Impact of vitamin D in children with chronic tonsillitis (immunohistochemical study of CD68 polarisation and proinflammatory cytokines estimation)

Abstract

Inflammatory processes are increasingly attributed to macrophage polarization. Proinflammatory macrophages promote T helper (Th) 1 response, tissue repair, and Th2 responses. Detection of macrophages in tissue sections is facilitated by CD68. Our study is focused on the expression of CD68 and the estimation of proinflammatory cytokines in children's patients with chronic tonsillitis secondary to vitamin D supplementation. This hospital-based Randomized prospective case-control study was conducted on 80 children with chronic tonsillitis associated with vitamin D deficiency where (40 received vitamin D 50,000 IU weekly for 3-6 months and 40 received 5 ml distilled water as placebo). The serum 25-hydroxyvitamin D [25(OH)D] was measured using an Enzyme-linked immunosorbent assay on all included children. Different histological and immunohistochemical studies for the detection of CD68 were done. There was a significantly lower serum level of 25(OH)D in the placebo group versus the vitamin D group (P < 0.001). The levels of pro-inflammatory cytokines, TNFα, and IL-2 significantly increased in the placebo group as compared to the vitamin D group (P < 0.001). The increased level of IL-4 and IL-10 in the placebo group as compared to the vitamin D group was insignificant (P = 0.32, 0.82) respectively. Vitamin D supplementation alleviated the deleterious effect of chronic tonsillitis on the histological structure of the tonsil. Tonsillar tissues of the children in the control and vitamin D groups demonstrated a highly statistically significantly lower number of CD68 immunoexpressing cells compared with those in the placebo group (P < 0.001). Low vitamin D may play a role in chronic tonsillitis. Vitamin D supplementation could help reduce the occurrence of chronic tonsillitis in susceptible children.

Figure 1

Photomicrographs of palatine tonsil sections stained with H&E showing: (a) Control group, covered with stratified squamous nonkeratinized or para keratinized surface epithelium (arrow). This epithelium is thrown into invaginations called crypts (C). There are many lymphatic follicles (F) in the subepithelial region. These follicles have a peripheral darkly stained zone (mantle zone or corona) and central pale stained zone; germinal center (G) [× 40, scale bar = 250 µm]. (b) Control group, there is a fibrous CT band (CT) surrounding the base of the palatine tonsil. Under this CT, there are numerous glands (Gl). Within the tonsil, multiple lymphatic follicles (F) can be seen with germinal center (G) [× 40, scale bar = 250 µm]. (c) Control group, showing the covering epithelium (arrow) of palatine tonsil. This epithelium is formed of the basal layer (B), intermediate layers (I), and superficial layer (S). Notice, the crypt (C) and lymphatic follicle (F) [× 100, scale bar = 200 µm]. (d) A higher magnification of the previous section showing the basal layer of the epithelium (B). It is formed of columnar cells with basal oval nuclei. Intermediate layers (I), multiple layers of polyhedral cells with central rounded nuclei. Superficial layer (S), multiple layers of flat cells with flat nuclei. Notice, the lymphatic follicle (F) which is formed of aggregation of lymphocytes with darkly stained nuclei [× 200, scale bar = 100 µm]. (e) Placebo group, showing thinning of the covering epithelium (arrow) with multiple areas of hemorrhage (Hg) can be seen within it. An apparent decrease in the number of lymphatic follicles (F), some of them have areas of hemorrhage (Hg). An apparent increase in connective tissue (CT) under the epithelium with multiple dilated congested blood vessels (Bv) within it [× 40, scale bar = 250 µm]. (f) Placebo group, showing follicular expansion and irregularity (F). Thickening of the CT around the tonsil (CT). Multiple areas of hemorrhage (Hg) and dilated congested blood vessels (Bv) can be seen. Notice, the gland (Gl) [× 40, scale bar = 250 µm]. (g) Placebo group, showing multiple vacuolated epithelial cells (arrow) within the epithelium. An apparent decrease in the number of lymphatic follicles (F). The apparent increase in connective tissue (CT) under the epithelium with dilated congested blood vessels (Bv) within it [× 100, scale bar = 200 µm]. (h) Placebo group, showing an area of erosion; ulceration (black arrow) within the epithelium. Some vacuolated cells appear within the epithelium (orange arrow). A marked mononuclear cellular infiltration (white arrow) can be seen [× 100, scale bar = 200 µm]. (i) Placebo group, showing many vacuolated cells within the covering epithelium (black arrow). Crypt (C) can be seen with desquamated cells within it. Lymphatic follicles can't be seen. A mononuclear cellular infiltration (white arrow) can be seen within the CT tissue. Notice, multiple dilated congested blood vessels (Bv) [× 100, scale bar = 200 µm]. (j) Placebo group, showing thinning of the covering epithelium (arrow) with a large area of hemorrhage (Hg) can be seen within it. Enlargement of the lymphatic follicle (F) under the epithelium is observed [× 100, scale bar = 200 µm]. (k) Placebo group, showing thinning of the covering epithelium (arrow) with multiple areas of hemorrhage (Hg) can be seen within it. Enlargement of the lymphatic follicle (F) under the epithelium is observed. Areas of hemorrhage (Hg) can be seen within this follicle. Notice, multiple dilated congested blood vessels (Bv) [× 100, scale bar = 200 µm]. (l) Vitamin D group, showing that the palatine tonsil restores its histological architecture, it appears the same as the control group. Notice, the epithelial covering (arrow) with crypt (C) and lymphatic follicles (F) with germinal centers (G) [× 40, scale bar = 250 µm]. (m) Vitamin D group, showing that the palatine tonsil restores its histological architecture, it appears the same as the control group but there are some congested blood vessels (Bv). Notice, the lymphatic follicles (F) with germinal centers (G), CT band, and glands (Gl) [× 40, scale bar = 250 µm]. (n) Vitamin D group, showing that the palatine tonsil restores its histological architecture, appears the same as the control group. The epithelial covering (black arrow) has some vacuolated cells (white arrow). Notice, the crypt (C) and lymphatic follicle (F) [× 100, scale bar = 200 µm].

