Experimental Autoimmune Breast Failure

Abstract

Mastitis is a substantial clinical problem in lactating women that may result in severe pain and abrupt termination of breastfeeding, thereby predisposing infants to long-term health risks. Many cases of mastitis involve no known infectious agent and may fundamentally be due to autoimmune-mediated inflammation of the breast. Herein, we develop a murine model of autoimmune mastitis and provide a detailed characterization of its resulting phenotype of breast failure and lactation insufficiency. To generate breast-specific autoimmunity, we immunized SWXJ mice with recombinant mouse α-lactalbumin, a lactation-dependent, breast-specific differentiation protein critical for production of lactose. Mice immunized with α-lactalbumin showed extensive T-cell–mediated inflammation in lactating normal breast parenchyma but none in nonlactating normal breast parenchyma. This targeted autoimmune attack resulted in breast failure characterized by lactation insufficiency and decreased ability to nurture offspring. Although immunization with α-lactalbumin had no effect on fertility and birth numbers, pups nursed by α-lactalbumin–immunized mice showed significantly disrupted growth often accompanied by kwashiorkor-like nutritional abnormalities, including alopecia, liver toxicity, and runting. This experimental model of autoimmune breast failure has useful applications for prophylactic breast cancer vaccination and for addressing inflammatory complications during breastfeeding. In addition, this model is suited for investigating nutritionally based “failure-to-thrive” issues, particularly regarding the long-term implications of postnatal nutritional deprivation. Mastitis is a substantial clinical problem in lactating women that may result in severe pain and abrupt termination of breastfeeding, thereby predisposing infants to long-term health risks. Many cases of mastitis involve no known infectious agent and may fundamentally be due to autoimmune-mediated inflammation of the breast. Herein, we develop a murine model of autoimmune mastitis and provide a detailed characterization of its resulting phenotype of breast failure and lactation insufficiency. To generate breast-specific autoimmunity, we immunized SWXJ mice with recombinant mouse α-lactalbumin, a lactation-dependent, breast-specific differentiation protein critical for production of lactose. Mice immunized with α-lactalbumin showed extensive T-cell–mediated inflammation in lactating normal breast parenchyma but none in nonlactating normal breast parenchyma. This targeted autoimmune attack resulted in breast failure characterized by lactation insufficiency and decreased ability to nurture offspring. Although immunization with α-lactalbumin had no effect on fertility and birth numbers, pups nursed by α-lactalbumin–immunized mice showed significantly disrupted growth often accompanied by kwashiorkor-like nutritional abnormalities, including alopecia, liver toxicity, and runting. This experimental model of autoimmune breast failure has useful applications for prophylactic breast cancer vaccination and for addressing inflammatory complications during breastfeeding. In addition, this model is suited for investigating nutritionally based “failure-to-thrive” issues, particularly regarding the long-term implications of postnatal nutritional deprivation. Mastitis is an inflammation of the breast that often causes women to stop breastfeeding. It is clinically characterized by breast engorgement, formation of noncaseating granulomatous inflammation with leukocytic infiltrates, and blocked milk ducts. If left untreated, mastitis can lead to lactation failure, recurrent inflammation, or breast abscess.1Lawrence R.A. Lawrence R.M. Medical complications of the mother.in: Lawrence R.A. Lawrence R.H. Breastfeeding: A Guide for the Medical Profession. ed 7. Elsevier Mosby, Maryland Heights, MO2011: 550-613Crossref Google Scholar Mastitis has been traditionally viewed to affect less than 3% of American women in their childbearing years.2Foxman B. D'Arcy H. Gillespie B. Bobo J.K. Schwartz K. Lactation mastitis: occurrence and medical management among 946 breastfeeding women in the United States.Am J Epidemiol. 2002; 155: 103-114Crossref PubMed Scopus (219) Google Scholar However, several studies indicate that mastitis may be much more prevalent, perhaps affecting up to 33% of lactating women.3Riordan J.M. Nichols F.H. A descriptive study of lactation mastitis in long-term breastfeeding women.J Hum Lact. 1990; 6: 53-58Crossref PubMed Scopus (83) Google Scholar, 4Jonsson S. Pulkkinen M.O. Mastitis today: incidence, prevention and treatment.Ann Chir Gynaecol Suppl. 