Alopecia in IL-10-deficient mouse pups is c-kit-dependent and can be triggered by iron deficiency

Abstract

Hair loss (alopecia) can result from a variety of metabolic, endocrine, immunologic, and environmental causes. This investigation was undertaken to determine the mechanisms underlying the sporadic development of alopecia in litters from C57BL/6 interleukin-10-deficient (Il10(-/-)) mice. All pups in affected litters demonstrated alopecia by postnatal days 17-19, with hair loss from their trunks but not from their head, base of tail, or feet. Histopathology revealed distorted hair follicles containing broken hair shafts and prominent dermal infiltrates containing increased numbers of activated mast cells. Hair re-growth began soon after weaning, suggesting that the alopecia was triggered by factors transmitted during lactation. Milk from Il10(-/-) dams induced macrophage secretion of pro-inflammatory cytokines in vitro regardless of whether or not their pups developed alopecia. Feeding dams a diet containing 3-6 ppm iron increased the percentage of litters with alopecia to 100% for pups with mast cells, with 0% alopecia in mast cell-deficient pups. When dams were fed a diet containing 131 ppm iron, significantly lower haemoglobin and hematocrit values were observed in pups from litters with alopecia (71%; 5 of 7 litters) compared to litters without alopecia. Genetic or pharmacologic inhibition of c-kit that resulted in depletion of mast cells in pups prevented hair loss in at-risk litters. These studies demonstrate that maternal iron-restricted diets enhance the incidence of alopecia in IL-10-deficient mouse pups and suggest mast cells as potential effector cells. Further studies are indicated to further explore the mechanisms involved and to determine how mast cells may contribute to alopecia in humans.

Figure 1. Alopecia in IL-10^−/− mice

Mouse is shown on day 27, representative of appearance on days 17 – 35.

Figure 2. Skin histology of Il10^−/− pups with alopecia

On day 19, the skin of mice with alopecia contains dilated follicles with bent and broken hairs (A), as well as a prominent inflammatory infiltrate that includes neutrophils (arrows, B) and mononuclear cells. Numerous degranulating mast cells are also present throughout the dermis (arrow points to relased granules). Panels A and B were stained with hematoxylin and eosin. Panel C was stained with toluidine blue. Bar in A – C represents 50 µm.

Figure 3. Skin histology from Il10^−/− pups with and without alopecia

Dilated follicles containing bent and broken hairs are evident in the skin of mice with alopecia (A), as well as a prominent inflammatory infiltrate that includes numerous degranulating mast cells (I). Il10^−/− mice that do not develop alopecia have normal follicles in catagen or telogen on day 19 (B). Mast cell depletion using anti-c-kit mAb prevents the development of alopecia in at-risk litters. (D). Skin from a saline-treated littermate with alopecia is shown in C for comparison. Feeding dams an iron-deficient diet enhances the incidence of alopecia in litters of Il10^−/− pups (E), but does not cause alopecia in mast cell-deficient Kit^W-sh/W-sh x Il10^−/− pups (F). The abnormal hair follicles seen when alopecia is triggered by an iron-deficient diet (H) are indistinguishable from those seen in sporadic alopecia (G). All skin shown was obtained on P19 and was stained with toluidine blue. The scale bar represents 100 µm in panels A – H and 20 µm in panel I.

Figure 4. Treatment with c-kit antibody or genetic mutation of c-kit protects against Il10^−/− pups against alopecia

A–C. Pups from an Il10^−/− dam were randomized to receive i.p. injections with anti-c-kit mAb ACK2 or saline every 3 days, beginning on day 5. In addition, one mast cell-deficient pup from a Kit^W-sh/W-sh x Il10^−/− dam was fostered to this litter on day 2 of life. Photographs were taken on day 17. Pharmacologic (ACK-2, panel B) or genetic lack of mast cells (panel C) prevented the spontaneous development of alopecia in pups nursed by this dam. D – F. Kit^+/W-sh x Il10^−/− mice were mated to generate black pups with normal numbers of mast cells (D), black and white-sashed pups with intermediate numbers of mast cells (E), and white pups lacking mast cells within the same litter (F). In this experiment, dams were placed on an iron-deficient (3 – 6 ppm) diet on day 16 of gestation. Similar results were obtained when a diet containing 131 ppm iron was initiated on day 0 of gestation. As shown, pups with mast cells (D, E) developed alopecia, while pups lacking mast cells (F) showed no evidence of hair loss. Photographs were obtained on day 21.

Figure 5. Milk from IL-10^−/− dams induces production of pro-inflammatory cytokines by RAW267.4 macrophages

Milk samples from dams whose litters did or did not develop alopecia were cultured with RAW267.4 macrophages and cytokine production was determined as described in Methods. On each graph, the mean ± SEM for each cytokine or chemokine is shown for milk samples from dams whose litters developed alopecia (n = 3) and from dams whose litters did not develop alopecia (n = 5). Note differences in scale between the 2 graphs. * indicates p < 0.05 compared with absence of lipoprotein lipase (LPL).