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 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

<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 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

Transfer of mesenteric lymph node (MLN) cells from <i>H hepaticus</i>-infected mice is sufficient to recapitulate prostate cancer in uninfected mice.

<p>Schematic illustration of MLN cells transfer experiments designed to test the hypothesis that local GI tract immune network events cause systematic immune imbalances that determine the fate of preneoplastic lesions in topographically distant epithelia such as those of the prostate gland. Trials used 6 mice per group. Experiments were done in duplicate.</p

Intestinal <i>H. hepaticus</i> infection triggers systemic elevations in pro-inflammatory cytokines.

<p><b>A</b>) Serum levels of pro-inflammatory cytokines were increased in <i>Apc</i>^<i>Min/+</i> mice after <i>H</i>. <i>hepaticus</i> infection. Eotaxin, IL-3, TNF-α and IL-1α were significantly elevated in comparison with age-matched uninfected <i>Apc</i>^<i>Min/+</i> controls. When compared with <i>H</i>. <i>hepaticus</i> infected <i>wt</i> mice, infected <i>Apc</i>^<i>Min/+</i> mice had significantly higher IL-17, IL-1α, IL-3, eotaxin and IL-9 concentration in their blood serum. Bio-Plex Cytokine Assay used sera of <i>n</i>=5 mice per group. ***p<0.001; *p<0.05. <b>B</b>) Paracortical areas in mesenteric lymph nodes. <i>H</i>. <i>hepaticus</i> infected <i>Apc</i>^<i>Min/+</i> mice at high risk of prostate cancer had large amounts of cytoplasmic and extracellular IL-17 that significantly decreased after depletion of TNF-α. IL-17-specific immunohistochemistry; Hematoxylin counterstain, DAB chromogen. Bars=25 μm. <b>C</b>) Morphometric assessment of IL-17 in immunohistochemically stained sections of mesenteric lymph nodes. Both <i>Apc</i>^<i>Min/+</i> genetic status and intestinal infection by <i>H</i>. <i>hepaticus</i> significantly correlate with a TNF-α-mediated increase of IL-17 expression in the mesenteric lymph nodes. ***p<0.001; **p<0.01; *p<0.05.</p

Clinically silent immune system alterations affect dorsolateral prostate (DLP) carcinogenesis in B6 <i>ApcMin</i>^/+ mice after GI infection with <i>H. hepaticus</i>.

<p><b>A</b>) By the age of 12 weeks, <i>Apc</i>^<i>Min/+</i> mice spontaneously develop focal low and high grade prostate intraepithelial neoplasia (LGPIN, white arrow-head; HGPIN, black arrow-head) but only rare microinvasive carcinoma (a). Aged-matched <i>Apc</i>^<i>Min/+</i> mice with <i>H</i>. <i>hepaticus</i>, develop significantly more PIN and microinvasiveCA lesions (asterisk) characterized by highly atypical prostate gland epithelial cells which break through the basal lamina and infiltrate the adjacent stroma (b). When mesenteric lymph node cells from <i>H</i>. <i>hepaticus</i>-infected <i>Apc</i>^<i>Min/+</i> mice are transferred into the peritoneal cavity of <i>H</i>. <i>hepaticus</i>-free <i>Apc</i>^<i>Min/+</i> mice the recipient mice develop preneoplastic and early neoplastic lesions (microinvasiveCA-asterisk) comparable to those found in donor mice (c). Depleting TNF- α from <i>H</i>. <i>hepaticus</i>-infected <i>Apc</i>^<i>Min/+</i> mice brings prostate neoplastic lesions (LGPIN, white arrow-head) occurrence to the level of <i>H</i>. <i>hepaticus</i>-free mice (d). Hematoxylin and Eosin. Bars=50 μm. <b>B</b>) Aberrant β-catenin and laminin immunostaining pattern in microinvasiveCA lesions in prostate cancer. DLP from <i>WT H</i>. <i>hepaticus-</i>positive mice (a and c), <i>H</i>. <i>hepaticus</i>-infected <i>Apc</i>^<i>Min/+</i> mouse (b) and <i>H</i>. <i>hepaticus</i>-free <i>Apc</i>^<i>Min/+</i> mouse transferred with mesenteric lymph node cells from <i>H</i>. <i>hepaticus</i>-infected <i>Apc</i>^<i>Min/+</i> donor (d). Normal DLP glands had a normal β-catenin lateral epithelial cell membrane staining pattern (a) and an intact basal lamina (c). In contrast, microinvasiveCA lesions were characterized by cytoplasmic and nuclear (arrow-heads) stabilization of β-catenin (b) and absence of laminin (asterisk) suggestive of basal membrane degradation in malignant epithelial cell foci of incipient invasion. Hematoxylin counterstain, DAB chromogen. Bars=25 μm. <b>C</b>) Bar graph showing frequency of PIN and microinvasiveCA in treatment groups. The most significantly elevated prostate cancer occurrence is denoted by asterisk. Standard error bars correspond to microinvasiveCA statistical comparisons. <b>D</b>) DLP of <i>H</i>. <i>hepaticus</i>-free <i>Apc</i>^<i>Min/+</i> mouse transferred with MLN cells from a <i>H</i>. <i>hepaticus</i>-infected <i>Apc</i>^<i>Min/+</i> donor, HGPIN. Most myeloid precursor cells with ring-shaped nuclei residing in the stroma have the typical granulocytic lineage phenotype (black arrow-head) while fewer appear to be myelo/monocytic in type (white arrow-head). Bar=16 μm.</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

Dietary supplementation with <i>L. reuteri</i> accelerates wound healing.

<p>(a) Microscopy of formalin-fixed, paraffinized wounded skin of aged C57BL/6 mice (at 3, 6, and 12 days post- wounding). Wound margins are delineated with yellow outlines. The healing time course is faster in mice consuming <i>L. reuteri</i> evidenced by reduced wound sizes. (b) Histopathology of wound healing timecourse shows wound epidermal gaps [indicated by black brackets]. Accelerated epidermal closure in mice consuming the purified lactic acid bacteria led to complete re-epithelialization of wounds in 8/12 female mice by the 6th day post-wounding. By contrast, zero of 12 control animals had complete epidermal wound closure at the same time-point. 12 days after biopsy, the newly formed epidermis in <i>L. reuteri</i>-treated mice was normal and lacked regenerative hyperplasia, indicating a rapid rate of epithelial remodeling. (c) Wound area at each of three time points decreases significantly in both male and female mice fed <i>L. reuteri</i>. Female mice fed <i>L. reuteri</i> exhibit more significant wound closure compared to controls versus male mice at 6 and 12 days. (d) Wound size diminishes more rapidly in mice fed <i>L. reuteri</i> with the increased rate of wound closure, accompanying a smaller epidermal gap in both male and female mice. Hematoxylin and Eosin. Scale bars: a =  250 µm. (3 day: Male: Control (n = 6), Control + LR (n = 7), Female: CD (n = 6), CD+LR (n = 8); 6 day: Male: Control (n = 12), Control + LR (n = 12); Female: Control (n = 12), Control + LR (n = 12); 12 day: Male: Control (n = 7), Control + LR (n = 7), Female: Control (n = 9), Control+LR (n = 8)).</p