1994; 208: 84-87PubMed Google Scholar, 5Fetherston C. Characteristics of lactation mastitis in a Western Australian cohort.Breastfeed Rev. 1997; 5: 5-11PubMed Google Scholar, 6Kinlay J.R. O'Connell D.L. Kinlay S. Incidence of mastitis in breastfeeding women during the six months after delivery: a prospective cohort study.Med J Aust. 1998; 169: 310-312PubMed Google Scholar Mastitis has been predominantly associated with Staphylococcus aureus infections.7Melish M.E. Campbell K.A. Coagulase-positive staphylococcal infections: breast abcesses.in: Feigin R.D. Cherry J.D. Textbook of Pediatric Infectious Diseases. ed 4. WB Saunders, Philadelphia1998: 1039-1066Google Scholar, 8Amir L.H. Harris H. Andriske L. An audit of mastitis in the emergency department.J Hum Lact. 1999; 15: 221-224Crossref PubMed Scopus (16) Google Scholar Several other etiopathogenic factors have also been implicated, including the use of illicit drugs, stress during labor, and trauma to the breast, all of which may cause or predispose to inflammation and related abnormalities.9Bair-Merritt M.H. Blackstone M. Feudtner C. Physical health outcomes of childhood exposure to intimate partner violence: a systematic review.Pediatrics. 2006; 117: e278-e290Crossref PubMed Scopus (206) Google Scholar Also, some cases of mastitis seem to involve no known infectious or other causative agent and, as such, suggest that breast-specific autoimmunity may be implicated. Indeed, idiopathic granulomatous mastitis is one form of this disorder that is often responsive to glucocorticoid treatment10Azlina A.F. Ariza Z. Arni T. Hisham A.N. Chronic granulomatous mastitis: diagnostic and therapeutic considerations.World J Surg. 2003; 27: 515-518Crossref PubMed Scopus (126) Google Scholar, 11Kim J. Tymms K.E. Buckingham J.M. Methotrexate in the management of granulomatous mastitis.ANZ J Surg. 2003; 73: 247-249Crossref PubMed Scopus (81) Google Scholar, 12Katz U. Molad Y. Ablin J. Ben-David D. Paran D. Gutman M. Langevitz P. Chronic idiopathic granulomatous mastitis.Ann NY Acad Sci. 2007; 1108: 603-608Crossref PubMed Scopus (24) Google Scholar, 13Patel R.A. Strickland P. Sankara I.R. Pinkston G. Many Jr, W. Rodriguez M. Idiopathic granulomatous mastitis: case reports and review of literature.J Gen Intern Med. 2010; 25: 270-273Crossref PubMed Scopus (75) Google Scholar and has been attributed to a localized immune response to an unidentified breast-specific antigen.14Brown K.L. Tang P.H. Postlactational tumoral granulomatous mastitis: a localized immune phenomenon.Am J Surg. 1979; 138: 326-329Abstract Full Text PDF PubMed Scopus (72) Google Scholar Inflamed breast tissues typically show substantial neutrophilic and lymphocytic infiltration involving T and B cells.15Milward T.M. Gough M.H. Granulomatous lesions in the breast presenting as carcinoma.Surg Gynecol Obstet. 1970; 130: 478-482PubMed Google Scholar, 16Tamiolakis D. Lambropoulou M. Koutlaki N. Manavis J. Alexiadis G. Tolparidou I. Papadopoulos N. Sivridis E. Anastasiadis P. Imprint cytology of non-specific granulomatous mastitis.Clin Exp Obstet Gynecol. 2001; 28: 176-178PubMed Google Scholar Immunohistochemical analyses of breast infiltrates often show a predominance of T cells17Carmalt H.L. Ramsey-Stewart G. Granulomatous mastitis.Med J Aust. 1981; 1: 356-359PubMed Google Scholar, 18Galea M.H. Robertson J.F. Ellis I.O. Elston C.W. Blamey R.W. Granulomatous lobular mastitis.Aust N Z J Surg. 1989; 59: 547-550Crossref PubMed Scopus (30) Google Scholar but may also show dense intralobular, perilobular, and perivascular B-cell infiltrates with lobular atrophy and sclerosis.19Schwartz I.S. Strauchen J.A. Lymphocytic mastopathy: an autoimmune disease of the breast?.Am J Clin Pathol. 