Dietary probiotics increase anagen hair follicles and proliferation of sebocytes in aged mice.

<p>(<b>A</b>) Quiescent (telogen-phase) hair follicles predominate in control-diet fed C57BL/6 mice. In contrast, the majority of the hair follicles are active (anagen-phase) in probiotic-fed mice of the same age. Active hair follicles undergoing rapid growth exhibit numerous proliferating cells (ki-67+). More rarely the regressing stage (catagen) contain caspase-3+ apoptotic cells. <i>Control Telogen</i> and <i>Probiotic Anagen</i>: Hematoxylin and Eosin. <i>Probiotic Anagen-ki-67</i> and <i>Probiotic Catagen-caspase-3</i>: DAB chromogen, Hematoxylin counterstain. Bars = 50 µm. Classification of fifty intact longitudinally-sectioned hair follicles per treatment group were evaluated according to their stage of cycling (in <b>B</b> and <b>C</b> below). The distribution pattern of hair-follicle staging differs significantly (P<0.0001) among probiotic- and control diet-fed (<b>B</b>) male and (<b>C</b>) female mice. Numbers on the y-axis of bar graphs represent the mean±SEM of hair-follicles classified in each hair cycle stage. The % percentage of hair follicles in telogen (T), anagen (A) or catagen (C) stage is illustrated in circular graphs. Probiotic-fed mice of both genders show an anagen stage predominance. (<b>D</b>) Dietary supplementation with probiotics lead to a significant (P<0.0001) increase of sebocytes in skin pilosebaceous units. The y-axis stands for the mean±SEM of sebocyte counts per X20 high power field image. (<b>E</b>) The quantitative assessment of cellular proliferation in sebaceous glands with ki-67-specific immunohistochemistry (circular image) reveals that dietary probiotics increase the proliferative capacity of sebocytes. Numbers on the y axis of bar graphs correspond to the mean±SEM of the index of proliferating sebocytes per total number of sebocytes in x40 high power fields. Circular image: DAB chromogen, Hematoxylin counterstain. Bar = 25 µm.</p

Vaginal pH correlates with Lactobacillus abundance and peak fertility in humans.

<p>(<b>A</b>) Bars show mean (+SEM) of vaginal pH for women as a function of number of years before or after peak fertility, which was taken to be 25 years of age for all subjects. (<b>B</b>) Vaginal pH is significantly associated with <i>Lactobacillus</i> abundance (Kendall’s Tau test; τ = −0.38; P = 3.2×10⁻⁸). Statistical differences between groups were assessed using the Mann-Whitney U test.</p

Dietary supplementation with probiotics affects skin histology of mice.

<p>(<b>A</b>) Probiotic-fed C57BL/6 male mice differ from their control diet-fed counterparts by having subcutaneous hair follicle profiles and a thicker skin. Hematoxylin and Eosin. Bars = 250 µm. Histomorphometrical analysis reveals significant (P<0.0001) probiotic diet-associated increase in (<b>B</b>) skin thickness and (<b>C</b>) subcutaneous hair follicles in both genders. The y-axis depicts the mean±SEM of histomorphometric counts in each experimental group.</p

<i>Lactobacillus reuteri</i> –induced benefits in hair quality require anti-inflammatory cytokine Interleukin-10.

<p>In contrast to wild type animals, feeding of L reuteri to aged C57BL/6 mice lacking interleukin (Il)-10 failed to improve subcutaneous hair follicle or sebocyte profile. (<b>A</b>) Histomorphometrical analysis in Il-10-deficient mice reveals insignificant differences in hair follicle activity and distribution (anagen-phase versus telogen-phase). Numbers on the y-axis of bar graphs represent the mean±SEM of hair-follicles classified in each hair cycle stage. (<b>B</b>) Likewise, sebocyte counts were not significantly different among <i>L. reuteri</i>- and control water-fed Il-10-deficient mice. Numbers on the y-axis of bar graphs represent the mean±SEM of sebocyte counts per X20 high power field image. (<b>C</b>) Evaluation of mucocutaneous pH shows eating probiotics induces more alkaline conditions in skin, oral cavity, rectum and vaginal mucosa of Il-10-deficient mice, contrasted with the more acidic conditions in WT female mice (Fig. 1D). (<b>D</b>) Depletion of Il-17A using anti-cytokine antibodies recapitulates the probiotic-induced glow of health features in the skin including hair follicle anagen phase predominance and vastly increased numbers of sebocytes in sebaceous glands. Numbers on the y-axis of bar graphs represent the mean±SEM of hair-follicles classified in each hair cycle stage. Numbers on the y-axis of bar graphs represent the mean±SEM of sebocyte counts per X20 high power field image.</p

Dietary supplementation with purified <i>Lactobacillus reuteri</i> in drinking water mimics effects of eating probiotic yogurt on skin histology of aged mice.

