J Mammary Gland Biol Neoplasia DOI 10.1007/s10911-014-9325-9
Inflammatory Mediators in Mastitis and Lactation Insufficiency Wendy V. Ingman & Danielle J. Glynn & Mark R. Hutchinson
Received: 6 May 2014 / Accepted: 18 June 2014 # Springer Science+Business Media New York 2014
Abstract Mastitis is a common inflammatory disease during lactation that causes reduced milk supply. A growing body of evidence challenges the central role of pathogenic bacteria in mastitis, with disease severity associated with markers of inflammation rather than infection. Inflammation in the mammary gland may be triggered by microbe-associated molecular patterns (MAMPs) as well as danger-associated molecular patterns (DAMPs) binding to pattern recognition receptors such as the toll-like receptors (TLRs) on the surface of mammary epithelial cells and local immune cell populations. Activation of the TLR4 signalling pathway and downstream nuclear factor kappa B (NFkB) is critical to mediating local mammary gland inflammation and systemic immune responses in mouse models of mastitis. However, activation of NFkB also induces epithelial cell apoptosis and reduced milk protein synthesis, suggesting that inflammatory mediators activated during mastitis promote partial involution. Perturbed milk flow, maternal stress and genetic predisposition are significant risk factors for mastitis, and could lead to a heightened TLR4-mediated inflammatory response, resulting in increased susceptibility and severity of mastitis disease in the context of low MAMP abundance. Therefore, heightened W. V. Ingman : D. J. Glynn Discipline of Surgery, School of Medicine, The Queen Elizabeth Hospital, University of Adelaide, Woodville, Australia W. V. Ingman : D. J. Glynn Robinson Research Institute, University of Adelaide, Adelaide, Australia W. V. Ingman : D. J. Glynn School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia M. R. Hutchinson (*) Discipline Physiology, School of Medical Sciences, University of Adelaide, Adelaide, Australia e-mail: [email protected]
host inflammatory signalling may act in concert with pathogenic or commensal bacterial species to cause both the inflammation associated with mastitis and lactation insufficiency. Here, we present an alternate paradigm to the widely held notion that breast inflammation is driven principally by infectious bacterial pathogens, and suggest there may be other therapeutic strategies, apart from the currently utilised antimicrobial agents, that could be employed to prevent and treat mastitis in women. Keywords Mastitis . Lactation insufficiency . Inflammation . Toll-like receptors Abbreviations DAMP Danger-associated molecular pattern IL Interleukin LPS Lipopolysaccharide MAMP Microbe-associated molecular pattern NOS Nitric oxide synthase NFkB Nuclear factor kappa b STAT5 Signal transducer and activator of transcription 5 TLR Toll-like receptor TNFA Tumour necrosis factor alpha
Mastitis and Lactation Insufficiency Lactation mastitis is a debilitating inflammatory breast disease in postpartum women. Prospective cohort studies have reported the cumulative incidence of lactation mastitis to be between 10 % and 27 % of breastfeeding women [1–5]. The disease causes localised pain, and is frequently accompanied by the rapid onset of systemic symptoms including fever, muscle aches, chills and fatigue [6]. In 3-11 % of cases, mastitis leads to a breast abscess [7], which must be surgically drained, and
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in severe disease there can be permanent disfiguration of the breast. Low milk supply frequently accompanies these symptoms [8, 9]. Collectively, the challenges posed by this disease lead many women to use supplementary formula to feed their infant, or cease breastfeeding altogether [8, 10]. Sub-clinical mastitis is characterised by increased interleukin 8 (IL8) and sodium in breast milk in the absence of clinical symptoms [11]. In a Ghanaian cohort, sub-clinical mastitis is associated with reduced infant weight gain of around 20 % in the first month postpartum [11, 12], which may be the result of a prolonged reduction in milk synthesis by the mother. The incidence of sub-clinical disease has not been well documented, however 45 % of Ghanaian women exhibit elevated inflammatory markers in their breast milk indicative of subclinical disease [13]. Although the prevalence of sub-clinical mastitis in Western women is not known, 56 % of Australian women who cease breastfeeding in the first six months postpartum cite low milk supply as the cause [14], and in some cases this might be the result of asymptomatic breast inflammation.
bacterium alone is not sufficient to trigger the disease. In addition, mastitis frequently presents as inflammation in the absence of abundant pathogenic bacteria, leading some clinical researchers to propose delineation of “infective” and “noninfective” forms of mastitis disease [25, 26]. The World Health Organization summarises the limitations of the current paradigm thus: “Many lactating women who have potentially pathogenic bacteria on their skin or in their milk do not develop mastitis. But: Many women who do develop mastitis do not have pathogenic organisms in their milk” [27]. In light of this evidence, it is not surprising that recent Cochrane systematic reviews have emphasized the uncertainty around management of mastitis with antimicrobial agents. Prophylactic antibiotic use is not effective in preventing mastitis [28] and there is insufficient evidence to support the use of antibiotics in treating the disease [29]. Meticulous hygiene by breastfeeding women also appears ineffective in preventing mastitis [4]. However, no alternative mechanistic basis for mastitis has been identified, and there continues to be considerable doubt around the aetiology of the disease [20, 30].
Bacterial Pathogens in Mastitis Inflammatory Mediators in Mastitis The relationship between mastitis in women and bacterial infection is largely inferred through studies in bovine species [15], and the identification of pathogenic bacteria including Staphylococcus aureus, Escherichia coli, and Group B streptococci in mastitis-affected human breast milk [16, 17]. Commensal bacteria including Staphylococcus epidermidis are also frequently observed in breast milk from women with mastitis [18], however their role in the aetiology of mastitis is not known. Other organisms such as Candida albicans have also been suggested to be involved, although no study has demonstrated a causative link [19]. Therefore the majority of clinical practitioners believe lactation mastitis is caused by infectious bacterial pathogens. However, there is now a growing body of scientific literature that challenges the current paradigm. A number of studies have shown inconsistencies between bacterial load and disease severity [17, 20, 21], and the immune system response to the insult appears to bear a more significant relationship to disease severity in comparison to the infection itself. In a study of 466 disease free women, breast milk from nearly a third of healthy women was found to harbour high (≥106 colony forming units/L) bacterial counts of Staphylococcus aureus and 10 % harboured Group B streptococci [17]. Furthermore, analysis of 192 women with mastitis indicated that there is no correlation between severity of symptoms of mastitis, including breast inflammation, fever and pain, and the abundance of pathogenic Staphylococcus aureus and Group B streptococci in breast milk [17]. These bacterial species are also commonly found in nasal passages and skin flora [22–24] suggesting that the presence of the
Bacteria may augment inflammation, however it is clear that other factors, as yet unidentified, are important components of mastitis. In women, disease severity is closely aligned with both systemic and local indicators of inflammation. Breast milk and blood samples collected between day 5 and 3 months postpartum in prospective studies show C-reactive protein in milk and serum, and sodium concentration in milk is significantly elevated during periods of mastitis [31, 32]. Furthermore, the severity of mastitis based on degree of local breast inflammation and systemic symptoms such as elevation in body temperature were closely correlated with serum concentration of C-reactive protein [32]. Therefore, specific inflammatory signalling pathways activated in the host tissue are likely to be key determinants of susceptibility to mastitis and severity of the disease. Women with mastitis exhibit increased serum cytokines including interleukins (IL) 1, 6 and 8, and tumour necrosis factor A (TNFA) [33, 34], indicative of activation of the transcription factor nuclear factor kappa B (NFkB) in the host. The NFkB family of transcription factors are ubiquitously expressed, and when activated, are central regulators of innate and adaptive immune responses, the cellular stress response and programmed cell death [35]. In the lactating mammary glands of mice, infection with Escherichia coli induces NFkB activation within 4 hours, peaking at 6 hours with a return to basal levels 24 hours post-infection [36]. Candidate receptors responsible for activating NFkB include the toll-like receptors (TLRs). TLRs expressed on the
