Tatiana Levkovich

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+ 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+ T cells alone. Specifically, bacterial effects depended upon active immune tolerance by induction of Foxp3+ 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+ 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.

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.

Unifying roles for regulatory T cells and inflammation in cancer

Activities of CD4+ regulatory (TREG) cells restore immune homeostasis during chronic inflammatory disorders. Roles for TREG cells in inflammation‐associated cancers, however, are paradoxical. It is widely believed that TREG function in cancer mainly to suppress protective anticancer responses. However, we demonstrate here that TREG cells also function to reduce cancer risk throughout the body by efficiently downregulating inflammation arising from the gastrointestinal (GI) tract. Building on a “hygiene hypothesis” model in which GI infections lead to changes in TREG that reduce immune‐mediated diseases, here we show that gut bacteria‐triggered TREG may function to inhibit cancer even in extraintestinal sites. Ability of bacteria‐stimulated TREG to suppress cancer depends on interleukin (IL)‐10, which serves to maintain immune homeostasis within bowel and support a protective antiinflammatory TREG phenotype. However, under proinflammatory conditions, TREG may fail to provide antiinflammatory protection and instead contribute to a T helper (Th)‐17‐driven procarcinogenic process; a cancer state that is reversible by downregulation of inflammation. Consequently, hygienic individuals with a weakened IL‐10 and TREG‐mediated inhibitory loop are highly susceptible to the carcinogenic consequences of elevated IL‐6 and IL‐17 and show more frequent inflammation‐associated cancers. Taken together, these data unify seemingly divergent disease processes such as autoimmunity and cancer and help explain the paradox of TREG and inflammation in cancer. Enhancing protective TREG functions may promote healthful longevity and significantly reduce risk of cancer.

Probiotic Bacteria Induce a ‘Glow of Health’

Radiant skin and hair are universally recognized as indications of good health. However, this ‘glow of health’ display remains poorly understood. We found that feeding of probiotic bacteria to aged mice induced integumentary changes mimicking peak health and reproductive fitness characteristic of much younger animals. Eating probiotic yogurt triggered epithelial follicular anagen-phase shift with sebocytogenesis resulting in thick lustrous fur due to a bacteria-triggered interleukin-10-dependent mechanism. Aged male animals eating probiotics exhibited increased subcuticular folliculogenesis, when compared with matched controls, yielding luxuriant fur only in probiotic-fed subjects. Female animals displayed probiotic-induced hyperacidity coinciding with shinier hair, a feature that also aligns with fertility in human females. Together these data provide insights into mammalian evolution and novel strategies for integumentary health.

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.

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.

Pathogenic Intestinal Bacteria Enhance Prostate Cancer Development via Systemic Activation of Immune Cells in Mice

A role for microbes has been suspected in prostate cancer but difficult to confirm in human patients. We show here that a gastrointestinal (GI) tract bacterial infection is sufficient to enhance prostate intraepithelial neoplasia (PIN) and microinvasive carcinoma in a mouse model. We found that animals with a genetic predilection for dysregulation of wnt signaling, Apc Min/+ mutant mice, were significantly susceptible to prostate cancer in an inflammation-dependent manner following infection with Helicobacter hepaticus. Further, early neoplasia observed in infected Apc Min/+ mice was transmissible to uninfected mice by intraperitoneal injection of mesenteric lymph node (MLN) cells alone from H. hepaticus-infected mutant mice. Transmissibility of neoplasia was preventable by prior neutralization of inflammation using anti-TNF-α antibody in infected MLN donor mice. Taken together, these data confirm that systemic inflammation triggered by GI tract bacteria plays a pivotal role in tumorigenesis of the prostate gland.

CD4+ lymphocytes modulate prostate cancer progression in mice

Chronic inflammation contributes to the development of prostate cancer in humans. Here, we show that male ApcMin/+ mice also develop prostate carcinoma with increasing age, mimicking that seen in humans in their 5th or 6th decade of life. Proinflammatory cytokines were significantly linked with cancer and increasing age in our mouse model; however, prostate and bowel tissues lacked evidence of inflammatory cell infiltrates other than mast cells. Lymphocytes protected against cancer, and protection from prostate cancer resided in antiinflammatory CD4+CD25+ regulatory (TREG) cells that downregulated inflammatory cytokines. Supplementation with syngeneic TREG cells collected from wild‐type mice reduced the levels of interleukin (IL)‐6 (p < 0.05) and IL‐9 (p < 0.001) and lowered prostate cancer risk (p < 0.05). Depletion of CD25+ cells in 2‐month‐old animals increased the expression of IL‐6 (p < 0.005) within prostate and increased the frequency of high‐grade prostatic intraepithelial neoplasia (p < 0.05) and microinvasive prostatic carcinoma (p < 0.05) in dorsolateral prostate. Depletion of CD25+ cells in young animals also increased the frequency of intestinal cancer in Min mice. Taken together, chronically elevated proinflammatory cytokines promoted carcinoma in ApcMin/+ mice. TREG lymphocytes downregulated inflammation‐associated carcinogenic processes and contributed to immune and epithelial homeostasis.

Beneficial bacteria inhibit cachexia

Muscle wasting, known as cachexia, is a debilitating condition associated with chronic inflammation such as during cancer. Beneficial microbes have been shown to optimize systemic inflammatory tone during good health; however, interactions between microbes and host immunity in the context of cachexia are incompletely understood. Here we use mouse models to test roles for bacteria in muscle wasting syndromes. We find that feeding of a human commensal microbe, Lactobacillus reuteri, to mice is sufficient to lower systemic indices of inflammation and inhibit cachexia. Further, the microbial muscle-building phenomenon extends to normal aging as wild type animals exhibited increased growth hormone levels and up-regulation of transcription factor Forkhead Box N1 [FoxN1] associated with thymus gland retention and longevity. Interestingly, mice with a defective FoxN1 gene (athymic nude) fail to inhibit sarcopenia after L. reuteri therapy, indicating a FoxN1-mediated mechanism. In conclusion, symbiotic bacteria may serve to stimulate FoxN1 and thymic functions that regulate inflammation, offering possible alternatives for cachexia prevention and novel insights into roles for microbiota in mammalian ontogeny and phylogeny.

Beneficial Bacteria Stimulate Youthful Thyroid Gland Activity

Healthful aging with active participation in society are global public health priorities. Sender physique and high productivity levels absent clinical disease are widely recognized features of healthful aging. During studies of obesity in mice, we found that feeding of a purified probiotic microbe, Lactobacillus reuteri, forestalled typical old age-associated weight gain and lethargy, and instead conveyed physical features of much younger mice. We hypothesized that these retained features of youth may be related to increased thyroid gland activity. We subsequently discovered elevated levels of serum T4 and larger thyroid glands in slender one-year-old recipients of probiotic microbes, when compared with their age-matched obese control subjects. Oral L. reuteri treatment also preserved thyroid follicle epithelial height, a key histologic feature of thyroid gland activity, which relied mechanistically upon bacteria-triggered anti-inflammatory CD25+ regulatory T cells. These data from animal models suggest that probiotic microbe supplementation may be used to stimulate beneficial host immune interactions with improved thyroid function and more healthful aging.