Hair loss results from follicular cyst (FC) formation
mediated by increased inflammation
In mice, hair follicle morphogenesis as well as the first
round of postnatal catagen (regression), telogen (quiescence),
exogen (shedding), and anagen (growth) development
follows a rather precise time scale (Paus and Cotsarelis
1999; Muller-Rover et al. 2001). To pinpoint the
hair cycling defect, we examined the histology of affected
skins throughout the postnatal time course. The
hair follicles exhibited normal morphology until P16–
P20. At P20, in pups from ?cre control mothers, catagen
started when the follicles regressed, followed by telogen
when the follicles became quiescent. Around P32, the
follicles underwent exogen, ejecting the hair shaft as the
next anagen began (Fig. 2A). In contrast, in pups from
+cre mutant mothers, a precocious formation of FCs was
observed around P20 with hair shaft ejection but without
the concurrent initiation of anagen (Fig. 2A). These aberrant
follicular structures persisted until around P32,
when the first postnatal anagen started in which all hair
layers showed inward proliferation comparable to controls.
The dermal papillae were well developed, and new
hair shafts were present in the follicles (Fig. 2A). Therefore,
the major defect was in the first postnatal catagen/
telogen. Furthermore, there appeared to be profound leukocyte
infiltration in the interfollicular region and subcutaneous
fat in the affected skin (Fig. 2B), suggesting the
presence of inflammation. Indeed, immunofluorescence
staining for CD11b/Mac-1 demonstrated a marked increase
in macrophage accumulation in the skin of the
pups nursed by +cre mutant mothers as early as P10 (Fig.
2C). Together, the histological evidence suggests that
the hair loss was due to FC formation during the first
postnatal catagen/telogen, possibly due to increased inflammation.
We performed gene expression analyses of the skins
from pups nursed by mutant or control mothers using
real-time quantitative PCR (RT-QPCR). First, we tested
two genes that are important for hair follicle development.
Cyclin D1 and GATA3 are critical for stem cell
differentiation in the outer root sheath (ORS) and inner
root sheath (IRS), respectively (Kaufman et al. 2003; Xu
et al. 2003). The expression of both genes was decreased
in the affected skin (+cre) during the stages of hair loss
(P20–P31) (Fig. 2D), but by P34, these differences resolved
along with the recovery of pelage. Reduced expression
of both genes indicated the lack of stem cell
proliferation and differentiation required for anagen.
To determine if there was increased inflammation in
the affected skin at the transcriptional level, the expression
of a set of inflammatory markers was measured (Fig.
3A). Expression of cytokines IL-1 and TNF, chemokines
MCP-1 and MCP-3, and the matrix metalloproteinase
MMP9 were all dramatically increased in the affected
skin compared with the control skin. IL-1 and
TNF are important mediators of the inflammatory response
that are mainly secreted by macrophages. MCP-1
and MCP-3 are chemotactic factors that attract monocytes,
basophils, or eosinophils. MMP9 plays an essential
role in local proteolysis of the extracellular matrix
and in leukocyte migration. Therefore, the affected
skin exhibited an inflammatory response hallmarked by
leukocyte infiltration and cytokine/chemokine secretion.
The arachidonic acid/COX/prostaglandin pathway has
been shown to be an important regulator of inflammatory
responses. Interestingly, this pathway is also involved
in the regulation of hair cycling. Two independent
studies have demonstrated that overexpression of
COX-2 in mouse skin leads to alopecia (Bol et al. 2002;
Muller-Decker et al. 2003). Therefore, we examined the
expression of several genes in this pathway. The expression
of both COX-1 and COX-2 genes were markedly
increased in the affected skin (Fig. 3A), as was that of the
prostaglandin transporter (PGT), which mediates the release
of newly synthesized prostaglandins from cells.
Western blot analyses further confirmed these gene expression
changes at the protein level (Fig. 3C). As shown
in Figure 3, A and C, COX-2 expression in the control
skin declined in catagen (P17–P20) and was barely detectable
in early telogen (P21), but increased again during
anagen (P28–P40) (Muller-Decker et al. 2003). In contrast,
COX-2 expression in the affected skin was significantly
elevated during catagen/telogen (P16–P25) (Fig.