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surface of macrophages, dendritic cells and epithelial cells are important mediators of inflammation [37]. TLR2, TLR4 and TLR5 bind bacterial byproducts such as lipopolysaccharide (LPS), bacterial lipopeptides and flagellins, collectively referred to as Microbe-Associated Molecular Patterns (MAMPs). Importantly, this terminology has recently been adapted from Pathogen-Associated Molecular Patterns, acknowledging that commensal microbiota contribute to critical TLR signalling in addition to foreign pathogen-derived patterns [38]. TLR activation initiates transcription, translation and release of a cascade of inflammatory cytokines, chemokines and adhesion molecules, and recruits other components of the innate immune system such as neutrophils to the infected site [39, 40]. Such innate immune responses contribute to the swelling and pain associated with mastitis, whilst systemic actions of pro-inflammatory cytokines IL1 and IL8 initiate the fever response [6, 27]. The role of TLR4 in mediating inflammation in mastitis is demonstrated in TLR4 deficient mouse models challenged with bacterial endotoxin LPS to the teat canal during lactation [39, 41]. TLR4 signalling deficiency in C3H/HeJ mice, due to a point mutation that limits MyD88-dependent NFkB activation, resulted in dampened inflammation in response to LPS [41], suggesting that TLR4 signalling is necessary for appropriate innate immune system responses to infection. However, LPS administered to mice carrying a null mutation in the gene encoding TLR4, on a Balb/c background, resulted in increased recruitment of neutrophils and macrophages to the mammary gland and dampened systemic inflammatory cytokines including CXCL1 and TNFA [39], suggesting that TLR4 affects the immune signalling fingerprint, rather than suppressing the response altogether. The difference in results reported in these two studies is likely due to the different background strain of mice used [42]. C3H and Balb/c mice exhibit striking differences in immune system responses that affect airway inflammation [43], and susceptibility to experimental autoimmune orchitis [44] and Clonorchis sinensis infection [45]. Interestingly, neutrophils are recruited in greater abundance in mice with a null mutation in the gene encoding inducible nitric oxide synthase (NOS2) [46]. Nitric oxide production, mediated by NOS2, is observed in TLR4-activated macrophages [47] suggesting that downstream effectors of TLR4 may alter the immune signalling fingerprint in response to LPS, as reported in Tlr4 null mutant Balb/c mice [39]. Further studies on the role of nitric oxide on mediating TLR4associated mammary gland inflammation are warranted. When C3H/HeJ TLR4 signalling deficient mice are challenged with Escherichia coli rather than the endotoxin LPS, the magnitude of mammary gland inflammation is comparable to similarly infected wildtype mice, however an increased abundance of bacteria within mammary epithelial cells is observed 24 and 48 hours post-infection [41]. TLR4expressing macrophages are significant in mediating
clearance of Escherichia coli when adoptively transferred to the teat canal, suggesting that alveolar macrophages expressing TLR4 are required to restrict bacterial invasion. However, the presence of intact TLR4 signalling in wildtype mice does not prevent the acute rapid growth of bacteria within the alveolar and ductal spaces in the infected mammary gland [41], and the impact of TLR4 on disease resolution is not known. Further mouse studies administering live bacteria to the lactating mouse mammary gland are required to better understand how TLR4-mediated inflammation affects susceptibility, severity and resolution of bacterial infections.
Lactation Insufficiency is a Consequence of TLR4-Mediated NFkB Activation In addition to mediating inflammation associated with bacterial infection, activation of the TLR4-signalling pathway also induces reduced milk synthesis. Administration of LPS to the teat canal during lactation results in decreased milk-secreting glandular area, and reduced nuclear phosphorylated signal transducer and activator of transcription 5 (STAT5) on day 7 following LPS administration [39]. However, this compromised capacity for lactation does not occur in LPS-treated Tlr4 null mutant mice [39]. Similarly, activation of NFkB in an inducible transgenic mouse model during normal lactation induces mammary epithelial cell apoptosis and a rapid decline in milk protein beta casein [48]. Lactation insufficiency in mice has also been observed as a consequence of inappropriate adaptive immune responses [49]. Therefore, reduced milk synthesis occurs due to immune responses in the host, irrespective of the presence of pathogenic bacteria, suggesting that perturbed milk supply during mastitis is a consequence of inflammatory mediators, rather than the bacterial infection itself. Inflammatory mediators are recognised as critical factors that promote mammary gland involution following weaning. As early as one hour following removal of pups from a lactating female, activated NFkB is detectable in mammary epithelial cells, and is highest 3 days following weaning [50]. Activated NFkB inhibits STAT5-mediated milk protein expression [51], and inhibition of NFkB activity through conditional deletion of upstream regulator IkB kinase 2/B delays involution-associated apoptosis and tissue remodelling [52]. NOS2, expressed in macrophages under the direction of activated NFkB, mediates nitric oxide production leading to apoptosis of mammary epithelial cells during involution [53], and this mechanism may be responsible for the delayed apoptosis observed in mice depleted of macrophages at the time of weaning [54]. Therefore, activation of NFkB and downstream effectors such as nitric oxide during mastitis is likely to lead to epithelial cell apoptosis and partial involution, and may be responsible for the reduced milk supply observed in women with mastitis. Similarly, sub-clinical mastitis in
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women is associated with poor weight gain in infants [11, 12] and might be the result of activated NFkB causing prolonged reduction in milk synthesis in the mother.
Risk Factors That may Affect Inflammatory Mediators in Mastitis Host inflammatory mediators such as NFkB are implicated as integral components of breast inflammation that affect lactation capacity. However, little is known as to how inflammation in the mammary gland can occur in the absence of abundant bacterial pathogens. Recently, advances in our understanding of TLRs have uncovered an expanded repertoire of TLR agonists beyond MAMPs, such that TLR activation is not only initiated by the presence of bacteria. A number of endogenous proteins and molecules induce TLR activation under sterile conditions, and are collectively referred to as Danger Associated Molecular Patterns (DAMPs) [55, 56]. The significance of the TLR4 signalling pathway and NFkB in mastitis, raises the possibility that in some cases, mastitis could be caused by inflammatory mediators activated by DAMPs, or heightened TLR4 immune responses in the absence of a high bacterial load. Perturbed milk flow is recognised as a major risk factor for mastitis [1, 3, 27, 57], and is caused by reduced infant feeding, poor attachment, oversupply of milk or a blocked mammary duct [27, 58]. Accumulation of milk within a duct may affect the abundance of co-stimulatory factors such as DAMPs, or downstream immune signalling pathways. An engorged milk duct is likely to be under severe mechanical stress, causing the release of a number of DAMPs including HMGB1 and HSP70 which augment inflammatory responses to MAMPs [59]. In addition, the abundance of lactate, a potent activator of the TLR4 signalling pathway and NFkB [60], increases in
Perturbed Mechanical stress milk flow DAMPs
bovine milk during mastitis [61] and may also act as a costimulatory inflammatory factor. The sensitivity of the mammary gland to both DAMPs and MAMPs may be increased due to maternal physical and psychological stress, which is also a significant risk factor for mastitis [1, 3, 57, 62]. Maternal stress and illness have the capacity to change the expression and signalling sensitivity of TLR4 [63, 64]. Importantly, physical and psychological stress-induced neuroendocrine responses can all feed forward to trigger even greater stress alterations of immune function. As discussed previously, peripheral activation of TLR4 results in pro-inflammatory cytokine secretion [40]. These cytokines can stimulate peripheral neuronal afferents or cross the blood– brain barrier to activate immunocompetent glial cells (microglia and astrocytes) within the brain, causing neuroinflammation, thus changing neuronal activity and ultimately behaviour [65]. Through these immune-to-brain communications, peripheral immune activation precipitates “sickness behaviour” [66], and is associated with depression [64, 67]. TLR4 activation can further modulate the stress response through activation of the hypothalamus-pituitary-adrenal axis [68]. Whilst stress is typically associated with decreased TLR4 responses, recent discoveries point to key proinflammatory actions of stress priming [63]. In fact the neuroendocrine response to stress is seen as a TLR dependent alarm of danger, akin to a DAMP [63]. Additionally, associated with periods of significant life changes and stress are multiple forms of sleep disruption, some of which cause alterations in TLR4dependent immune responses. For example, long term sleep fragmentation leads to increased TLR4-dependent fevering [69]. As such, the neuroendocrine response associated with multiple forms of stress and sleep disruption will significantly impact sensitivity to inflammatory stimuli, affecting susceptibility, severity and recovery from mastitis.