1990; 93: 725-730PubMed Google Scholar However, breast inflammation in the absence of any infectious pathogens or other contributing factors distinguishes idiopathic mastitis and a putative autoimmune etiology. In the present study, we define the detailed phenotype of breast failure by creating a murine experimental autoimmune mastitis. We selected mouse α-lactalbumin as the target antigen to induce autoimmune disruption of breast function because it is a differentiation protein involved in regulation of lactose biosynthesis and because its expression is breast specific and lactation dependent.20Nagamatsu Y. Oka T. Purification and characterization of mouse α-lactalbumin and preparation of its antibody.Biochem J. 1980; 185: 227-237Crossref PubMed Scopus (31) Google Scholar, 21Vilotte J.L. Soulier S. Mercier J.C. Sequence of the murine α-lactalbumin-encoding cDNA: interspecies comparison of the coding frame and deduced pre-protein.Gene. 1992; 112: 251-255Crossref PubMed Scopus (25) Google Scholar, 22Vilotte J.L. Soulier S. Isolation and characterization of the mouse alpha-lactalbumin-encoding gene: interspecies comparison, tissue- and stage-specific expression.Gene. 1992; 119: 287-292Crossref PubMed Scopus (31) Google Scholar, 23Ren J. Stuart D.I. Acharya K.R. α-Lactalbumin possesses a distinct zinc binding site.J Biol Chem. 1993; 268: 19292-19298Abstract Full Text PDF PubMed Google Scholar We found that immunization of SWXJ mice with recombinant mouse α-lactalbumin causes extensive T-cell–mediated inflammation in lactating normal breast parenchyma and none in nonlactating normal breast parenchyma. This targeted autoimmune attack results in breast failure characterized by lactation insufficiency and decreased ability to nurture offspring. Although we observed no effect on fertility, pups nursed by α-lactalbumin–immunized mice consistently showed significant growth inhibition, often complicated by kwashiorkor-like nutritional abnormalities and runting. This experimental model has useful applications for prophylactic breast cancer vaccination, for addressing inflammatory complications during breastfeeding, and for investigating nutritionally based “failure-to-thrive” issues, particularly regarding the long-term implications of nutritional deprivation during the early stages of development. mRNA was extracted (Qiagen Inc., Valencia, CA) from lactating mouse mammary tissue taken from 8-week-old female SWXJ mice, and α-lactalbumin cDNA was generated by RT-PCR using α-lactalbumin–specific primers (Table 1). The cDNA for mouse α-lactalbumin was inserted into the pQE-82L expression vector (Qiagen Inc.) for producing a 6X-His–tagged fusion protein. SG13009 Escherichia coli (Qiagen Inc.) was transformed and screened for expression with horseradish peroxidase–conjugated His antibody (Qiagen Inc.). High-level expression colonies were selected after induction with isopropyl thiogalactopyronidase and treatment with His antibody. Plasmids from selected colonies were purified using the Plasmid Maxi Kit (Qiagen Inc.) and sequenced for verifying proper orientation and alignment. His-tagged α-lactalbumin was purified under denaturing conditions using a nickel-nitrilo triacetic acid affinity chromatography column (Qiagen Inc.). The column was washed, and multiple elution fractions were collected and electrophoresed on 15% Tris-HCl SDS-PAGE denaturing gels with Kaleidoscope prestained molecular weight markers (Bio-Rad Laboratories, Hercules, CA). The gels were blotted onto Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA), blocked with 3% bovine serum albumin, and stained using polyclonal goat anti-mouse α-lactalbumin primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and horseradish peroxidase–labeled donkey anti-goat IgG secondary antibody (Santa Cruz Biotechnology). Detection was performed using ECL chemiluminescence reagents (GE Healthcare, Buckinghamshire, UK) and HyBlot autoradiography film (Denville Scientific, Metuchen, NJ). Before immunization or use in cultures, His-tagged α-lactalbumin was further purified by reverse-phase high-performance liquid chromatography to yield endotoxin-free protein.24Dudley A. McKinstry W. Thomas D. Best J. Jenkins A. Removal of endotoxin by reverse phase HPLC abolishes anti-endothelial cell activity of bacterially expressed plasminogen kringle 5.Biotechniques. 2003; 35: 724-726PubMed Google ScholarTable 1Primer Pairs for Quantitative RT-PCR, Cloning of α-Lactalbumin, and Conventional RT-PCRProteinSequenceAmplicon length (bp)Quantitative RT-PCRIFN-γ Forward5′-TCAAGTGGCATAGATGTGGAAGAA-3′192 Reverse5′-TGGCTCTGCAGGATTTTCATG-3′IL-10 Forward5′-GGTTGCCAAGCCTTATCGGA-3′190 Reverse5′-ACCTGCTCCACTGCCTTGCT-3′α-Casein Forward5′-AAGTTTCCCCAGCACAGCAATCT-3′248 Reverse5′-CCAAAGGGGAAAGGCATCATACT-3′α-Lactalbumin Forward5′-TCAACGACAACGGCAGCACAGAGT-3′292 Reverse5′-CACAGGGGCAGGAGCAGCAAGGAC-3′GAPDH of RT-PCRIFN-γ in a SWXJ mice were generated by with to of mice were in the with of recombinant mouse α-lactalbumin in of of of and of mice were immunized with the were by the and of the and were performed in with the on and use of To the phenotype of breast