Dietary Microbes Modulate Transgenerational Cancer Risk

Environmental factors are suspected in the increase of obesity and cancer in industrialized countries but are poorly understood. Here, we used animal models to test how future generations may be affected by Westernized diets. We discover long-term consequences of grandmothers' in utero dietary exposures, leading to high rates of obesity and frequent cancers of lung and liver in two subsequent generations of mice. Transgenerational effects were transplantable using diet-associated bacteria communities alone. Consequently, feeding of beneficial microbes was sufficient to lower transgenerational risk for cancer and obesity regardless of diet history. Targeting microbes may be a highly effective population-based approach to lower risk for cancer.National Institutes of Health (U.S.) (RO1CA108854)National Institutes of Health (U.S.) (U01 CA164337)National Institutes of Health (U.S.) (P30-ES002109

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

Dietary supplementation with L. reuteri protects from age-associated Western diet obesity.

<p>Specifically, the abdominal (epididymal) fat mass is significantly reduced in probiotic-consuming Swiss mice (<b>a</b>). The slenderizing effect of <i>L. reuteri</i> is also observed in the subcutaneous fat depot. The subcutaneous fat layer (SF) is significantly thicker and has many CLS (inset) in “fast food”-fed mice in contrast to mice eating the same diet and <i>L. reuteri</i>. There is thicker dermis and increased subcutaneous hair follicle profiles in the left inset of the “fast food”+probiotic skin image (<b>b</b>). Fad pad weight and subcutaneous fat thickness histomorphometric analyses show that probiotics protect from age-associated obesity irrespective of baseline diet (<b>c</b>). Eating probiotics benefits aged Swiss mice as well as the young animals, evident here from the body weight analysis of 7- and 9-months-old male and female mice (<b>d</b>). Skin histology: Hematoxylin and eosin, Bars = 250 µm.</p

Mice exhibit an Interleukin 10-dependent Treg cell-mediated gut microbiota-immunity-obesity axis.

<p>Genetically-inbred C57BL/6 strain mice similarly benefit from probiotic protection against Western diet-associated obesity and fat pathology, including CLS and focal pyogranulomatous inflammation as shown here in males at 5 months of age (<b>a</b>). <i>Interleukin (Il) 10</i>-deficient C57BL/6 male mice eating Westernized chow failed to benefit from oral <i>L. reuteri</i> supplementation. Mice from both experimental groups were obese and had increased CLS that were often seen coalescing to form focally diffuse areas of adipocyte necrosis (<b>b</b>). Fat histology: Hematoxylin and eosin. Bars = 250 µm (a) and 100 µm (b).</p

Anti-obesity protection of oral probiotics in outbred Swiss mice requires CD25⁺ immune cells.

<p>Depletion of CD4⁺CD25⁺ Treg cells entirely inhibits probiotic-induced protection from age-associated obesity and abdominal fat pathology (<b>a</b>). Probiotics protect from weight gain unless mice were simultaneously treated with anti-CD25 antibody, in which case animals rapidly became obese. Frequency of prototype crown-like structures was increased in abdominal fat after depletion of CD25⁺ cells but not in sham IgG-treated control animals (<b>b</b>). Fat histology: Hematoxylin and eosin. Bars = 100 µm.</p

Diet-associated fat pathology is transferable to naïve animals using purified T-lymphocytes.