Pathogenic or commensal bacteria
MAMP DAMP
Mammary epithelium
Maternal stress Sleep deprivation
TLR4 and downstream mediators
DAMP MAMP-mediated NFkB activation Fig. 1 TLR4-dependent inflammatory mediators affect lactation mastitis and milk supply. Perturbed milk flow, maternal stress, genetic predisposition and sleep deprivation can lead to accumulation of DAMPs and heightened TLR4 signalling in the mammary gland, leading to increased
Genetic predisposition
TLR4
Inflammation Reduced milk synthesis
susceptibility to mastitis and increased severity of the disease. Importantly, activation of NFkB could lead to partial mammary gland involution and may be responsible for the reduced milk supply associated with mastitis
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Susceptibility to mastitis in the bovine is affected by polymorphisms in the Tlr4 gene [70, 71], and a high proliferative response of peripheral blood mononuclear cells exposed to LPS in vitro is associated with increased resistance to mastitis [72]. Although similar studies have not been conducted in women, this suggests that genetic factors can affect inflammatory signalling pathways in the mammary gland, and may affect mastitis disease susceptibility in women, in combination with environmental factors discussed above. Therefore, heightened TLR4 signalling in the host due to perturbed milk flow, maternal stress, sleep deprivation or genetic predisposition could trigger breast inflammation in response to an otherwise innocuous bacterial load, leading to NFkB-mediated reduced milk supply (Fig. 1). If this proposed paradigm is correct, there may be opportunities to develop novel treatment and prevention strategies for lactation mastitis and sub-clinical disease based upon dampening inflammatory mediators in the breast.
New Directions for Treatments of Inflammatory Breast Disease TLRs are ancient innate signalling mediators, largely redundant in effective elimination of bacterial pathogens [73]. Indeed, greater local accumulation of neutrophils and macrophages in the mammary gland occurs in the absence of TLR4 [39], suggesting that TLR4 signalling promotes a greater systemic immune response to a mammary gland insult which is not necessarily beneficial for resolution of the disease. However, this remains speculative, and the effect of TLR4 inhibition on resolution of bacterial infection in the mammary gland has not been studied. Nonetheless, new therapeutic approaches to mastitis that dampen TLR4 signalling are now being explored, with some naturally occurring TLR4 antagonists including curcumin, alpinetin and chlorogenic acid showing promising results in mouse models [74–76], and could be used in conjunction with existing antimicrobial treatments. However, the effects of these TLR4 antagonists on resolution of inflammation and lactation capacity are yet to be explored. The World Health Organization recommends six months exclusive breastfeeding postpartum [77, 78], however in Western countries breastfeeding rates are alarmingly low. Despite most women’s intention to breastfeed, the 2010 Australian National Infant Feeding Survey reported that 85 % of infants are not exclusively breastfed during their first six months of life, and therefore not provided with the recommended optimal nutrition [14]. This leaves these infants at increased risk of respiratory and gastrointestinal diseases as babies, and non-communicable diseases including heart disease, obesity, diabetes, cancer, allergies, asthma, mental illness and chronic lung, liver and renal diseases as both children and adults [79, 80]. Targeting host inflammatory mediators as a
therapeutic approach to the treatment of mastitis might circumvent the associated reduced milk supply. There may also be opportunities to apply preventative strategies to high risk women in the form of supplements or pharmacological agents to reduce the risk of mastitis, and improve the low milk supply and poor infant weight gain associated with sub-clinical disease. These treatment and preventative approaches have the potential to promote optimal lactation and greatly improve the percentage of exclusively breastfed infants.
Conclusion Mastitis disease aetiology is poorly understood, and the relationship between infective pathogenic bacteria and mastitis is unclear. We have presented mechanistic evidence for a new paradigm for mastitis, where the principal drivers of the disease are inflammatory mediators in the host. These same inflammatory mediators may also lead to lactation insufficiency in both clinical and sub-clinical mastitis. The proposed model incorporates known mastitis risk factors including perturbed milk flow and maternal stress, leading to heightened TLR4 signalling. Together with commensal or pathogenic bacterial species, these factors converge on the NFkB signalling pathway in the mammary gland, causing local and systemic inflammation and reduced milk synthesis.
Funding WVI is a NBCF/THRF Fellow, MRH is an ARC Fellow [ID:110100297].
References 1. Tang, L., et al., Mastitis in Chinese Breastfeeding Mothers: A Prospective Cohort Study. Breastfeed Med, 2013. 2. Scott JA et al. Occurrence of lactational mastitis and medical management: a prospective cohort study in Glasgow. Int Breastfeed J. 2008;3:21. 3. Kinlay JR, O’Connell DL, Kinlay S. Risk factors for mastitis in breastfeeding women: results of a prospective cohort study. Aust N Z J Public Health. 2001;25(2):115–20. 4. Foxman B et al. Lactation mastitis: occurrence and medical management among 946 breastfeeding women in the United States. Am J Epidemiol. 2002;155(2):103–14. 5. Vogel A, Hutchison BL, Mitchell EA. Mastitis in the first year postpartum. Birth. 1999;26(4):218–25. 6. Wambach KA. Lactation mastitis: a descriptive study of the experience. J Hum Lact. 2003;19(1):24–34. 7. Amir LH et al. Incidence of breast abscess in lactating women: report from an Australian cohort. BJOG. 2004;111(12):1378–81. 8. Fetherston C. Characteristics of lactation mastitis in a Western Australian cohort. Breastfeed Rev. 1997;5(2):5–11. 9. Michie C, Lockie F, Lynn W. The challenge of mastitis. Arch Dis Child. 2003;88(9):818–21.
J Mammary Gland Biol Neoplasia 10. Wockel A et al. Inflammatory breast diseases during lactation: health effects on the newborn-a literature review. Mediators Inflamm. 2008;2008:298760. 11. Filteau SM et al. Milk cytokines and subclinical breast inflammation in Tanzanian women: effects of dietary red palm oil or sunflower oil supplementation. Immunology. 1999;97(4): 595–600. 12. Morton JA. The clinical usefulness of breast milk sodium in the assessment of lactogenesis. Pediatrics. 1994;93(5):802–6. 13. Aryeetey RN et al. Subclinical mastitis is common among Ghanaian women lactating 3 to 4 months postpartum. J Hum Lact. 2008;24(3): 263–7. 14. AIHW, 2010 Australian national infant feeding survey: indicator results. 2011: Cat. no. PHE 156. Australian Institute for Health and Welfare, Canberra. 15. Barkema HW et al. Invited review: The role of contagious disease in udder health. J Dairy Sci. 2009;92(10):4717–29. 16. Osterman KL, Rahm VA. Lactation mastitis: bacterial cultivation of breast milk, symptoms, treatment, and outcome. J Hum Lact. 2000;16(4):297–302. 17. Kvist LJ et al. The role of bacteria in lactational mastitis and some considerations of the use of antibiotic treatment. Int Breastfeed J. 2008;3:6. 18. Delgado S et al. Staphylococcus epidermidis strains isolated from breast milk of women suffering infectious mastitis: potential virulence traits and resistance to antibiotics. BMC Microbiol. 2009;9:82. 19. Carmichael AR, Dixon JM. Is lactation mastitis and shooting breast pain experienced by women during lactation caused by Candida albicans? Breast. 2002;11(1):88–90. 20. Fetherston C. Mastitis in lactating women: physiology or pathology? Breastfeed Rev. 2001;9(1):5–12. 21. Lindberg E et al. Long-time persistence of superantigen-producing Staphylococcus aureus strains in the intestinal microflora of healthy infants. Pediatr Res. 2000;48(6):741–7. 22. Kluytmans J, van Belkum A, Verbrugh H. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev. 1997;10(3):505–20. 23. Zdrazilek J. Staphylococcus aureus at a maternity ward. I. Colonization of mothers and neonates and survival of various S. aureus types in colonized individuals. J Hyg Epidemiol Microbiol Immunol. 1988;32(1):49–57. 24. Barbosa-Cesnik C, Schwartz K, Foxman B. Lactation mastitis. JAMA. 2003;289(13):1609–12. 25. Thomsen AC, Espersen T, Maigaard S. Course and treatment of milk stasis, noninfectious inflammation of the breast, and infectious mastitis in nursing women. Am J Obstet Gynecol. 1984;149(5):492–5. 26. Thomsen AC, Hansen KB, Moller BR. Leukocyte counts and microbiologic cultivation in the diagnosis of puerperal mastitis. Am J Obstet Gynecol. 1983;146(8):938–41. 27. WHO, Mastitis: causes and management. WHO/FCH/CAH/00, ed. D.o.C.a.A.h.a. Development. 2000, Geneva, Switerzland: World Health Organization. 28. Crepinsek, M.A., et al., Interventions for preventing mastitis after childbirth. Cochrane Database Syst Rev, 2012;10: p. CD007239. 29. Jahanfar, S., C.J. Ng, and C.L. Teng, Antibiotics for mastitis in breastfeeding women. Cochrane Database Syst Rev, 2013. 2: p. CD00545. 30. Kvist LJ. Toward a clarification of the concept of mastitis as used in empirical studies of breast inflammation during lactation. J Hum Lact. 2010;26(1):53–9. 31. Fetherston CM, Lai CT, Hartmann PE. Relationships between symptoms and changes in breast physiology during lactation mastitis. Breastfeed Med. 2006;1(3):136–45. 32. Fetherston CM, Wells JI, Hartmann PE. Severity of mastitis symptoms as a predictor of C-reactive protein in milk and blood during lactation. Breastfeed Med. 2006;1(3):127–35.
