Is there a 'gut-brain-skin axis'?

Emerging evidence arising from interdisciplinary research supports the occurrence of communication axes between organs, such as the brain-gut or brain-skin axis. The latter is employed in response to stress challenge, along which neurogenic skin inflammation and hair growth inhibition is mediated. We now show that ingestion of a Lactobacillus strain in mice dampens stress-induced neurogenic skin inflammation and the hair growth inhibition. In conclusion, we are introducing a hypothesis, encouraged by our pilot observations and resting upon published prior evidence from the literature, which amalgamates previously proposed partial concepts into a new, unifying model, i.e. the gut-brain-skin axis. This concept suggests that modulation of the microbiome by deployment of probiotics can not only greatly reduce stress-induced neurogenic skin inflammation but even affect a very complex cutaneous phenomenon of (mini-) organ transformation, i.e. hair follicle cycling. These observations raise the intriguing prospect that feeding of just the right kind of bacteria can exert profound beneficial effects on skin homoeostasis, skin inflammation, hair growth and peripheral tissue responses to perceived stress.

Stress-induced neurogenic inflammation in murine skin skews dendritic cells towards maturation and migration: key role of intercellular adhesion molecule-1/leukocyte function-associated antigen interactions.

The skin continuously serves as a biosensor of multiple exogenous stressors and integrates the resulting responses with an individual's central and peripheral endogenous response systems to perceived stress; it also acts to protect against external challenges such as wounding and infection. We have previously shown in mice that stress induces nerve growth factor- and substance P-dependent neurogenic inflammation, which includes the prominent clustering of MHC class II(+) cells. Because the contribution of dendritic cells (DCs) in response to stress is not well understood, we examined the role of DCs in neurogenic inflammation in murine skin using a well-established murine stress model. We show that sound stress increases the number of intradermal langerin(+) and CD11c(+) DCs and induces DC maturation, as indicated by the up-regulated expression of CD11c, MHC class II, and intercellular adhesion molecule-1 (ICAM-1). Blocking of ICAM-1/leukocyte function-associated antigen-1 interactions significantly abrogated the stress-induced numeric increase, maturation, and migration of dermal DCs in vivo and also reduced stress-induced keratinocyte apoptosis and endothelial cell expression of ICAM-1. In conclusion, stress exposure causes a state of immune alertness in the skin. Such adaptation processes may ensure protection from possible infections on wounding by stressors, such as attack by predators. However, present-day stressors have changed and such adaptations appear redundant and may overrun skin homeostasis by inducing immune dermatoses.

Electron paramagnetic resonance (EPR) spectroscopy for investigating murine telogen skin after spontaneous or depilation-induced hair growth.

BACKGROUND: Depilation has greatly promoted our understanding of hair follicle biology, however, only marginally of telogen (the "resting" stage of the hair cycle). Since electron paramagnetic resonance (EPR) spectroscopy provides an instructive technique for analyzing hair biology, it may be useful for telogen research. OBJECTIVES: To identify differences in murine telogen skin after a spontaneous and depilation-induced hair follicle cycling, and to analyze applicability of EPR to investigate telogen. METHODS: Spontaneous or depilation-induced hair cycling in C57BL/6 mice. EPR spectroscopy of unshaven skin and of shaved hair shafts, microscopical examination of plucked or shed hair shafts, standardized histomorphometry. RESULTS: Melanin EPR signals did not differ qualitatively between the two examined types of skin, nor did depilation change the hair length. However, unmanipulated telogen skin revealed greater thickness, stronger EPR signals, 25% more hair shafts, and lower melanin content of individual hair shafts, as creating a much more intricate mosaic of telogen hair follicles with various numbers of hair shafts (0-3) than the skin after depilation-induced hair growth. In both types of skin empty pilary canals were found. Both groups of animals lost hair shafts which were typical of exogen (the actively controlled process of hair shedding). CONCLUSIONS: EPR spectroscopy can be profitably employed to study telogen. Murine telogen skin reveals a kenogen-like phenomenon (the "lag" phase following telogen and exogen when hair follicles remain empty, i.e. are devoid of hair shafts). Murine skin thickness in telogen and individual hair shaft pigmentation depend on the way of hair growth induction. Telogens after a spontaneous or depilation-induced hair growth are biologically distinct.