3A,C). This was also the case in the K5-COX2 and K14-
COX2 transgenic mice that developed alopecia (Bol et al.
2002; Muller-Decker et al. 2003). Together with the increased
expression of COX-1, this pattern of COX expression
was unscheduled compared with the normal
controls and correlated with the precocious FC formation
and hair loss. The increased expression of these in-flammatory genes and macrophage infiltration began between
P7 and P13, preceding the hair loss (Figs. 3B, 2C),
suggesting the inflammation was likely the cause rather
than the consequence of the hair loss. Furthermore, the
expression of most inflammatory markers in the affected
skin decreased by P48, indicating that the inflammation
slowly resolved after the pups were weaned to a standard
diet (Fig. 3A).
Detailed examination of the pups nursed by gf/fTie2cre
mothers revealed that their livers often appeared paler
compared with control pups. Oil Red O staining of liver
sections indicated increased lipid accumulation (Fig.
3D). Hepatic steatosis is often linked to inflammation,
and indeed, expression of several inflammatory markers
were also elevated in liver (Fig. 3E), suggesting the presence
of inflammation at multiple tissues and manifested
in the skin as hair loss.
Hair loss can be rescued by COX inhibitors or foster
mothers
To test if the increased inflammation and COX signaling
was the cause of the hair cycling defect, we performed
rescue experiments by topical treatment with the COX-
1/2 inhibitor aspirin. Gene expression analyses indicated
that aspirin treatment resulted in the inhibition of inflammation
in the skin of pups from gf/fTie2cre mothers
(Fig. 3F). By P29, all DMSO-treated pups exhibited complete
hair loss over the trunk, while all the aspirin treated
pups retained fairly normal pelage on the back(Fig. 3G), although some hair loss was observed on the
scalp (Fig. 3G, red arrow) and the chest (data not shown)
that was not directly treated. Aspirin treatment did not
have any visible effect on the skin of pups from the control
mothers. This demonstrated that the hair loss was
the result of COX-mediated inflammation.
To further determine if the phenotype was maternally
dependent, we performed foster mother experiments. All
pups born and nursed by gf/fTie2cre mothers exhibited
hair loss, while the pups born from gf/fTie2cre mothers
but fostered by gf/f mothers were rescued at 100% penetrance
(eight out of eight pups, Fig. 3H). Reciprocally,
all pups born and nursed by gf/f mothers showed normal
pelage, while the pups born from gf/f mothers but fostered
by gf/fTie2cre mothers developed hair loss at 75%
penetrance (six out of eight pups). Thus, the phenotype
was indeed maternally dependent. The fact that the hair
cycling defect could be reversed by fostering demonstrates
that it was due to postnatal factors such as milk,
rather than prenatal factors such as the placenta. In addition,
fostering (Fig. 3H), but not topical aspirin treatment
(Fig. 3G), also exhibited a rescuing effect on the
growth retardation. Together, these results suggest that
components in the milk of gf/fTie2cre mothers were able
to elicit inflammatory responses in the pups.
..........................................................
The hair
loss was typically confined to the trunk; however, in
severe cases, it could extend to the scalp (Fig. 3G, left). It
is known that the duration of the hair cycle and the
lengths of hair shafts are different among scalp, dorsal,
and ventricle follicles, with the scalp hair having the
longest cycle (Saitoh et al. 1970; Stenn and Paus 2001).
Therefore, hair follicles from different skin regions have
varying sensitivity to the inflammatory insult from the
“toxic milk,†resulting in differences in the timing and
severity of the hair loss. The acute onset and recovery
after weaning suggests that this is a specific type of alopecia
equivalent to “telogen effluvium†in human patients.
Our findings will enhance the understanding of
the roles for lipid metabolites and inflammation in the
etiology of alopecia and other skin disorders, and facilitate
the development of novel pharmacological strategies
for the prevention and treatment of these diseases.