33. Zheng J, Watson AD, Kerr DE. Genome-wide expression analysis of lipopolysaccharide-induced mastitis in a mouse model. Infect Immun. 2006;74(3):1907–15. 34. Mizuno K et al. Mastitis is Associated with IL-6 Levels and Milk Fat Globule Size in Breast Milk. Journal of Human Lactation. 2012;28(4):529–34. 35. Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol. 2007;8(1):49–62. 36. Notebaert S et al. In vivo imaging of NF-kappaB activity during Escherichia coli-induced mammary gland infection. Cell Microbiol. 2008;10(6):1249–58. 37. Kawai T, Akira S. Pathogen recognition with Toll-like receptors. Curr Opin Immunol. 2005;17(4):338–44. 38. Sirard JC, Bayardo M, Didierlaurent A. Pathogen-specific TLR signaling in mucosa: mutual contribution of microbial TLR agonists and virulence factors. Eur J Immunol. 2006;36(2):260–3. 39. Glynn DJ, Hutchinson MR, Ingman WV. Toll-Like Receptor 4 Regulates Lipopolysaccharide-Induced Inflammation and Lactation Insufficiency in a Mouse Model of Mastitis. Biol Reprod. 2014;90(5):91. 40. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010;11(5): 373–84. 41. Gonen E et al. Toll-like receptor 4 is needed to restrict the invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine model of acute mastitis. Cell Microbiol. 2007;9(12):2826–38. 42. Goodridge HS et al. Phosphorylcholine mimics the effects of ES-62 on macrophages and dendritic cells. Parasite Immunol. 2007;29(3): 127–37. 43. Ichinose T et al. Mouse strain differences in eosinophilic airway inflammation caused by intratracheal instillation of mite allergen and diesel exhaust particles. J Appl Toxicol. 2004;24(1):69–76. 44. Tokunaga Y et al. Genetic susceptibility to the induction of murine experimental autoimmune orchitis (EAO) without adjuvant. I. Comparison of pathology, delayed type hypersensitivity, and antibody. Clin Immunol Immunopathol. 1993;66(3):239–47. 45. Uddin MH et al. Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice. Parasitol Int. 2012;61(1):118–23. 46. Elazar S et al. Neutrophil recruitment in endotoxin-induced murine mastitis is strictly dependent on mammary alveolar macrophages. Vet Res. 2010;41(1):10. 47. Chen LC et al. Role of TLR-4 in liver macrophage and endothelial cell responsiveness during acute endotoxemia. Exp Mol Pathol. 2007;83(3):311–26. 48. Connelly L et al. Activation of nuclear factor kappa B in mammary epithelium promotes milk loss during mammary development and infection. J Cell Physiol. 2010;222(1):73–81. 49. Kesaraju P et al. Experimental autoimmune breast failure: a model for lactation insufficiency, postnatal nutritional deprivation, and prophylactic breast cancer vaccination. Am J Pathol. 2012;181(3):775–84. 50. Clarkson RW et al. NF-kappaB inhibits apoptosis in murine mammary epithelia. J Biol Chem. 2000;275(17):12737–42. 51. Geymayer S, Doppler W. Activation of NF-kappaB p50/p65 is regulated in the developing mammary gland and inhibits STAT5-mediated beta-casein gene expression. FASEB J. 2000;14(9):1159–70. 52. Baxter FO et al. IKKbeta/2 induces TWEAK and apoptosis in mammary epithelial cells. Development. 2006;133(17):3485–94. 53. Zaragoza R et al. Nitric oxide triggers mammary gland involution after weaning: remodelling is delayed but not impaired in mice lacking inducible nitric oxide synthase. Biochem J. 2010;428(3):451–62. 54. O’Brien J et al. Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution. Development. 2012;139(2):269–75.
J Mammary Gland Biol Neoplasia 55. Buchanan MM et al. Toll-like receptor 4 in CNS pathologies. J Neurochem. 2010;114(1):13–27. 56. McDermott MF, Tschopp J. From inflammasomes to fevers, crystals and hypertension: how basic research explains inflammatory diseases. Trends Mol Med. 2007;13(9):381–8. 57. Fetherston C. Risk factors for lactation mastitis. J Hum Lact. 1998;14(2):101–9. 58. Abou-Dakn M et al. Inflammatory Breast Diseases during Lactation: Milk Stasis, Puerperal Mastitis, Abscesses of the Breast, and Malignant Tumors - Current and Evidence-Based Strategies for Diagnosis and Therapy. Breast Care (Basel). 2010;5(1):33–7. 59. El Mezayen R et al. Endogenous signals released from necrotic cells augment inflammatory responses to bacterial endotoxin. Immunol Lett. 2007;111(1):36–44. 60. Samuvel DJ et al. Lactate boosts TLR4 signaling and NF-kappaB pathway-mediated gene transcription in macrophages via monocarboxylate transporters and MD-2 up-regulation. J Immunol. 2009;182(4):2476–84. 61. Davis SR et al. Milk L-lactate concentration is increased during mastitis. J Dairy Res. 2004;71(2):175–81. 62. Wockel A et al. Predictors of inflammatory breast diseases during lactation–results of a cohort study. Am J Reprod Immunol. 2010;63(1):28–37. 63. Frank MG, Watkins LR, Maier SF. Stress-induced glucocorticoids as a neuroendocrine alarm signal of danger. Brain Behav Immun. 2013;33:1–6. 64. Garate I et al. Origin and consequences of brain Toll-like receptor 4 pathway stimulation in an experimental model of depression. J Neuroinflammation. 2011;8:151. 65. McCusker RH, Kelley KW. Immune-neural connections: how the immune system’s response to infectious agents influences behavior. J Exp Biol. 2013;216(Pt 1):84–98. 66. Hines DJ et al. Prevention of LPS-induced microglia activation, cytokine production and sickness behavior with TLR4 receptor interfering peptides. PLoS One. 2013;8(3):e60388. 67. Miller AH, Maletic V, Raison CL. Inflammation and its discontents: the role of cytokines in the pathophysiology of major depression. Biol Psychiatry. 2009;65(9):732–41.
68. Bienkowski MS, Rinaman L. Noradrenergic inputs to the paraventricular hypothalamus contribute to hypothalamicpituitary-adrenal axis and central Fos activation in rats after acute systemic endotoxin exposure. Neuroscience. 2008;156(4):1093–102. 69. Ringgold KM et al. Prolonged sleep fragmentation of mice exacerbates febrile responses to lipopolysaccharide. J Neurosci Methods. 2013;219(1):104–12. 70. Sharma BS et al. Association of toll-like receptor 4 polymorphisms with somatic cell score and lactation persistency in Holstein bulls. J Dairy Sci. 2006;89(9):3626–35. 71. Wang X et al. Genetic polymorphism of TLR4 gene and correlation with mastitis in cattle. J Genet Genomics. 2007;34(5):406–12. 72. Catalani, E., et al., Short communication: Lymphoproliferative response to lipopolysaccharide and incidence of infections in periparturient dairy cows. J Dairy Sci, 2013. 73. Palm NW, Medzhitov R. Immunostimulatory activity of haptenated proteins. Proc Natl Acad Sci U S A. 2009;106(12):4782–7. 74. Fu Y et al. Curcumin attenuates inflammatory responses by suppressing TLR4-mediated NF-kappaB signaling pathway in lipopolysaccharide-induced mastitis in mice. Int Immunopharmacol. 2014;20(1):54–8. 75. Ruifeng G et al. Chlorogenic acid attenuates lipopolysaccharideinduced mice mastitis by suppressing TLR4-mediated NF-kappaB signaling pathway. Eur J Pharmacol. 2014;729:54–8. 76. Chen H et al. Alpinetin attenuates inflammatory responses by interfering toll-like receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharide-induced mastitis in mice. Int Immunopharmacol. 2013;17(1):26–32. 77. WHO. Global strategy for infant and young child feeding. Geneva: World Health Organization; 2003. 78. NHMRC. Infant Feeding Guidelines. Canberra: National Health and Medical Research Council; 2012. 79. Geddes DT, Prescott SL. Developmental origins of health and disease: the role of human milk in preventing disease in the 21 (st) century. J Hum Lact. 2013;29(2):123–7. 80. Black RE et al. Maternal and child undernutrition: global and regional exposures and health consequences. Lancet. 2008;371(9608):243–60.