<p>Wild type C57BL/6 mice fed purified <i>L. reuteri</i> in drinking water differ significantly from their regular water-fed counterparts by having thicker skin and an increased subcutaneous hair follicle and sebocyte profile. (<b>A</b>) Histomorphometrical analysis reveals significant (P<0.0001) probiotic diet-associated increase in skin thickness and (<b>B</b>) number of subcutaneous hair follicles (P<0.05) in both genders. The y-axis depicts the mean±SEM of histomorphometric counts in each experimental group. (<b>C</b>) In mice treated with <i>L. reuteri</i> in drinking water, the majority of the hair follicles are active (anagen-phase). In contrast, control mice of the same age have predominantly quiescent (telogen-phase) hair follicles. The distribution pattern of hair-follicle staging differs significantly (P<0.0001) among <i>L. reuteri</i>- and control diet-fed in both male and female mice. Numbers on the y-axis of bar graphs represent the mean±SEM of hair follicles classified in each hair cycle stage. (<b>D</b>) Further, adding purified <i>L. reuteri</i> in drinking water lead to a significant (P<0.0001) increase of sebocytes in skin pilosebaceous units. The y-axis stands for the mean±SEM of sebocyte counts per X20 high power field image. Numbers on the y-axis of bar graphs correspond to the mean±SEM of the index of proliferating sebocytes per total number of sebocytes in x40 high power fields.</p

Systemic depletion of IL-17 recapitulates reproductive fitness attributes of <i>L. reuteri.</i>

<p>Compared to sham IgG-treated control mice, 12 month-old mice depleted of IL-17A had <b>a.</b> increased paired testicular weights; <b>b.</b> increased cross-sectional ST areas; <b>c.</b> increased germ cell nuclear volumes; <b>d and e.</b> increased interstitial Leydig cell areas. Hematoxylin and eosin. Bars = 100 µm; <b>f.</b> increased Leydig cell nuclear volumes and total number per testis and <b>g.</b> decreased ST atrophy. *p<0.05, **p<0.001, ***p<0.0001.</p

<i>L. reuteri</i> consumption increases the size of interstitial Leydig cell areas and Leydig cell numbers.

<p><b>a.</b> Representative histology of Swiss mice testes at the age of 12 months. Leydig cell areas in the testicular interstitium of probiotic-fed mice are of increased size compared to the corresponding areas of control mice. Hematoxylin and eosin. Bars = 100 µm <b>b.</b> Histomorphometrical counts of Leydig cell areas at different time-points revealed the statistical significance of the <i>L. reuteri</i> effect. Numbers on the y-axis of bar graphs correspond to the mean±SEM of Leydig area size. <b>c.</b> Point-counting stereology counts reveal that <i>L. reuteri</i>-fed mouse testes have significantly increased nuclear volumes and an increased number of Leydig cells per testis. Numbers on the y axis of bar graphs correspond to the mean±SEM of “Nuclear volume of germ cells per unit area” or the “absolute nuclear volume of germ cells”; *p<0.05, **p<0.001, ***p<0.0001.</p

Consumption of probiotic microbes increases the weight of mouse testes.

<p><b>a.</b> Gross appearance of outbred Swiss male mice with oral <i>Lactobacillus reuteri</i> treatment at 12 months of age. Testes of <i>L. reuteri</i>-fed mice are larger compared to control mice. <b>b–e.</b> Results of statistical analyses of testes weight at different time-points after starting treatment, including the (<b>b</b>) 5, (<b>c</b>) 7, (<b>d</b>) 9 and (<b>e</b>) 12 months of age time-points. <i>L. reuteri</i> consistently increased testicular weight in both control- and new western-diet-fed mice compared to age- and diet-matched controls. Dietary supplementation with <i>L. reuteri</i>, but not with <i>E coli</i> K12, increased testicular weight in control-diet-fed mice at age 7 months when compared to age- and diet-matched controls (<b>c</b>). Numbers on the y-axis of bar graphs correspond to the mean±SEM of testes weight or the testicular weight; *p<0.05, **p<0.001.</p

Depletion of CD25+ cells abolishes the <i>L. reuteri</i> effect in wound healing while depletion of IL-17 restores the wound healing benefit.

<p>(a) Male and female C57BL/6 mice (n = 8 per group) depleted of CD25+ cells by anti-CD25 antibody have larger wounds when compared with sham isotype IgG-treated control mice, despite uniform <i>L. reuteri</i> consumption in both groups. (b) The wounds of CD25 cell-depleted mice do not show the histopathological evidence of the typical <i>L. reuteri</i>-induced accelerated wound repair process, namely sham IgG exhibit complete epidermal closure and mature granulation tissue filling of the wound gap at 6 days after biopsy. (c) (d) Depletion of IL-17A benefits wound healing closure. Hematoxylin and Eosin (b and d). Scale bars =  250 µm.</p

<i>L. reuteri</i> increases Leydig cell proliferation and serum testosterone levels.

<p><b>a.</b> At the age of 5 months a small number of proliferating (Ki-67+) Leydig cells did exist in the testicular interstitium (arrows). Proliferating Leydig cells were significantly more in probiotic-fed mice. DAB chromogen, Hematoxylin counterstain. Bars = 25 µm. The y-axis of the bar graph stands for the mean±SEM of Ki-67+ Leydig cells. <b>b.</b> Serum testosterone levels are significantly higher when consuming <i>L. reuteri</i>, as compared to their diet-matched controls. Numbers on the y-axis corresponds to the mean±SEM of serum testosterone level in ng/ml.; *p<0.05, **p<0.001.</p