Beneficial bacteria stimulate host immune cells to counteract dietary and genetic predisposition to mammary cancer in mice

Recent studies suggest health benefits including protection from cancer after eating fermented foods such as probiotic yogurt, though the mechanisms are not well understood. Here we tested mechanistic hypotheses using two different animal models: the first model studied development of mammary cancer when eating a Westernized diet, and the second studied animals with a genetic predilection to breast cancer. For the first model, outbred Swiss mice were fed a Westernized chow putting them at increased risk for development of mammary tumors. In this Westernized diet model, mammary carcinogenesis was inhibited by routine exposure to Lactobacillus reuteri ATCC-PTA-6475 in drinking water. The second model was FVB strain erbB2 (HER2) mutant mice, genetically susceptible to mammary tumors mimicking breast cancers in humans, being fed a regular (non-Westernized) chow diet. We found that oral supplement with these purified lactic acid bacteria alone was sufficient to inhibit features of mammary neoplasia in both models. The protective mechanism was determined to be microbially-triggered CD4+CD25+ lymphocytes. When isolated and transplanted into other subjects, these L. reuteri-stimulated lymphocytes were sufficient to convey transplantable anti-cancer protection in the cell recipient animals. These data demonstrate that host immune responses to environmental microbes significantly impact and inhibit cancer progression in distal tissues such as mammary glands, even in genetically susceptible mice. This leads us to conclude that consuming fermentative microbes such as L. reuteri may offer a tractable public health approach to help counteract the accumulated dietary and genetic carcinogenic events integral in the Westernized diet and lifestyle.National Institutes of Health (U.S.) (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant RO1CA108854)National Institutes of Health (U.S.) (Grant U01 CA164337

Probiotic Microbes Sustain Youthful Serum Testosterone Levels and Testicular Size in Aging Mice

The decline of circulating testosterone levels in aging men is associated with adverse health effects. During studies of probiotic bacteria and obesity, we discovered that male mice routinely consuming purified lactic acid bacteria originally isolated from human milk had larger testicles and increased serum testosterone levels compared to their age-matched controls. Further investigation using microscopy-assisted histomorphometry of testicular tissue showed that mice consuming Lactobacillus reuteri in their drinking water had significantly increased seminiferous tubule cross-sectional profiles and increased spermatogenesis and Leydig cell numbers per testis when compared with matched diet counterparts This showed that criteria of gonadal aging were reduced after routinely consuming a purified microbe such as L. reuteri. We tested whether these features typical of sustained reproductive fitness may be due to anti-inflammatory properties of L. reuteri, and found that testicular mass and other indicators typical of old age were similarly restored to youthful levels using systemic administration of antibodies blocking pro-inflammatory cytokine interleukin-17A. This indicated that uncontrolled host inflammatory responses contributed to the testicular atrophy phenotype in aged mice. Reduced circulating testosterone levels have been implicated in many adverse effects; dietary L. reuteri or other probiotic supplementation may provide a viable natural approach to prevention of male hypogonadism, absent the controversy and side-effects of traditional therapies, and yield practical options for management of disorders typically associated with normal aging. These novel findings suggest a potential high impact for microbe therapy in public health by imparting hormonal and gonad features of reproductive fitness typical of much younger healthy individuals.National Institutes of Health (U.S.) (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant U01 CA164337)National Institutes of Health (U.S.) (Grant RO1CA108854