Inflammatory Mediators in Mastitis and Lactation Insufficiency Wendy V. Ingman & Danielle J. Glynn & Mark R. Hutchinson
Received: 6 May 2014 / Accepted: 18 June 2014 # Springer Science+Business Media New York 2014
Abstract Mastitis is a common inflammatory disease during lactation that causes reduced milk supply. A growing body of evidence challenges the central role of pathogenic bacteria in mastitis, with disease severity associated with markers of inflammation rather than infection. Inflammation in the mammary gland may be triggered by microbe-associated molecular patterns (MAMPs) as well as danger-associated molecular patterns (DAMPs) binding to pattern recognition receptors such as the toll-like receptors (TLRs) on the surface of mammary epithelial cells and local immune cell populations. Activation of the TLR4 signalling pathway and downstream nuclear factor kappa B (NFkB) is critical to mediating local mammary gland inflammation and systemic immune responses in mouse models of mastitis. However, activation of NFkB also induces epithelial cell apoptosis and reduced milk protein synthesis, suggesting that inflammatory mediators activated during mastitis promote partial involution. Perturbed milk flow, maternal stress and genetic predisposition are significant risk factors for mastitis, and could lead to a heightened TLR4-mediated inflammatory response, resulting in increased susceptibility and severity of mastitis disease in the context of low MAMP abundance. Therefore, heightened W. V. Ingman : D. J. Glynn Discipline of Surgery, School of Medicine, The Queen Elizabeth Hospital, University of Adelaide, Woodville, Australia W. V. Ingman : D. J. Glynn Robinson Research Institute, University of Adelaide, Adelaide, Australia W. V. Ingman : D. J. Glynn School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia M. R. Hutchinson (*) Discipline Physiology, School of Medical Sciences, University of Adelaide, Adelaide, Australia e-mail: [email protected]
host inflammatory signalling may act in concert with pathogenic or commensal bacterial species to cause both the inflammation associated with mastitis and lactation insufficiency. Here, we present an alternate paradigm to the widely held notion that breast inflammation is driven principally by infectious bacterial pathogens, and suggest there may be other therapeutic strategies, apart from the currently utilised antimicrobial agents, that could be employed to prevent and treat mastitis in women. Keywords Mastitis . Lactation insufficiency . Inflammation . Toll-like receptors Abbreviations DAMP Danger-associated molecular pattern IL Interleukin LPS Lipopolysaccharide MAMP Microbe-associated molecular pattern NOS Nitric oxide synthase NFkB Nuclear factor kappa b STAT5 Signal transducer and activator of transcription 5 TLR Toll-like receptor TNFA Tumour necrosis factor alpha
Mastitis and Lactation Insufficiency Lactation mastitis is a debilitating inflammatory breast disease in postpartum women. Prospective cohort studies have reported the cumulative incidence of lactation mastitis to be between 10 % and 27 % of breastfeeding women [1–5]. The disease causes localised pain, and is frequently accompanied by the rapid onset of systemic symptoms including fever, muscle aches, chills and fatigue [6]. In 3-11 % of cases, mastitis leads to a breast abscess [7], which must be surgically drained, and
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in severe disease there can be permanent disfiguration of the breast. Low milk supply frequently accompanies these symptoms [8, 9]. Collectively, the challenges posed by this disease lead many women to use supplementary formula to feed their infant, or cease breastfeeding altogether [8, 10]. Sub-clinical mastitis is characterised by increased interleukin 8 (IL8) and sodium in breast milk in the absence of clinical symptoms [11]. In a Ghanaian cohort, sub-clinical mastitis is associated with reduced infant weight gain of around 20 % in the first month postpartum [11, 12], which may be the result of a prolonged reduction in milk synthesis by the mother. The incidence of sub-clinical disease has not been well documented, however 45 % of Ghanaian women exhibit elevated inflammatory markers in their breast milk indicative of subclinical disease [13]. Although the prevalence of sub-clinical mastitis in Western women is not known, 56 % of Australian women who cease breastfeeding in the first six months postpartum cite low milk supply as the cause [14], and in some cases this might be the result of asymptomatic breast inflammation.
bacterium alone is not sufficient to trigger the disease. In addition, mastitis frequently presents as inflammation in the absence of abundant pathogenic bacteria, leading some clinical researchers to propose delineation of “infective” and “noninfective” forms of mastitis disease [25, 26]. The World Health Organization summarises the limitations of the current paradigm thus: “Many lactating women who have potentially pathogenic bacteria on their skin or in their milk do not develop mastitis. But: Many women who do develop mastitis do not have pathogenic organisms in their milk” [27]. In light of this evidence, it is not surprising that recent Cochrane systematic reviews have emphasized the uncertainty around management of mastitis with antimicrobial agents. Prophylactic antibiotic use is not effective in preventing mastitis [28] and there is insufficient evidence to support the use of antibiotics in treating the disease [29]. Meticulous hygiene by breastfeeding women also appears ineffective in preventing mastitis [4]. However, no alternative mechanistic basis for mastitis has been identified, and there continues to be considerable doubt around the aetiology of the disease [20, 30].
Bacterial Pathogens in Mastitis Inflammatory Mediators in Mastitis The relationship between mastitis in women and bacterial infection is largely inferred through studies in bovine species [15], and the identification of pathogenic bacteria including Staphylococcus aureus, Escherichia coli, and Group B streptococci in mastitis-affected human breast milk [16, 17]. Commensal bacteria including Staphylococcus epidermidis are also frequently observed in breast milk from women with mastitis [18], however their role in the aetiology of mastitis is not known. Other organisms such as Candida albicans have also been suggested to be involved, although no study has demonstrated a causative link [19]. Therefore the majority of clinical practitioners believe lactation mastitis is caused by infectious bacterial pathogens. However, there is now a growing body of scientific literature that challenges the current paradigm. A number of studies have shown inconsistencies between bacterial load and disease severity [17, 20, 21], and the immune system response to the insult appears to bear a more significant relationship to disease severity in comparison to the infection itself. In a study of 466 disease free women, breast milk from nearly a third of healthy women was found to harbour high (≥106 colony forming units/L) bacterial counts of Staphylococcus aureus and 10 % harboured Group B streptococci [17]. Furthermore, analysis of 192 women with mastitis indicated that there is no correlation between severity of symptoms of mastitis, including breast inflammation, fever and pain, and the abundance of pathogenic Staphylococcus aureus and Group B streptococci in breast milk [17]. These bacterial species are also commonly found in nasal passages and skin flora [22–24] suggesting that the presence of the
Bacteria may augment inflammation, however it is clear that other factors, as yet unidentified, are important components of mastitis. In women, disease severity is closely aligned with both systemic and local indicators of inflammation. Breast milk and blood samples collected between day 5 and 3 months postpartum in prospective studies show C-reactive protein in milk and serum, and sodium concentration in milk is significantly elevated during periods of mastitis [31, 32]. Furthermore, the severity of mastitis based on degree of local breast inflammation and systemic symptoms such as elevation in body temperature were closely correlated with serum concentration of C-reactive protein [32]. Therefore, specific inflammatory signalling pathways activated in the host tissue are likely to be key determinants of susceptibility to mastitis and severity of the disease. Women with mastitis exhibit increased serum cytokines including interleukins (IL) 1, 6 and 8, and tumour necrosis factor A (TNFA) [33, 34], indicative of activation of the transcription factor nuclear factor kappa B (NFkB) in the host. The NFkB family of transcription factors are ubiquitously expressed, and when activated, are central regulators of innate and adaptive immune responses, the cellular stress response and programmed cell death [35]. In the lactating mammary glands of mice, infection with Escherichia coli induces NFkB activation within 4 hours, peaking at 6 hours with a return to basal levels 24 hours post-infection [36]. Candidate receptors responsible for activating NFkB include the toll-like receptors (TLRs). TLRs expressed on the
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surface of macrophages, dendritic cells and epithelial cells are important mediators of inflammation [37]. TLR2, TLR4 and TLR5 bind bacterial byproducts such as lipopolysaccharide (LPS), bacterial lipopeptides and flagellins, collectively referred to as Microbe-Associated Molecular Patterns (MAMPs). Importantly, this terminology has recently been adapted from Pathogen-Associated Molecular Patterns, acknowledging that commensal microbiota contribute to critical TLR signalling in addition to foreign pathogen-derived patterns [38]. TLR activation initiates transcription, translation and release of a cascade of inflammatory cytokines, chemokines and adhesion molecules, and recruits other components of the innate immune system such as neutrophils to the infected site [39, 40]. Such innate immune responses contribute to the swelling and pain associated with mastitis, whilst systemic actions of pro-inflammatory cytokines IL1 and IL8 initiate the fever response [6, 27]. The role of TLR4 in mediating inflammation in mastitis is demonstrated in TLR4 deficient mouse models challenged with bacterial endotoxin LPS to the teat canal during lactation [39, 41]. TLR4 signalling deficiency in C3H/HeJ mice, due to a point mutation that limits MyD88-dependent NFkB activation, resulted in dampened inflammation in response to LPS [41], suggesting that TLR4 signalling is necessary for appropriate innate immune system responses to infection. However, LPS administered to mice carrying a null mutation in the gene encoding TLR4, on a Balb/c background, resulted in increased recruitment of neutrophils and macrophages to the mammary gland and dampened systemic inflammatory cytokines including CXCL1 and TNFA [39], suggesting that TLR4 affects the immune signalling fingerprint, rather than suppressing the response altogether. The difference in results reported in these two studies is likely due to the different background strain of mice used [42]. C3H and Balb/c mice exhibit striking differences in immune system responses that affect airway inflammation [43], and susceptibility to experimental autoimmune orchitis [44] and Clonorchis sinensis infection [45]. Interestingly, neutrophils are recruited in greater abundance in mice with a null mutation in the gene encoding inducible nitric oxide synthase (NOS2) [46]. Nitric oxide production, mediated by NOS2, is observed in TLR4-activated macrophages [47] suggesting that downstream effectors of TLR4 may alter the immune signalling fingerprint in response to LPS, as reported in Tlr4 null mutant Balb/c mice [39]. Further studies on the role of nitric oxide on mediating TLR4associated mammary gland inflammation are warranted. When C3H/HeJ TLR4 signalling deficient mice are challenged with Escherichia coli rather than the endotoxin LPS, the magnitude of mammary gland inflammation is comparable to similarly infected wildtype mice, however an increased abundance of bacteria within mammary epithelial cells is observed 24 and 48 hours post-infection [41]. TLR4expressing macrophages are significant in mediating
clearance of Escherichia coli when adoptively transferred to the teat canal, suggesting that alveolar macrophages expressing TLR4 are required to restrict bacterial invasion. However, the presence of intact TLR4 signalling in wildtype mice does not prevent the acute rapid growth of bacteria within the alveolar and ductal spaces in the infected mammary gland [41], and the impact of TLR4 on disease resolution is not known. Further mouse studies administering live bacteria to the lactating mouse mammary gland are required to better understand how TLR4-mediated inflammation affects susceptibility, severity and resolution of bacterial infections.