<p>Adoptive transfer of purified CD4+ cells from C57BL/6 <i>wild type</i> donor mice eating probiotics into C57BL/6 <i>Rag2</i>-null mice was sufficient to significantly reduce recipient body fat depots such as subcutaneous fat, as well as ameliorate abdominal fat pathology (<b>a</b>). Control diet-fed mice used as donors for lymphocyte transfer experiments showed IL10-dependent <i>L.reuteri</i> benefits including significantly less CLS in their abdominal fat (<b>b</b>). This immune-mediated protection requires Il10, as adoptive transfer of CD4⁺ cells from probiotic-fed Il10-deficient donors did not protect the recipient mice from obesity and associated fat pathology (<b>c</b>)<b>.</b> Purified wild type CD4⁺ FoxP3⁺ Treg cells from mice eating <i>L. reuteri</i> were sufficient for beneficial effects in <i>Rag2</i>-null recipient mice rescuing them from obesity-associated pathology (<b>d</b>). Skin and fat histology: Hematoxylin and eosin. Bars skin = 250 µm, edididymal fat = 50 µm.</p

Proposed mechanistic overview.

<p>In order to explain the ‘fast food’ <i>versus</i> ‘yogurt’ age-associated weight disparity observed in human subjects, we propose that Western-style dietary habits alter gut microbiota fueling an IL-6 driven, IL-17-dominant, systemic smoldering inflammatory milieu, ultimately leading to a vicious circle of obesity and inflammation. On the other hand, individuals consuming probiotic yogurt enrich their gut with probiotic bacteria that stimulate the anti-inflammatory arm of the immune system. Potent IL-10-associated Treg responses in these individuals rescue them from the inflammation-obesity cycle, thus increasing likelihood of a leaner physique.</p

Eating probiotics blocks a gut microbiota-immunity-obesity axis.

<p><i>L. reuteri</i> protects mice from Western diet-associated obesity. Data are shown in male outbred Swiss mice at the age of 5 months. Numerous crown-like structures (CLS) caused by adipocyte death-related inflammation, and focal pyogranulomatous inflammation (PGI) arise in abdominal fat of ‘fast food’-fed but not probiotic-fed animals. Probiotics increase anti-inflammatory Foxp3+ regulatory (Treg) cells and reduce pro-inflammatory Il17 protein to restore immune balance coinciding with a slender physique (<b>a and b</b>), without restructuring GI microbial communities (<b>c</b>). In the same mice, serum cytokine analysis shows that the pro-inflammatory Il-17-associated effect of obesity is systemic, and that <i>L. reuteri</i> negates this effect up-regulating the anti-inflammatory cytokine Il-10 (<b>d</b>). Humans frequently eating ‘fast food’ also show an elevated ratio of pro-inflammatory IL17+/anti-inflammatory Foxp3+ Treg in peripheral blood cells compared to subjects never eating ‘fast food’ (<b>e</b>)<b>.</b> Probiotic-consuming slim mice chose similar calories when compared with obese animals, regardless of baseline diet, highlighting potential for translational medicine (<b>f</b>). Fat histology: Hematoxylin and eosin, Bars = 50 µm; MLN Immunohistochemistry: Diaminobenzidine chromogen, hematoxylin counterstain, Bars = 8.3 µm.</p

Microbial Symbionts Accelerate Wound Healing via the Neuropeptide Hormone Oxytocin

Wound healing capability is inextricably linked with diverse aspects of physical fitness ranging from recovery after minor injuries and surgery to diabetes and some types of cancer. Impact of the microbiome upon the mammalian wound healing process is poorly understood. We discover that supplementing the gut microbiome with lactic acid microbes in drinking water accelerates the wound-healing process to occur in half the time required for matched control animals. Further, we find that Lactobacillus reuteri enhances wound-healing properties through up-regulation of the neuropeptide hormone oxytocin, a factor integral in social bonding and reproduction, by a vagus nerve-mediated pathway. Bacteria-triggered oxytocin serves to activate host CD4+Foxp3+CD25+ immune T regulatory cells conveying transplantable wound healing capacity to naive Rag2-deficient animals. This study determined oxytocin to be a novel component of a multi-directional gut microbe-brain-immune axis, with wound-healing capability as a previously unrecognized output of this axis. We also provide experimental evidence to support long-standing medical traditions associating diet, social practices, and the immune system with efficient recovery after injury, sustained good health, and longevity.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 R01CA08854