1 General introduction
1.3.2 Where do breast milk bacteria come from? 13
Until recently, it was generally believed that bacteria in human milk originated from the skin surrounding the nipple or from the oral cavity of the infant as a result of retrograde backflow during suckling (Ramsay et al., 2004). However, the presence of obligate anaerobes and species associated with the gut, respiratory tract and vagina would suggest additional mechanisms.
This begs the question then, as to what other mechanisms could be responsible for bacterial homing to the breast? Mucosal surfaces are defined as areas of the body exposed to the external environment with a single layer of epithelial cells protecting the lumen from the rest of the body. The mammary glands, along with the GIT, respiratory tract, urogenital tract and glandular tissue (salivary, lacrimal) are considered mucosal surfaces with their own specialized immune system that maintains homeostasis (McGhee and Fujihashi, 2012). It has long been recognized that there is a common mucosal system that allows for trafficking and homing of immune cells between mucosal sites. This has been established through studies showing that (i) immunization at one mucosal site confers protection at another mucosal site (Weisz-Carrington et al., 1979; Wilson and Obradovic, 2014) and that (ii) adoptive transfer of lymphoblasts from the mesenteric lymph nodes (MLN) or bronchial lymph nodes (BLN) into syngeneic recipients home to other mucosal surfaces such as the breast, bronchus, cervix and gut (McDermott and Bienenstock, 1979). It was also shown in the 1970s that cells isolated from the MLN would preferentially home to the breast during lactation (Roux et al., 1977) which is probably due the increased expression of CCL28 during this time (Meurens et al., 2006; Wilson and Butcher, 2004). CCL28 is a chemokine expressed on mammary epithelial cells,
responsible for the recruitment of IgA plasmoblasts and T cells to the mammary glands from the gut or the airways (Lazarus et al., 2003; Wang et al., 2000). This migration and homing of cells from the GIT and the respiratory tract to the breast has been termed the entero-mammary and bronchial-mammary pathway respectively.
While these entero-mammary and bronchial-mammary pathways are important in delivering antigen specific immune cells to the immunologically immature infant in order to provide passive immunity against gastrointestinal and respiratory infections, it could also be a mode by which bacteria migrate to the mammary glands. Indeed, during pregnancy and lactation, increased numbers of bacteria have been detected in the mammary glands compared to virgin mice with these bacteria co-localizing with dendritic cells (Donnet-Hughes et al., 2010). This transfer of bacteria from the GIT to the breast would explain the commonality of strains between a mother’s faeces and her breast milk (Albesharat et al., 2011; Jost et al., 2014) and the detection of probiotic strains in human milk after oral ingestion (Arroyo et al., 2010; Jiménez et al., 2008) (Figure 1-2).
1.3.2.1
Does trafficking occur in the “resting” breast?
Trafficking of bacteria via dendritic cells to the mammary glands has only been proposed in the context of pregnancy and lactation but the fact that immune cells still migrate to the mammary glands in non-gravid and non-lactating women suggests that bacterial translocation to the breast could be happening continuously throughout a woman’s life.VCAM-1, expressed on mammary epithelial cells, is responsible for B cell migration to the breast during pregnancy (Low et al., 2010). However it is also expressed at high levels irrespective of pregnancy and lactation and it is the main mechanism by which
mouse mammary tumour virus infected lymphocytes reach the non-lactating/non-gravid gland from the gastrointestinal tract (Finke and Acha-Orbea, 2001). High proportions of intralobular B cells and luminal deposits of IgA have also been observed in breast tissue of non-gravid/non-lactating women (Brandtzaeg, 1983; Drife et al., 1976). Most importantly, dendritic cells, which have been the proposed vehicle for bacterial transfer from the GIT to the mammary gland, are present in high numbers in breast lobules of non-lactating/non-pregnant women (Degnim et al., 2014).
Figure 1-2. Possible mechanism of bacterial translocation from the gut to the breast
The lumen of the gut is inhabited by trillions of bacteria and is separated from the interior by a thin layer of cells called the epithelium. DCs can sample bacteria directly from the lumen by the extension of dendrites or can capture bacteria that have translocated across specialized epithelial cells called ‘M cells.’ DCs laden with bacteria can either prime T cells in the Peyer’s patches or migrate to the MLN to prime T-cell reactions there. Unlike primed T cells, DCs do not normally move past the MLN. However, during pregnancy and lactation, it is believed that bacteria-laden DCs migrate out of the MLN and into the mammary glands. These glands are composed of approximately 15–20 lobes that circle around the nipple, albeit not as definitively visible during surgery as depicted in this diagram. Inside these lobes are lobules and at the end of each lobule are tiny alveoli that produce milk. The milk is carried via the ducts into the nipple. DC: Dendritic cell; MLN: Mesenteric lymph node. Figure taken from (Urbaniak et al., 2012)
Human microbiome and breast cancer
1.4
While the breast (via milk) is so vital to the health of the offspring, ironically, it is the cause of one of the deadliest cancers in women. In Canada alone, it is estimated that by the end of this year, 25,400 women will be diagnosed with breast cancer with 5,000 of them dying from the disease (Canadian Cancer Society, 2016). To put into perspective, this equates to 68 women being diagnosed and 14 women dying each day. Based on current trends, it is predicted that these numbers will increase rather than decrease over the next 20 years.
The etiology of breast cancer is still unknown but thought to be due to a combination of both genetic and environmental factors. Support for environmental factors comes from migration studies showing an increased incidence of breast cancer amongst migrants and their descendants, after they move from a region of low breast cancer risk to a region of high risk (Le et al., 2002; Shimizu et al., 1991). While many environmental predictors have been proposed, such as smoking and diet, one factor not considered at the time of entrance into my PhD program, was the potential of microbes, specifically in the breast, to influence the risk of breast cancer development. The rationale for this idea is presented below.
1.4.1
Causative role of bacteria in breast cancer
development
Almost 40 years ago, it was reported that the spontaneous rate of various tumour formations, over a 10 -year period, was higher in conventional rats compared to germ- free ones, with mammary tumours being the second most common tumour recorded (Sacksteder, 1976). The role of bacteria in mammary carcinoma was again delineated a
few years later, when it was shown that conventional rats had an increased risk of mammary tumours compared to germ free ones after sub-cutaneous injection with the carcinogen, 3,2'-dimethyl-4-aminobiphenyl (DMAB) (Reddy and Watanabe, 1978).
One mechanism by which bacteria may contribute to disease onset is through production of carcinogenic compounds from dietary components. Numerous studies using germ-free and conventional animals have shown that risk-related components of the human diet interact with the gut microbiota to induce DNA adducts and colon cancer (Hambly et al., 1997; Reddy et al., 1975; Rumney et al., 1993). One of these risk-related dietary components, PhIP (2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), has been detected in the milk of lactating rats and human milk (DeBruin and Josephy, 2002; Scott et al., 2007).
Another mechanism by which bacteria may promote breast cancer is through chronic inflammation, which is now widely accepted to be associated with the development of cancer (Mantovani et al., 2008). The effect of pathogen-induced inflammation is however not limited to the site of infection. It has been shown that C57BL/6 ApcMin/+ mice, which are genetically predisposed to develop mammary carcinomas, fail to develop disease when housed under specific pathogen free conditions (Moser et al., 1993) but upon gastric administration of Helicobacter hepaticus they develop mammary adenosquamous carcinoma as a result of innate immune induction of inflammation (Rao et al., 2006a, 2006b).