Lactation Insufficiency is a Consequence of TLR4-Mediated NFkB Activation In addition to mediating inflammation associated with bacterial infection, activation of the TLR4-signalling pathway also induces reduced milk synthesis. Administration of LPS to the teat canal during lactation results in decreased milk-secreting glandular area, and reduced nuclear phosphorylated signal transducer and activator of transcription 5 (STAT5) on day 7 following LPS administration [39]. However, this compromised capacity for lactation does not occur in LPS-treated Tlr4 null mutant mice [39]. Similarly, activation of NFkB in an inducible transgenic mouse model during normal lactation induces mammary epithelial cell apoptosis and a rapid decline in milk protein beta casein [48]. Lactation insufficiency in mice has also been observed as a consequence of inappropriate adaptive immune responses [49]. Therefore, reduced milk synthesis occurs due to immune responses in the host, irrespective of the presence of pathogenic bacteria, suggesting that perturbed milk supply during mastitis is a consequence of inflammatory mediators, rather than the bacterial infection itself. Inflammatory mediators are recognised as critical factors that promote mammary gland involution following weaning. As early as one hour following removal of pups from a lactating female, activated NFkB is detectable in mammary epithelial cells, and is highest 3 days following weaning [50]. Activated NFkB inhibits STAT5-mediated milk protein expression [51], and inhibition of NFkB activity through conditional deletion of upstream regulator IkB kinase 2/B delays involution-associated apoptosis and tissue remodelling [52]. NOS2, expressed in macrophages under the direction of activated NFkB, mediates nitric oxide production leading to apoptosis of mammary epithelial cells during involution [53], and this mechanism may be responsible for the delayed apoptosis observed in mice depleted of macrophages at the time of weaning [54]. Therefore, activation of NFkB and downstream effectors such as nitric oxide during mastitis is likely to lead to epithelial cell apoptosis and partial involution, and may be responsible for the reduced milk supply observed in women with mastitis. Similarly, sub-clinical mastitis in
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women is associated with poor weight gain in infants [11, 12] and might be the result of activated NFkB causing prolonged reduction in milk synthesis in the mother.
Risk Factors That may Affect Inflammatory Mediators in Mastitis Host inflammatory mediators such as NFkB are implicated as integral components of breast inflammation that affect lactation capacity. However, little is known as to how inflammation in the mammary gland can occur in the absence of abundant bacterial pathogens. Recently, advances in our understanding of TLRs have uncovered an expanded repertoire of TLR agonists beyond MAMPs, such that TLR activation is not only initiated by the presence of bacteria. A number of endogenous proteins and molecules induce TLR activation under sterile conditions, and are collectively referred to as Danger Associated Molecular Patterns (DAMPs) [55, 56]. The significance of the TLR4 signalling pathway and NFkB in mastitis, raises the possibility that in some cases, mastitis could be caused by inflammatory mediators activated by DAMPs, or heightened TLR4 immune responses in the absence of a high bacterial load. Perturbed milk flow is recognised as a major risk factor for mastitis [1, 3, 27, 57], and is caused by reduced infant feeding, poor attachment, oversupply of milk or a blocked mammary duct [27, 58]. Accumulation of milk within a duct may affect the abundance of co-stimulatory factors such as DAMPs, or downstream immune signalling pathways. An engorged milk duct is likely to be under severe mechanical stress, causing the release of a number of DAMPs including HMGB1 and HSP70 which augment inflammatory responses to MAMPs [59]. In addition, the abundance of lactate, a potent activator of the TLR4 signalling pathway and NFkB [60], increases in
Perturbed Mechanical stress milk flow DAMPs
bovine milk during mastitis [61] and may also act as a costimulatory inflammatory factor. The sensitivity of the mammary gland to both DAMPs and MAMPs may be increased due to maternal physical and psychological stress, which is also a significant risk factor for mastitis [1, 3, 57, 62]. Maternal stress and illness have the capacity to change the expression and signalling sensitivity of TLR4 [63, 64]. Importantly, physical and psychological stress-induced neuroendocrine responses can all feed forward to trigger even greater stress alterations of immune function. As discussed previously, peripheral activation of TLR4 results in pro-inflammatory cytokine secretion [40]. These cytokines can stimulate peripheral neuronal afferents or cross the blood– brain barrier to activate immunocompetent glial cells (microglia and astrocytes) within the brain, causing neuroinflammation, thus changing neuronal activity and ultimately behaviour [65]. Through these immune-to-brain communications, peripheral immune activation precipitates “sickness behaviour” [66], and is associated with depression [64, 67]. TLR4 activation can further modulate the stress response through activation of the hypothalamus-pituitary-adrenal axis [68]. Whilst stress is typically associated with decreased TLR4 responses, recent discoveries point to key proinflammatory actions of stress priming [63]. In fact the neuroendocrine response to stress is seen as a TLR dependent alarm of danger, akin to a DAMP [63]. Additionally, associated with periods of significant life changes and stress are multiple forms of sleep disruption, some of which cause alterations in TLR4dependent immune responses. For example, long term sleep fragmentation leads to increased TLR4-dependent fevering [69]. As such, the neuroendocrine response associated with multiple forms of stress and sleep disruption will significantly impact sensitivity to inflammatory stimuli, affecting susceptibility, severity and recovery from mastitis.