A pivotal role for galectin-1 in fetomaternal tolerance.

A successful pregnancy requires synchronized adaptation of maternal immune-endocrine mechanisms to the fetus. Here we show that galectin-1 (Gal-1), an immunoregulatory glycan-binding protein, has a pivotal role in conferring fetomaternal tolerance. Consistently with a marked decrease in Gal-1 expression during failing pregnancies, Gal-1-deficient (Lgals1-/-) mice showed higher rates of fetal loss compared to wild-type mice in allogeneic matings, whereas fetal survival was unaffected in syngeneic matings. Treatment with recombinant Gal-1 prevented fetal loss and restored tolerance through multiple mechanisms, including the induction of tolerogenic dendritic cells, which in turn promoted the expansion of interleukin-10 (IL-10)-secreting regulatory T cells in vivo. Accordingly, Gal-1's protective effects were abrogated in mice depleted of regulatory T cells or deficient in IL-10. In addition, we provide evidence for synergy between Gal-1 and progesterone in the maintenance of pregnancy. Thus, Gal-1 is a pivotal regulator of fetomaternal tolerance that has potential therapeutic implications in threatened pregnancies.

Neuronal plasticity of the "brain-skin connection": stress-triggered up-regulation of neuropeptides in dorsal root ganglia and skin via nerve growth factor-dependent pathways.

Emerging research indicates that central-nervous stress perception is translated to peripheral tissues such as the skin not only via classical stress hormones but also via neurotrophins and neuropeptides. This can result in neurogenic inflammation, which is likely to contribute to the triggering and/aggravation of immunodermatoses. Although the existence of such a "brain-skin connection" is supported by steadily increasing experimental evidence, it remains unclear to which extent perceived stress affects the sensory "hardwiring" between skin and its afferent neurons in the corresponding dorsal root ganglia (DRG). In this paper, we provide experimental evidence in a murine model of stress (exposure of C57BL/6 mice to sound stress) that stress exposure, or intracutaneous injection of recombinant nerve growth factor (NGF) to mimic the skin's response to stress, up-regulate the percentage of substance P (SP)+ or calcitonin gene-related peptide (CGRP)+ sensory neurons in skin-innervating DRG. Further, we show that the number of SP+ or CGRP+ sensory nerve fibers in the dermis of stressed C57BL/6 mice is significantly increased. Finally, we document that neutralization of NGF activity abrogates stress-induced effects on the percentage of SP+ and CGRP+ sensory neurons in skin-innervating DRG as well as on dermal sensory nerve fibers. These data suggest that high stress perception results in an intense cross talk between the skin and skin-innervating DRG, which increases the likelihood of NGF-dependent neurogenic skin inflammation by enhancing sensory skin innervation.

High expression of survivin and down-regulation of Stat-3 characterize the feto-maternal interface in failing murine pregnancies during the implantation period.

The materno-fetal interface has for long been considered as an immune privileged biological site and thus understanding the mechanisms underlying fetal survival have been the focus of intense research. In adults, survivin and Stat-3 proteins are involved in tolerance as well as the induction of apoptosis. However, the role of these molecules in pregnancy and development has not been addressed. We have evaluated the expression of survivin and Stat-3 in allogeneic mouse models of low abortions (CBA/J x Balb/c), abortion prone (CBA/J x DBA/2J) and stress-triggered abortions from DBA/2J-mated CBA/J mice. We show that survivin is over-expressed in abortion-prone mating on gestation day 7.5. This effect was also found in stress-exposed mice, whereas expression was low in normal pregnancy mice. The phosphorylated Stat-3 (p-Stat-3) was down regulated in high abortion mating compared with low abortion mating, CBA/J x Balb/c. The level of apoptosis was similar in the three groups studied. Our results suggest that high expression of survivin and low expression of p-Stat-3 are involved in pregnancy loss in mice.