Figure 2

Photomicrographs of palatine tonsil sections stained with Sirius red stain showing: (a) Control group, minimal collagen fibers deposition around the follicles (arrow) [× 40, scale bar = 250 µm]. (b) Control group, few collagens’ fibers depositions around the follicles (arrow) [× 100, scale bar = 200 µm]. (c) Placebo group, thick collagen bands in the subepithelial region (arrow) [× 40, scale bar = 250 µm]. (d) Placebo group marked deposition of thick collagen bands in the subepithelial region and between the follicles (arrows) [× 40, scale bar = 250 µm]. (e) Placebo group, deposition of thick collagen fibers in the subepithelial region (arrow) [× 100, scale bar = 200 µm]. (f) Placebo group marked deposition of thick collagen fibers within the tonsil (arrow) [× 100, scale bar = 200 µm]. (g) Vitamin D group, mild collagen fibers deposition in the subepithelial region and around the follicles (arrows) [× 40, scale bar = 250 µm]. (h) Vitamin D group, mild collagen fibers deposition around the follicles (arrow) [× 100, scale bar = 200 µm].

Figure 3

Photomicrographs of palatine tonsil sections stained with immunohistochemical stain for CD68 [Avidine biotin peroxidase stain with Hx counter stain X 400, scale bar = 50 µm] showing: (a) Control group, few CD68 positive cells, macrophages at the subepithelial level (arrow). (b) Placebo group, numerous CD68 positive cells, and macrophages present in the epithelial covering and at the subepithelial level (arrows). (c) Placebo group, numerous CD68 positive cells, macrophages at the subepithelial level (arrow). (d) Placebo group, numerous CD68 positive cells, macrophages at the subepithelial level (arrow). (e) Vitamin D group, some CD68 positive cells, macrophages at the subepithelial level (arrow).

Figure 4

Effect of vitamin D supplementation on area percent collagen fibers in tonsillar tissue, n = 10. Bars not sharing the common superscript letters differ significantly at a P < 0.001 by ANOVA followed by post hoc Tukey's multiple comparisons tests.

Figure 5

Effect of vitamin D supplementation on the number of CD68 + ve cells in tonsillar tissue, n = 10. Bars not sharing the common superscript letters differ significantly at a P < 0.001 by ANOVA followed by post hoc Tukey's multiple comparisons tests.