Pathogenic or commensal bacteria
MAMP DAMP
Mammary epithelium
Maternal stress Sleep deprivation
TLR4 and downstream mediators
DAMP MAMP-mediated NFkB activation Fig. 1 TLR4-dependent inflammatory mediators affect lactation mastitis and milk supply. Perturbed milk flow, maternal stress, genetic predisposition and sleep deprivation can lead to accumulation of DAMPs and heightened TLR4 signalling in the mammary gland, leading to increased
Genetic predisposition
TLR4
Inflammation Reduced milk synthesis
susceptibility to mastitis and increased severity of the disease. Importantly, activation of NFkB could lead to partial mammary gland involution and may be responsible for the reduced milk supply associated with mastitis
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Susceptibility to mastitis in the bovine is affected by polymorphisms in the Tlr4 gene [70, 71], and a high proliferative response of peripheral blood mononuclear cells exposed to LPS in vitro is associated with increased resistance to mastitis [72]. Although similar studies have not been conducted in women, this suggests that genetic factors can affect inflammatory signalling pathways in the mammary gland, and may affect mastitis disease susceptibility in women, in combination with environmental factors discussed above. Therefore, heightened TLR4 signalling in the host due to perturbed milk flow, maternal stress, sleep deprivation or genetic predisposition could trigger breast inflammation in response to an otherwise innocuous bacterial load, leading to NFkB-mediated reduced milk supply (Fig. 1). If this proposed paradigm is correct, there may be opportunities to develop novel treatment and prevention strategies for lactation mastitis and sub-clinical disease based upon dampening inflammatory mediators in the breast.
New Directions for Treatments of Inflammatory Breast Disease TLRs are ancient innate signalling mediators, largely redundant in effective elimination of bacterial pathogens [73]. Indeed, greater local accumulation of neutrophils and macrophages in the mammary gland occurs in the absence of TLR4 [39], suggesting that TLR4 signalling promotes a greater systemic immune response to a mammary gland insult which is not necessarily beneficial for resolution of the disease. However, this remains speculative, and the effect of TLR4 inhibition on resolution of bacterial infection in the mammary gland has not been studied. Nonetheless, new therapeutic approaches to mastitis that dampen TLR4 signalling are now being explored, with some naturally occurring TLR4 antagonists including curcumin, alpinetin and chlorogenic acid showing promising results in mouse models [74–76], and could be used in conjunction with existing antimicrobial treatments. However, the effects of these TLR4 antagonists on resolution of inflammation and lactation capacity are yet to be explored. The World Health Organization recommends six months exclusive breastfeeding postpartum [77, 78], however in Western countries breastfeeding rates are alarmingly low. Despite most women’s intention to breastfeed, the 2010 Australian National Infant Feeding Survey reported that 85 % of infants are not exclusively breastfed during their first six months of life, and therefore not provided with the recommended optimal nutrition [14]. This leaves these infants at increased risk of respiratory and gastrointestinal diseases as babies, and non-communicable diseases including heart disease, obesity, diabetes, cancer, allergies, asthma, mental illness and chronic lung, liver and renal diseases as both children and adults [79, 80]. Targeting host inflammatory mediators as a
therapeutic approach to the treatment of mastitis might circumvent the associated reduced milk supply. There may also be opportunities to apply preventative strategies to high risk women in the form of supplements or pharmacological agents to reduce the risk of mastitis, and improve the low milk supply and poor infant weight gain associated with sub-clinical disease. These treatment and preventative approaches have the potential to promote optimal lactation and greatly improve the percentage of exclusively breastfed infants.
Conclusion Mastitis disease aetiology is poorly understood, and the relationship between infective pathogenic bacteria and mastitis is unclear. We have presented mechanistic evidence for a new paradigm for mastitis, where the principal drivers of the disease are inflammatory mediators in the host. These same inflammatory mediators may also lead to lactation insufficiency in both clinical and sub-clinical mastitis. The proposed model incorporates known mastitis risk factors including perturbed milk flow and maternal stress, leading to heightened TLR4 signalling. Together with commensal or pathogenic bacterial species, these factors converge on the NFkB signalling pathway in the mammary gland, causing local and systemic inflammation and reduced milk synthesis.
Funding WVI is a NBCF/THRF Fellow, MRH is an ARC Fellow [ID:110100297].
References 1. Tang, L., et al., Mastitis in Chinese Breastfeeding Mothers: A Prospective Cohort Study. Breastfeed Med, 2013. 2. Scott JA et al. Occurrence of lactational mastitis and medical management: a prospective cohort study in Glasgow. Int Breastfeed J. 2008;3:21. 3. Kinlay JR, O’Connell DL, Kinlay S. Risk factors for mastitis in breastfeeding women: results of a prospective cohort study. Aust N Z J Public Health. 2001;25(2):115–20. 4. Foxman B et al. Lactation mastitis: occurrence and medical management among 946 breastfeeding women in the United States. Am J Epidemiol. 2002;155(2):103–14. 5. Vogel A, Hutchison BL, Mitchell EA. Mastitis in the first year postpartum. Birth. 1999;26(4):218–25. 6. Wambach KA. Lactation mastitis: a descriptive study of the experience. J Hum Lact. 2003;19(1):24–34. 7. Amir LH et al. Incidence of breast abscess in lactating women: report from an Australian cohort. BJOG. 2004;111(12):1378–81. 8. Fetherston C. Characteristics of lactation mastitis in a Western Australian cohort. Breastfeed Rev. 1997;5(2):5–11. 9. Michie C, Lockie F, Lynn W. The challenge of mastitis. Arch Dis Child. 2003;88(9):818–21.
J Mammary Gland Biol Neoplasia 10. Wockel A et al. Inflammatory breast diseases during lactation: health effects on the newborn-a literature review. Mediators Inflamm. 2008;2008:298760. 11. Filteau SM et al. Milk cytokines and subclinical breast inflammation in Tanzanian women: effects of dietary red palm oil or sunflower oil supplementation. Immunology. 1999;97(4): 595–600. 12. Morton JA. The clinical usefulness of breast milk sodium in the assessment of lactogenesis. Pediatrics. 1994;93(5):802–6. 13. Aryeetey RN et al. Subclinical mastitis is common among Ghanaian women lactating 3 to 4 months postpartum. J Hum Lact. 2008;24(3): 263–7. 14. AIHW, 2010 Australian national infant feeding survey: indicator results. 2011: Cat. no. PHE 156. Australian Institute for Health and Welfare, Canberra. 15. Barkema HW et al. Invited review: The role of contagious disease in udder health. J Dairy Sci. 2009;92(10):4717–29. 16. Osterman KL, Rahm VA. Lactation mastitis: bacterial cultivation of breast milk, symptoms, treatment, and outcome. J Hum Lact. 2000;16(4):297–302. 17. Kvist LJ et al. The role of bacteria in lactational mastitis and some considerations of the use of antibiotic treatment. Int Breastfeed J. 2008;3:6. 18. Delgado S et al. Staphylococcus epidermidis strains isolated from breast milk of women suffering infectious mastitis: potential virulence traits and resistance to antibiotics. BMC Microbiol. 2009;9:82. 19. Carmichael AR, Dixon JM. Is lactation mastitis and shooting breast pain experienced by women during lactation caused by Candida albicans? Breast. 2002;11(1):88–90. 20. Fetherston C. Mastitis in lactating women: physiology or pathology? Breastfeed Rev. 2001;9(1):5–12. 21. Lindberg E et al. Long-time persistence of superantigen-producing Staphylococcus aureus strains in the intestinal microflora of healthy infants. Pediatr Res. 2000;48(6):741–7. 22. Kluytmans J, van Belkum A, Verbrugh H. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev. 1997;10(3):505–20. 23. Zdrazilek J. Staphylococcus aureus at a maternity ward. I. Colonization of mothers and neonates and survival of various S. aureus types in colonized individuals. J Hyg Epidemiol Microbiol Immunol. 1988;32(1):49–57. 24. Barbosa-Cesnik C, Schwartz K, Foxman B. Lactation mastitis. JAMA. 2003;289(13):1609–12. 25. Thomsen AC, Espersen T, Maigaard S. Course and treatment of milk stasis, noninfectious inflammation of the breast, and infectious mastitis in nursing women. Am J Obstet Gynecol. 1984;149(5):492–5. 26. Thomsen AC, Hansen KB, Moller BR. Leukocyte counts and microbiologic cultivation in the diagnosis of puerperal mastitis. Am J Obstet Gynecol. 1983;146(8):938–41. 27. WHO, Mastitis: causes and management. WHO/FCH/CAH/00, ed. D.o.C.a.A.h.a. Development. 2000, Geneva, Switerzland: World Health Organization. 28. Crepinsek, M.A., et al., Interventions for preventing mastitis after childbirth. Cochrane Database Syst Rev, 2012;10: p. CD007239. 29. Jahanfar, S., C.J. Ng, and C.L. Teng, Antibiotics for mastitis in breastfeeding women. Cochrane Database Syst Rev, 2013. 2: p. CD00545. 30. Kvist LJ. Toward a clarification of the concept of mastitis as used in empirical studies of breast inflammation during lactation. J Hum Lact. 2010;26(1):53–9. 31. Fetherston CM, Lai CT, Hartmann PE. Relationships between symptoms and changes in breast physiology during lactation mastitis. Breastfeed Med. 2006;1(3):136–45. 32. Fetherston CM, Wells JI, Hartmann PE. Severity of mastitis symptoms as a predictor of C-reactive protein in milk and blood during lactation. Breastfeed Med. 2006;1(3):127–35.
