary Proteins Is Modulated by Orchiectomy

Abstract

Mast cells (MCs) play a crucial role during Leishmania infections, which is transmitted through the bite of an infected sand fly that injects saliva together with the parasite. Sand fly saliva is a complex fluid that modulates the host immune response. In addition, hormonal factors modulate the host immune response and alter susceptibility to infections. Thus, to assess the impact of male sex hormones on the mast-cell (MC) response to Leishmania infections, we orchiectomized male mice, infected them with the parasite in the presence of sand fly salivary proteins, and analyzed the inflammatory response of MCs. Our results showed that the MC response to the parasite and vector salivary proteins differed between orchiectomized and sham-operated mice. In orchiectomized mice, MC showed a retarded activation pattern, associated with slower degranulation and weaker TNF-α, histamine, and tryptase staining in response to the infection with Leishmania mexicana combined with vector-salivary proteins, as compared to sham mice. Furthermore, neutrophil infiltration was slower in orchiectomized mice, and numbers of infected macrophages and lesion sizes were smaller. Our results show that, during Leishmania infection, male sex hormones modulate the mast-cell response against the parasite and salivary proteins of the sand fly vector, inducing an intense inflammatory response. Their absence in orchiectomized mice retards the inflammatory response, enabling better control of the infection and slower disease progression.

Keywords: Leishmania mexicana; mast cells; sand fly salivary proteins; sexual hormones.

Figure 1

Kinetics of inflammatory cell infiltration in pinna skin from sham (SH) and orchiectomized (Or) BALB/c mice in response to intradermal inoculation of L. mexicana together with salivary-gland lysates. In sham mice, the dermal accumulation of inflammatory cells, principally neutrophils, was evident at 30 min after challenge (SH 30 min), and remained intense up to 48 h (SH 48 h). In contrast, in orchiectomized mice, dermal neutrophil infiltration was delayed, beginning at 24 h after challenge and remaining intense throughout 48 h (Or 48 h). Neutrophils were identified by their multilobed nuclei typically consisting of 3 to 5 segments. Yellow ovals show vasodilation and vascular congestion, and black stars show edema. Photomicrographs are representative of three independent experiments with five replicates for each group. Hematoxylin–eosin staining was used. Scale bars are of different sizes because photomicrographs had small magnification differences due to the use of optovar.

Figure 2

Mast-cell (MC) numbers from pinna skin samples of sham (SH) and orchiectomized (Or) mice after intradermal inoculation of L. mexicana with salivary gland lysates. Toluidine blue-stained MCs counted in 20 mm² of tissue sections f rom (A) sham-operated and (B) orchiectomized mice. In sham-operated mice (A,*), a significantly lower number of MCs were found in tissues in basal conditions (BSH), as compared to at 24, 48, and 72 h postinfection (24SH, 48SH, 72SH). CSH, 30SH and 8SH were significantly lower as compared to 24SH and 48SH. In orchiectomized mice (B,*), significantly lower numbers of MCs were found in basal and control conditions (BOr and Cor), as compared to 8, 24, 48 and 72 h post infection (8Or, 24Or, 48Or and 72OR). At 30 min (30Or) post infection, the MC numbers were significantly lower as compared to 24 and 48 h (24Or, 48Or) in orchiectomized animals. Nonparametric ANOVA ordinary multiple comparisons was employed (Significance p < 0.0001). (Abbreviations: BSH: basal sham; CSH: control sham; 30SH: 30 min sham; 8SH: 8 h sham; 24SH: 24 h sham, 48SH: 48 h sham; 72SH: 72 h sham. BOr (basal orchiectomized; COr control orchiectomized; 30Or: 30 min orchiectomized, 8–72 Or: 8–72 h orchiectomized). Three independent experiments with five replicates for each group.* p ˂ 0.001.

Figure 3

Photomicrographs of mast-cell (MCs) degranulation/activation in pinna skin from sham (SH) and orchiectomized (Or) mice after challenge with L. mexicana, together with salivary gland lysates. Toluidine blue-stained sections from inoculated ears showed a differential MC degranulation pattern between the groups of SH and Or mice. Overall, Or mice showed intact dark purple MCs without apparent degranulation at 30 min. At 8, 24, and 48 h, increase in released granules was observed (see Or 8, 24, and 48 h). In contrast, in SH mice, evident MC degranulation/activation was found at 30 min with dermal scattered granules. Degranulation increased at SH 8, 24, and 48 h. Images are representative of three independent experiments with five replicates for each group. Mast cells were identified by cytoplasmic granules showing metachromatic properties (alkaline toluidine blue and red–violet stain of secretory granules) and expelled granules with pink stain.

Figure 4

Comparative immunohistochemical assessment of histamine, tryptase, and TNF-α release in pinna skin mast cells (MCs) from sham (SH) and orchiectomized (Or) BALB/c mice, at 30 min in response to the dermal inoculation of L. mexicana, combined with salivary gland lysate. The immunoreactivity of inflammatory factors in MCs was more intense in Or mice than in SH mice. However, a diffuse and intense interstitial mark was observed only in SH mice. Images representative of three independent experiments with five replicates for each group.

Figure 5

Comparative immunohistochemical assessment of histamine, tryptase, and TNF-α release in pinna skin mast cells (MCs) from sham (SH) and orchiectomized (Or) BALB/c mice, at 24 h in response to the injection of L. mexicana, combined with salivary gland lysate. Immunoreactivity of inflammatory factors in MCs was similar between Or and SH mice. However, diffuse and intense mark remained only in SH mice. Images representative of three independent experiments with five replicates for each group.

Figure 6

Comparative immunohistochemical assessment of release of histamine, tryptase, and TNF-α in pinna skin mast cells (MCs) from sham (SH) and orchiectomized (Or) BALB/c mice, at 72 h in response to the injection of L. mexicana combined with salivary gland lysate. Immunoreactivity of inflammatory factors in MCs from Or mice. Images representative of three independent experiments with five replicates for each group.

Figure 7

Comparative sizes of pinna skin ulcered nodules at 4 and 8 weeks (W) after L. mexicana promastigote infections, including counts of infected macrophages. Orchiectomized mice (Or) produced smaller lesions as compared to sham-operated (SH) mice. Lower numbers of infected macrophages were evidenced in Or mice (42.8 ± 0.2 and 21.6 ± 0.4/20 mm²) and smaller lesions (0.58 ± 0.084 mm and 0.62 ± 0.074 mm) after 4 or 8 weeks, respectively, as compared to SH mice (71.2 ± 0.3 and 79.8 ± 0.2/20 mm²) and (1 ± 0.1 mm and 1.1 ± 0.04 mm), at the same time points. Orchiectomized mice showed strict control of parasitic infection at 8 weeks. Images representative of three independent experiments with five replicates for each group. Significant differences (*) between SH mice and Or mice (p ≤ 0.03).