failure, α-lactalbumin–immunized female SWXJ mice and female mice immunized with were with the SWXJ several the were of To the of recombinant mouse α-lactalbumin, female SWXJ mice were immunized with of and and were into cell in and in in with bovine serum of of and CA) a of α-lactalbumin was in to with of anti-mouse CA) and no of was with specific and by onto Inc., of were by expressed as of experimental with antigen by of antigen were by of of in for a of in in the of of antigen in a of antibody and recombinant were and anti-mouse and anti-mouse and anti-mouse and and anti-mouse and was using a model (Bio-Rad were as and were as in the of the using known of after immunization with α-lactalbumin or was extracted from lactating mammary tissue using the (Qiagen Inc.). cDNA was using reverse for of of murine reverse and of The was to for by for Quantitative RT-PCR was performed using for murine α-lactalbumin, and (Table 1). were in CA) in of of and of were in an and were to the using as The as as the of expression to expression for tissue Conventional RT-PCR was performed using with primers as (Table 1). the RT-PCR were performed in of using and of were electrophoresed on a and were stained with and were on a using a (Bio-Rad of serum and serum were by the using and CA). after immunization with recombinant mouse α-lactalbumin, and T were purified from by using a cell Inc., CA). of fractions were by using and were consistently found to be mammary were in Scientific, and in were in blocked with normal goat serum Laboratories, with a of anti-mouse with of goat IgG with in and by treatment with and CA). were with Scientific, in an of by and in Scientific, for under SWXJ female mice were immunized with of α-lactalbumin in or of recombinant as an after were for with of recombinant mouse α-lactalbumin, and to were into lactating female SWXJ In addition, T and T were selected from by as and into lactating female to after was from lactating mammary and was by for expression as using for murine and (Table 1). of mRNA expression and serum liver was by the growth was by of We selected α-lactalbumin as the target because it is a differentiation protein expressed and in lactating mammary but in nonlactating breast Y. Oka T. Purification and characterization of mouse α-lactalbumin and preparation of its antibody.Biochem J. 1980; 185: 227-237Crossref PubMed Scopus (31) Google Scholar, 21Vilotte J.L. Soulier S. Mercier J.C. Sequence of the murine α-lactalbumin-encoding cDNA: interspecies comparison of the coding frame and deduced pre-protein.Gene. 1992; 112: 251-255Crossref PubMed Scopus (25) Google Scholar, 22Vilotte J.L. Soulier S. Isolation and characterization of the mouse alpha-lactalbumin-encoding gene: interspecies comparison, tissue- and stage-specific expression.Gene. 1992; 119: 287-292Crossref PubMed Scopus (31) Google Scholar, 23Ren J. Stuart D.I. Acharya K.R. α-Lactalbumin possesses a distinct zinc binding site.J Biol Chem. 1993; 268: 19292-19298Abstract Full Text PDF PubMed Google Scholar α-Lactalbumin cDNA was generated by reverse of extracted from lactating mouse breast tissue and inserted into an E. coli expression vector for producing recombinant fusion with of 6X-His–tagged α-lactalbumin in E. coli was by isopropyl thiogalactopyronidase to in phase and purified by nickel-nitrilo triacetic acid affinity chromatography involving The molecular weight of α-lactalbumin was by using Kaleidoscope prestained protein and the protein was further purified use by reverse-phase high-performance liquid chromatography to yield an endotoxin-free A. McKinstry W. Thomas D. Best J. Jenkins A. Removal of endotoxin by reverse phase HPLC abolishes anti-endothelial cell activity of bacterially expressed plasminogen kringle 5.Biotechniques. 2003; 35: 724-726PubMed Google Scholar after immunization of female SWXJ mice with recombinant mouse α-lactalbumin, showed in to α-lactalbumin and were to recombinant generated in E. coli in a J.M. D. Thomas Robertson of T in with autoimmune 2006; Google Scholar to α-lactalbumin involved production with a response with production of and and production of and to α-lactalbumin involved and T as by of purified from after immunization We associated with α-lactalbumin and nonlactating SWXJ female mice were immunized with α-lactalbumin in or with and breast tissues were to after Immunohistochemical using antibody showed T in breast tissues from lactating mice immunized with α-lactalbumin but from lactating mice immunized with or from nonlactating mice immunized