Towards a "free radical theory of graying": melanocyte apoptosis in the aging human hair follicle is an indicator of oxidative stress induced tissue damage.

Oxidative stress is generated by a multitude of environmental and endogenous challenges such as radiation, inflammation, or psychoemotional stress. It also speeds the aging process. Graying is a prominent but little understood feature of aging. Intriguingly, the continuous melanin synthesis in the growing (anagen) hair follicle generates high oxidative stress. We therefore hypothesize that hair bulb melanocytes are especially susceptible to free radical-induced aging. To test this hypothesis, we subjected human scalp skin anagen hair follicles from graying individuals to macroscopic and immunohistomorphometric analysis and organ culture. We found evidence of melanocyte apoptosis and increased oxidative stress in the pigmentary unit of graying hair follicles. The "common" deletion, a marker mitochondrial DNA-deletion for accumulating oxidative stress damage, occurred most prominently in graying hair follicles. Cultured unpigmented hair follicles grew better than pigmented follicles of the same donors. Finally, cultured pigmented hair follicles exposed to exogenous oxidative stress (hydroquinone) showed increased melanocyte apoptosis in the hair bulb. We conclude that oxidative stress is high in hair follicle melanocytes and leads to their selective premature aging and apoptosis. The graying hair follicle, therefore, offers a unique model system to study oxidative stress and aging and to test antiaging therapeutics in their ability to slow down or even stop this process.

Zinc as an ambivalent but potent modulator of murine hair growth in vivo- preliminary observations.

Oral zinc (Zn(2+)) is often employed for treating hair loss, even in the absence of zinc deficiency, although its mechanisms of action and efficacy are still obscure. In the current study, we explored the in vivo effects of oral zinc using the C57BL/6 mouse model for hair research. Specifically, we investigated whether continuous administration of high-dose ZnSO(4) x 7H(2)O (20 mg/ml) in drinking water affects hair follicle (HF) cycling, whether it retards or inhibits chemotherapy-induced alopecia (CIA) and whether it modulates the subsequent hair re-growth pattern. Here, we show that high doses of oral zinc significantly inhibit hair growth by retardation of anagen development. However, oral zinc also significantly retards and prolongs spontaneous, apoptosis-driven HF regression (catagen). Oral zinc can also retard, but not prevent, the onset of CIA in mice. Interestingly, Zn(2+) treatment of cyclophosphamide-damaged HFs also significantly accelerates the re-growth of normally pigmented hair shafts, which reflects a promotion of HF recovery. However, if given for a more extended time period, zinc actually retards hair re-growth. Thus, high-dose oral zinc is a powerful, yet ambivalent hair growth modulator in mice, whose ultimate effects on the HF greatly depend on the timing and duration of zinc administration. The current study also encourages one to explore whether oral zinc can mitigate chemotherapy-induced hair loss in humans and/or can stimulate hair re-growth.

Reduced uterine indoleamine 2,3-dioxygenase versus increased Th1/Th2 cytokine ratios as a basis for occult and clinical pregnancy failure in mice and humans.