Microbial Reprogramming Inhibits Western Diet-Associated Obesity

A recent epidemiological study showed that eating ‘fast food’ items such as potato chips increased likelihood of obesity, whereas eating yogurt prevented age-associated weight gain in humans. It was demonstrated previously in animal models of obesity that the immune system plays a critical role in this process. Here we examined human subjects and mouse models consuming Westernized ‘fast food’ diet, and found CD4[superscript +] T helper (Th)17-biased immunity and changes in microbial communities and abdominal fat with obesity after eating the Western chow. In striking contrast, eating probiotic yogurt together with Western chow inhibited age-associated weight gain. We went on to test whether a bacteria found in yogurt may serve to lessen fat pathology by using purified Lactobacillus reuteri ATCC 6475 in drinking water. Surprisingly, we discovered that oral L. reuteri therapy alone was sufficient to change the pro-inflammatory immune cell profile and prevent abdominal fat pathology and age-associated weight gain in mice regardless of their baseline diet. These beneficial microbe effects were transferable into naïve recipient animals by purified CD4[superscript +] T cells alone. Specifically, bacterial effects depended upon active immune tolerance by induction of Foxp3[superscript +] regulatory T cells (Treg) and interleukin (Il)-10, without significantly changing the gut microbial ecology or reducing ad libitum caloric intake. Our finding that microbial targeting restored CD4[superscript +] T cell balance and yielded significantly leaner animals regardless of their dietary ‘fast food’ indiscretions suggests population-based approaches for weight management and enhancing public health in industrialized societies.National Institutes of Health (U.S.) (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant RO1CA108854)National Institutes of Health (U.S.) (Grant P01 AI045757)National Institutes of Health (U.S.) (Grant U19 AI046130)National Institutes of Health (U.S.) (Grant U19 AI070352)National Institutes of Health (U.S.) (Grant P01 AI039671)National Institute of Neurological Disorders and Stroke (U.S.) (Jacob Javits Merit Award NS2427)The Penates FoundationNancy Taylor Foundation for Chronic Diseases, Inc

Airway epithelial cell necroptosis contributes to asthma exacerbation in a mouse model of house dust mite-induced allergic inflammation

Regulation of epithelial cell death has emerged as a key mechanism controlling immune homeostasis in barrier surfaces. Necroptosis is a type of regulated necrotic cell death induced by receptor interacting protein kinase 3 (RIPK3) that has been shown to cause inflammatory pathologies in different tissues. The role of regulated cell death and particularly necroptosis in lung homeostasis and disease remains poorly understood. Here we show that mice with Airway Epithelial Cell (AEC)-specific deficiency of Fas-associated with death domain (FADD), an adapter essential for caspase-8 activation, developed exacerbated allergic airway inflammation in a mouse model of asthma induced by sensitization and challenge with house dust mite (HDM) extracts. Genetic inhibition of RIPK1 kinase activity by crossing to mice expressing kinase inactive RIPK1 as well as RIPK3 or MLKL deficiency prevented the development of exaggerated HDM-induced asthma pathology in FADDAEC-KO mice, suggesting that necroptosis of FADD-deficient AECs augmented the allergic immune response. These results reveal a role of AEC necroptosis in amplifying airway allergic inflammation and suggest that necroptosis could contribute to asthma exacerbations caused by respiratory virus infections inducing AEC death

Airway epithelial cell necroptosis contributes to asthma exacerbation in a mouse model of house dust mite-induced allergic inflammation

Regulation of epithelial cell death has emerged as a key mechanism controlling immune homeostasis in barrier surfaces. Necroptosis is a type of regulated necrotic cell death induced by receptor interacting protein kinase 3 (RIPK3) that has been shown to cause inflammatory pathologies in different tissues. The role of regulated cell death and particularly necroptosis in lung homeostasis and disease remains poorly understood. Here we show that mice with Airway Epithelial Cell (AEC)-specific deficiency of Fas-associated with death domain (FADD), an adapter essential for caspase-8 activation, developed exacerbated allergic airway inflammation in a mouse model of asthma induced by sensitization and challenge with house dust mite (HDM) extracts. Genetic inhibition of RIPK1 kinase activity by crossing to mice expressing kinase inactive RIPK1 as well as RIPK3 or MLKL deficiency prevented the development of exaggerated HDM-induced asthma pathology in FADD(AEC-KO) mice, suggesting that necroptosis of FADD-deficient AECs augmented the allergic immune response. These results reveal a role of AEC necroptosis in amplifying airway allergic inflammation and suggest that necroptosis could contribute to asthma exacerbations caused by respiratory virus infections inducing AEC death

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

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