with α-lactalbumin or We molecular in lactating breast tissues from mice immunized with after immunization with α-lactalbumin or from lactating mammary tissue was for expression by We found that lactating breast tissue from α-lactalbumin–immunized mice showed significantly expression of but the induction of a response In addition, we found significantly expression of the differentiation α-lactalbumin and a response of the breast tissue to more milk in the of an autoimmune expression of lactation in nonlactating breast tissue from mice immunized with α-lactalbumin or expression of lactation as a for breast It is to that after immunization of nonlactating female mice with α-lactalbumin, we any in expression of a expression is to the of T α-lactalbumin autoimmune inflammation is to the lactating breast and all other normal we observed inflammation in lactating breast tissue we that α-lactalbumin–immunized mice may lactation abnormalities that affect after immunization with α-lactalbumin or were with the for the and were for after birth several for in from α-lactalbumin–immunized consistently showed a failure to as by significantly decreased several for The decreased were accompanied by decreased fertility were no significant in the of mice immunized with α-lactalbumin and immunized with The observed failure often with pups and liver with a severe nutritional often observed in during with 2007; 27: PubMed Scopus (16) Google Scholar in pups from immunized with α-lactalbumin and in pups from In addition, severe of the in α-lactalbumin–immunized mice, by the pups to This breast failure phenotype that observed in J.L. Soulier S. Mercier J.C. and of Acad A. 1994; 5: Scopus Google Scholar and in growth mice immunized with M. E. A. A. S. P. S. of mammary in mice induction of autoimmunity by Google Scholar We breast failure was by T or B To this we and from α-lactalbumin–immunized mice into and found that the growth phenotype be into lactating with but with B or we found that significantly a for autoimmune breast failure in lactating breast tissues from and but in lactating breast tissues from T or T results indicate that breast failure is by α-lactalbumin–specific or T and by B or antibody. In the present study, we and characterized a mouse model for autoimmune breast the breast-specific lactation protein α-lactalbumin causes T-cell–mediated mastitis in lactating The targeted breast-specific inflammation causes failure of the breast characterized by lactation resulting in decreased ability to nurture offspring. nursed by α-lactalbumin–immunized mice show significantly growth often accompanied by kwashiorkor-like nutritional abnormalities, including alopecia, liver toxicity, and runting. It is to that nonlactating mice immunized with α-lactalbumin were by inflammatory associated with the breast failure showed no of infiltrates in breast tissues and no or molecular of any inflammatory in breast and other Indeed, the of α-lactalbumin immunization in nonlactating mice has the for a prophylactic breast cancer P. J.M. D. An autoimmune-mediated for prophylactic breast cancer Med. 2010; PubMed Scopus Google Scholar α-lactalbumin immune the of and breast in mice inflammatory expressed or that and no expressed may for in the of a prophylactic cancer cancer vaccination by Med. PubMed Scopus Google Scholar The present results and the of immunization expressed lactation as breast cancer that lactation is after This experimental model of autoimmune breast failure may have useful applications for addressing inflammatory complications during lactation and the associated to breastfeeding. This inflammatory mastitis in a substantial of lactating women the of and often to of breastfeeding. It is that early postnatal an in the long-term health of L. G. 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A. T. S. A. P. S. S. of and in autoimmune N Acad Sci. 1998; PubMed Scopus Google Scholar expression in tissues of with M. C. Chronic inflammation in and PubMed Scopus Google Scholar and of of in tissues during inflammatory stages of autoimmune J.A. M. J.M. mRNA expression distinguishes distinct of from murine autoimmune 2001; PubMed Scopus Google Scholar This expression of differentiation to a response by the tissue to In the case of autoimmune the expression for lactation for the lactation in the autoimmune targeted breast and may have for breast this autoimmune mastitis model may as a for bovine a and disease in resulting in H. 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