PROBLEM: Indoleamine 2,3-dioxygenase (IDO) expression in fetal trophoblast and decidual antigen-presenting cells has been proposed to inactivate maternal T cells and thereby prevent rejection of the "fetal allograft" in early pregnancy. Psychic stress has been proposed to cause miscarriages as well as infertility, at the same time in pregnancy when blockade of IDO causes loss, but the suggested mechanism of stress-triggered loss has been an increased ratio of pro-rejection Th1-type cytokines to anti-rejection Th2/3 cytokines. Could stress act by reducing IDO expression? METHODS: Using DBA/2-mated A/J mice where stress causes early pregnancy failure, we examined the role of stress in reducing IDO versus increasing Th1/Th2 ratio in deciduas. IDO loss was also examined in human decidua associated with pregnancy failure. RESULTS: A post-implantation sonic stress increased the pregnancy failure rate, increased the Th1/Th2 ratio, but did not reduce IDO. IDO was reduced, and Th1/Th2 ratios increased in A/J mice pre-immunized against paternal DBA/2 antigens, and concomitant stress increased these effects. The rate of pregnancy failure was not further increased consistent with recent discoveries of factors that limit the impact of Th1 cytokines at the feto-maternal interface. In deciduas from spontaneous miscarriage patients, IDO(+) cell frequencies were low in only 30% of patients. CD3(+) T-cell numbers and percentage terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labelling (TUNEL)(+) apoptotic T cells were increased, but the level of IDO did not correlate with likelihood of apoptosis. CONCLUSIONS: Loss of an allogeneic embryo in early pregnancy is more likely to be due to a high Th1/Th2 ratio than loss of putative protection by IDO.

Splenic eumelanin differs from hair eumelanin in C57BL/6 mice.

The presence of melanin in spleens of black C57BL/6 mice has been known for long. Although its origin and biological functions are still obscure, the relation of splenic melanin to the hair follicle and skin pigmentation was suggested. Here, we demonstrated using for the first time electron paramagnetic resonance spectroscopy that black-spotted C57BL/6 spleens contain eumelanin. Its presence here is a "yes or no" phenomenon, as even in the groups which revealed the highest percentage of spots single organs completely devoid of the pigment were found. Percentage of the spotted spleens decreased, however, with the progress of telogen after spontaneously-induced hair growth. The paramagnetic properties of the spleen eumelanin differed from the hair shaft or anagen VI skin melanin. The splenic melanin revealed narrower signal, and its microwave power saturability betrayed more heterogenous population of paramagnetic centres than in the skin or hair shaft pigment. Interestingly, the pigment of dry hair shafts and of the wet tissue of depilated anagen VI skin revealed almost identical properties. The properties of splenic melanin better resembled the synthetic dopa melanin (water suspension, and to a lesser degree -- powder sample) than the skin/hair melanin. All these findings may indicate a limited degradation of splenic melanin as compared to the skin/hair pigment. The splenic eumelanin may at least in part originate from the skin melanin phagocyted in catagen by the Langerhans cells or macrophages and transported to the organ.

A guide to assessing damage response pathways of the hair follicle: lessons from cyclophosphamide-induced alopecia in mice.

After chemical, biological, or physical damage, growing (i.e. anagen) hair follicles develop abnormalities that are collectively called hair follicle dystrophy. Comparatively lower follicular damage induces the "dystrophic anagen" response pathway (=prolonged, dystrophic anagen, followed by severely retarded follicular recovery). More severe follicular damage induces the dystrophic catagen pathway (=immediate anagen termination, followed by a dystrophic, abnormally shortened telogen and maximally fast follicular recovery). In order to recognize these distinct damage response strategies of the hair follicle in a clinical or histopathological context, we have used the well-established C57BL/6J mouse model of cyclophosphamide-induced alopecia to define pragmatic classification criteria for hair follicle dystrophy (e.g., structure and pigmentation of the hair shaft, location, and volume of ectopic melanin granules, distension of follicular canal, number of TdT-mediated dUTP nick end labeling positive keratinocytes in the hair bulb; neural cell-adhesion molecule immunoreactivity and alkaline phosphatase activity as markers for the level of damage to the follicular papilla). These classification criteria for hair follicle dystrophy are useful not only in chemotherapy-induced alopecia models, but also in the screening of drug-treated or mutant mice in a highly standardized, accurate, sensitive, reproducible, easily applicable, and quantifiable manner.

Mast cell deficient and neurokinin-1 receptor knockout mice are protected from stress-induced hair growth inhibition.