33. Zheng J, Watson AD, Kerr DE. Genome-wide expression analysis of lipopolysaccharide-induced mastitis in a mouse model. Infect Immun. 2006;74(3):1907–15. 34. Mizuno K et al. Mastitis is Associated with IL-6 Levels and Milk Fat Globule Size in Breast Milk. Journal of Human Lactation. 2012;28(4):529–34. 35. Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol. 2007;8(1):49–62. 36. Notebaert S et al. In vivo imaging of NF-kappaB activity during Escherichia coli-induced mammary gland infection. Cell Microbiol. 2008;10(6):1249–58. 37. Kawai T, Akira S. Pathogen recognition with Toll-like receptors. Curr Opin Immunol. 2005;17(4):338–44. 38. Sirard JC, Bayardo M, Didierlaurent A. Pathogen-specific TLR signaling in mucosa: mutual contribution of microbial TLR agonists and virulence factors. Eur J Immunol. 2006;36(2):260–3. 39. Glynn DJ, Hutchinson MR, Ingman WV. Toll-Like Receptor 4 Regulates Lipopolysaccharide-Induced Inflammation and Lactation Insufficiency in a Mouse Model of Mastitis. Biol Reprod. 2014;90(5):91. 40. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010;11(5): 373–84. 41. Gonen E et al. Toll-like receptor 4 is needed to restrict the invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine model of acute mastitis. Cell Microbiol. 2007;9(12):2826–38. 42. Goodridge HS et al. Phosphorylcholine mimics the effects of ES-62 on macrophages and dendritic cells. Parasite Immunol. 2007;29(3): 127–37. 43. Ichinose T et al. Mouse strain differences in eosinophilic airway inflammation caused by intratracheal instillation of mite allergen and diesel exhaust particles. J Appl Toxicol. 2004;24(1):69–76. 44. Tokunaga Y et al. Genetic susceptibility to the induction of murine experimental autoimmune orchitis (EAO) without adjuvant. I. Comparison of pathology, delayed type hypersensitivity, and antibody. Clin Immunol Immunopathol. 1993;66(3):239–47. 45. Uddin MH et al. Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice. Parasitol Int. 2012;61(1):118–23. 46. Elazar S et al. Neutrophil recruitment in endotoxin-induced murine mastitis is strictly dependent on mammary alveolar macrophages. Vet Res. 2010;41(1):10. 47. Chen LC et al. Role of TLR-4 in liver macrophage and endothelial cell responsiveness during acute endotoxemia. Exp Mol Pathol. 2007;83(3):311–26. 48. Connelly L et al. Activation of nuclear factor kappa B in mammary epithelium promotes milk loss during mammary development and infection. J Cell Physiol. 2010;222(1):73–81. 49. Kesaraju P et al. Experimental autoimmune breast failure: a model for lactation insufficiency, postnatal nutritional deprivation, and prophylactic breast cancer vaccination. Am J Pathol. 2012;181(3):775–84. 50. Clarkson RW et al. NF-kappaB inhibits apoptosis in murine mammary epithelia. J Biol Chem. 2000;275(17):12737–42. 51. Geymayer S, Doppler W. Activation of NF-kappaB p50/p65 is regulated in the developing mammary gland and inhibits STAT5-mediated beta-casein gene expression. FASEB J. 2000;14(9):1159–70. 52. Baxter FO et al. IKKbeta/2 induces TWEAK and apoptosis in mammary epithelial cells. Development. 2006;133(17):3485–94. 53. Zaragoza R et al. Nitric oxide triggers mammary gland involution after weaning: remodelling is delayed but not impaired in mice lacking inducible nitric oxide synthase. Biochem J. 2010;428(3):451–62. 54. O’Brien J et al. Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution. Development. 2012;139(2):269–75.
J Mammary Gland Biol Neoplasia 55. Buchanan MM et al. Toll-like receptor 4 in CNS pathologies. J Neurochem. 2010;114(1):13–27. 56. McDermott MF, Tschopp J. From inflammasomes to fevers, crystals and hypertension: how basic research explains inflammatory diseases. Trends Mol Med. 2007;13(9):381–8. 57. Fetherston C. Risk factors for lactation mastitis. J Hum Lact. 1998;14(2):101–9. 58. Abou-Dakn M et al. Inflammatory Breast Diseases during Lactation: Milk Stasis, Puerperal Mastitis, Abscesses of the Breast, and Malignant Tumors - Current and Evidence-Based Strategies for Diagnosis and Therapy. Breast Care (Basel). 2010;5(1):33–7. 59. El Mezayen R et al. Endogenous signals released from necrotic cells augment inflammatory responses to bacterial endotoxin. Immunol Lett. 2007;111(1):36–44. 60. Samuvel DJ et al. Lactate boosts TLR4 signaling and NF-kappaB pathway-mediated gene transcription in macrophages via monocarboxylate transporters and MD-2 up-regulation. J Immunol. 2009;182(4):2476–84. 61. Davis SR et al. Milk L-lactate concentration is increased during mastitis. J Dairy Res. 2004;71(2):175–81. 62. Wockel A et al. Predictors of inflammatory breast diseases during lactation–results of a cohort study. Am J Reprod Immunol. 2010;63(1):28–37. 63. Frank MG, Watkins LR, Maier SF. Stress-induced glucocorticoids as a neuroendocrine alarm signal of danger. Brain Behav Immun. 2013;33:1–6. 64. Garate I et al. Origin and consequences of brain Toll-like receptor 4 pathway stimulation in an experimental model of depression. J Neuroinflammation. 2011;8:151. 65. McCusker RH, Kelley KW. Immune-neural connections: how the immune system’s response to infectious agents influences behavior. J Exp Biol. 2013;216(Pt 1):84–98. 66. Hines DJ et al. Prevention of LPS-induced microglia activation, cytokine production and sickness behavior with TLR4 receptor interfering peptides. PLoS One. 2013;8(3):e60388. 67. Miller AH, Maletic V, Raison CL. Inflammation and its discontents: the role of cytokines in the pathophysiology of major depression. Biol Psychiatry. 2009;65(9):732–41.
68. Bienkowski MS, Rinaman L. Noradrenergic inputs to the paraventricular hypothalamus contribute to hypothalamicpituitary-adrenal axis and central Fos activation in rats after acute systemic endotoxin exposure. Neuroscience. 2008;156(4):1093–102. 69. Ringgold KM et al. Prolonged sleep fragmentation of mice exacerbates febrile responses to lipopolysaccharide. J Neurosci Methods. 2013;219(1):104–12. 70. Sharma BS et al. Association of toll-like receptor 4 polymorphisms with somatic cell score and lactation persistency in Holstein bulls. J Dairy Sci. 2006;89(9):3626–35. 71. Wang X et al. Genetic polymorphism of TLR4 gene and correlation with mastitis in cattle. J Genet Genomics. 2007;34(5):406–12. 72. Catalani, E., et al., Short communication: Lymphoproliferative response to lipopolysaccharide and incidence of infections in periparturient dairy cows. J Dairy Sci, 2013. 73. Palm NW, Medzhitov R. Immunostimulatory activity of haptenated proteins. Proc Natl Acad Sci U S A. 2009;106(12):4782–7. 74. Fu Y et al. Curcumin attenuates inflammatory responses by suppressing TLR4-mediated NF-kappaB signaling pathway in lipopolysaccharide-induced mastitis in mice. Int Immunopharmacol. 2014;20(1):54–8. 75. Ruifeng G et al. Chlorogenic acid attenuates lipopolysaccharideinduced mice mastitis by suppressing TLR4-mediated NF-kappaB signaling pathway. Eur J Pharmacol. 2014;729:54–8. 76. Chen H et al. Alpinetin attenuates inflammatory responses by interfering toll-like receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharide-induced mastitis in mice. Int Immunopharmacol. 2013;17(1):26–32. 77. WHO. Global strategy for infant and young child feeding. Geneva: World Health Organization; 2003. 78. NHMRC. Infant Feeding Guidelines. Canberra: National Health and Medical Research Council; 2012. 79. Geddes DT, Prescott SL. Developmental origins of health and disease: the role of human milk in preventing disease in the 21 (st) century. J Hum Lact. 2013;29(2):123–7. 80. Black RE et al. Maternal and child undernutrition: global and regional exposures and health consequences. Lancet. 2008;371(9608):243–60.