Despite the lack of insight on distinct mediators in the skin orchestrating the pathophysiological response to stress, hair loss has often been reported to be caused by stress. Recently we revealed the existence of a "brain-hair follicle axis" by characterizing the neurokinin (NK) substance P (SP) as a central element in the stress-induced threat to the hair follicle, resulting in premature onset of catagen accompanied by mast cell activation in the skin. However, our understanding of possible SP-mast cell interactions in the skin in response to stress was limited since the receptor by which SP activates skin mast cells and the extent of mast cell mediated aggravation of SP remained to be elucidated. We now employed NK-1 receptor knockout mice (NK-1R(-/-)) and mast cell deficient W/W(v) mice and observed that stress-triggered premature induction of catagen and hair follicle apoptosis does not occur in NK1(-/-) and W/W(v) mice. Furthermore, the activation status of mast cells was less in stressed NK1(-/-) mice than in wild-type control. Additionally, stress-induced upregulation of SP positive nerve fibers was absent in both NK-1R and W/W(v) mice. These results indicate that the cross-talk between SP and mast cell activation via NK-1R appears to be the most important pathway in the regulation of hair follicle cycling upon stress response.

Hair cycle control by estrogens: catagen induction via estrogen receptor (ER)-alpha is checked by ER beta signaling.

Although 17beta-estradiol (E2) is recognized as a potent hair growth modulator, our knowledge of estrogen function, signaling, and target genes in hair biology is still very limited. Between the two recognized estrogen receptors (ERs), ER alpha and ER beta, only ER alpha had been detected in murine skin. Here we show that ER alpha, ER beta, and ER beta ins are all expressed throughout the murine hair cycle, both at the protein and RNA level, but show distinct expression patterns. We confirm that topical E2 arrests murine pelage hair follicles in telogen and demonstrate that E2 is a potent inducer of premature catagen development. The ER antagonist ICI 182.780 does not induce anagen prematurely but accelerates anagen development and wave spreading in female mice. ER beta knockout mice display accelerated catagen development along with an increase in the number of apoptotic hair follicle keratinocytes. This suggests that, contrary to previous concepts, ER beta does indeed play a significant role in murine hair growth control: whereas the catagen-promoting properties of E2 are mediated via ER alpha, ER beta mainly may function as a silencer of ER alpha action in hair biology. These findings illustrate the complexity of hair growth modulation by estrogens and suggest that one key to more effective hair growth manipulation with ER ligands lies in the use of selective ER alpha or -beta antagonists/agonists. Our study also underscores that the hair cycling response to estrogens offers an ideal model for studying the controls and dynamics of wave propagation in biological systems.

Neurogenic inflammation in stress-induced termination of murine hair growth is promoted by nerve growth factor.

Recently, we have revealed the existence of a "brain-hair follicle axis" in murine skin and have identified the neuropeptide substance P (SP) as a key mediator of stress-induced hair growth inhibition in vivo. Published evidence suggests that increased numbers of SP-immunoreactive sensory fibers, as seen in the dermis of stressed mice in anagen-catagen transition, are a result of transient high levels of nerve growth factor (NGF). Thus, we now aimed at dissecting the role of NGF in stress-triggered hair growth termination in our murine model. By real time PCR and immunohistochemistry, stress-exposed mice showed an up-regulation of NGF and its low-affinity receptor p75NTR; the NGF high-affinity receptor TrkA was moderately down-regulated. On neutralization of NGF, premature onset of catagen, apoptosis, and increased number/activation of perifollicular mast cells and antigen-presenting cells, which reflects the skin response to stress, was significantly abrogated. Stress or subcutaneous injection of recombinant NGF (to mimic stress) resulted in an increased percentage of SP(+) neurons in dorsal root ganglia, as measured by retrograde tracing. Taken together, these data suggest that NGF is a central element in the perifollicular neurogenic inflammation that develops during the murine skin response to stress and antagonizing NGF may be a promising therapeutic approach to counter the negative effect of stress on hair growth.

Topical estrogen accelerates hair regrowth in mice after chemotherapy-induced alopecia by favoring the dystrophic catagen response pathway to damage.

Estrogen receptor ligands are important modulators of skin physiology and are involved in the control of normal hair follicle cycling. Here, we have studied the effects of topically applied 17-beta-estradiol on pathologic hair follicle cycling as seen during chemotherapy-induced alopecia, one of the major unresolved problems of clinical oncology. For this study we employed a well-established murine model that mimics chemotherapy-induced alopecia in humans. For precisely quantifying the area of hair loss and hair regrowth in this model in vivo, we developed a simple planimetric assay (dotmatrix planimetry). We show that topical 17-beta-estradiol significantly alters the cycling response of murine follicles to cyclophosphamide, whereas the estrogen antagonist ICI 182.780 exerted no such effects. Initially, topical 17-beta-estradiol enhanced chemotherapy-induced alopecia significantly by forcing the follicles into the dystrophic catagen response pathway to hair follicle damage, whereas follicles treated by ICI 182.780 or vehicle shifted into the dystrophic anagen response pathway. Consequently, the regrowth of normally pigmented hair shafts after chemotherapy-induced alopecia was significantly accelerated in the 17-beta-estradiol treated group. Our data encourage one to explore topical estrogens as a potential stimulant for hair re-growth after chemotherapy-induced alopecia.

Stress inhibits hair growth in mice by induction of premature catagen development and deleterious perifollicular inflammatory events via neuropeptide substance P-dependent pathways.

It has been much disputed whether or not stress can cause hair loss (telogen effluvium) in a clinically relevant manner. Despite the paramount psychosocial importance of hair in human society, this central, yet enigmatic and controversial problem of clinically applied stress research has not been systematically studied in appropriate animal models. We now show that psychoemotional stress indeed alters actual hair follicle (HF) cycling in vivo, ie, prematurely terminates the normal duration of active hair growth (anagen) in mice. Further, inflammatory events deleterious to the HF are present in the HF environment of stressed mice (perifollicular macrophage cluster, excessive mast cell activation). This provides the first solid pathophysiological mechanism for how stress may actually cause telogen effluvium, ie, by hair cycle manipulation and neuroimmunological events that combine to terminate anagen. Furthermore, we show that most of these hair growth-inhibitory effects of stress can be reproduced by the proteotypic stress-related neuropeptide substance P in nonstressed mice, and can be counteracted effectively by co-administration of a specific substance P receptor antagonist in stressed mice. This offers the first convincing rationale how stress-induced hair loss in men may be pharmacologically managed effectively.

Topical minoxidil counteracts stress-induced hair growth inhibition in mice.

Stress has long been suspected as a possible cause of hair loss in various species, even though convincing experimental evidence has not been available. Recently, we have shown in a murine model that sonic stress alters hair growth and cycling in vivo, and have postulated the existence of a 'brain-hair follicle axis' (BHA). In order to study whether a clinically available and widely used topically active hair growth stimulator mitigates stress-triggered hair growth inhibition in this stress model, we have applied a 5% minoxidil solution. Female CBA/J mice were depilated and randomized in to two groups: control (n = 20) and sonic stress (n = 20). These groups were further divided and either treated daily with 5% minoxidil solution or vehicle alone. The stress group was exposed to sonic stress for 24 h starting 14 days after anagen induction by depilation. All mice were sacrificed 16 days after the depilation and assessed by quantitative histomorphometry. Sonic stress significantly increased the number of hair follicles with apoptotic cells and inhibited intrafollicular keratinocyte proliferation. In addition, the number of clusters of perifollicular MHC class II+ cells and degranulated perifollicular mast cells was significantly enhanced in the stressed mice. In accordance with previous findings, all stressed mice showed an advanced hair cycle progression towards catagen. All of these stress-induced hair growth inhibitory changes along the BHA were down-regulated by topical minoxidil application. This encourages one to explore clinically whether topical minoxidil is a safe and effective pharmacologic tool for the management of stress-associated telogen effluvium in humans.