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*Corresponding author’s e-mail: drgeetapatho@gmail.com1Animal Disease Research Centre, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.2Department of Veterinary Pathology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.

Indian J. Anim. Res., 53(11) 2019: 1440-1444Print ISSN:0367-6722 / Online ISSN:0976-0555

Immunohistochemical detection of alpha-smooth muscle actin and S-100 inbovine mammary gland with mastitisGeeta Devi Leishangthem*1, Nittin Dev Singh2 and Amarjit Singh2

Animal Disease Research Centre,Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.Received: 28-6-2018 Accepted: 20-02-2019 DOI: 10.18805/ijar.B-3674

ABSTRACTFibrosis is a common finding in chronic mastitis. Epithelial-mesenchymal transition (EMT) plays a prominent role infibrogenesis in adult tissues. The aim of this study was to investigate whether EMT occurs in bovine mastitis by assessingthe expression or localization of alpha-smooth muscle actin (alpha-SMA), S-100 protein and caspase-3. Two normalbovine mammary gland tissue samples and five mastitic samples were used in the study. Antibodies to alpha-SMA, S-100and caspase-3 were used for immunohistochemical studies. In mastitis there was increased expression of S-100 in thealveolar epithelial cells. Alpha-SMA was highly expressed in the areas where there is fibrosis in chronic mastitis. Furthercaspase-3 was observed in alveolar cells in mastitic tissue. Overexpression of S-100 and alpha-SMA directly correlates tothe severity of mastitis.S-100 and alpha-SMA may serve as biomarkers to know the extent of tissue structural changes inmastitis. EMT could be one of the mechanistic pathways underlying the pathogenesis of fibrosis in mastitis.

Key words: Alpha-smooth muscle actin, Bovine mastitis, Caspase, Epithelial to mesenchymal transition, S-100.

INTRODUCTIONBovine mastitis is the inflammation of mammary

gland caused mainly by bacterial infections and is associatedwith irreversible mammary tissue damage which later mightpermanently lead to fibrosis (Sharma and Jeong, 2013) andthus the affected quarter are ultimately left nonfunctional orblind quarter.

Bovine mastitis may be broadly classified as acuteand chronic mastitis. Gram negative coliform bacteria mostfrequently cause acute inflammation and eventually severemastitis (Burvenich et al., 2003). On other hand, chronicmastitis characterized by presence of fibrous connectivetissue proliferation and infiltrating mononuclearinflammatory cells is caused by gram positive bacteria(Hensen et al., 2000). This fibrosis may be due toaccumulation of fibroblast. Fibroblast may be originatedfrom various cells one such is epithelial cells throughepithelial-mesenchymal transition (EMT) (Kalluri andNeilson, 2003).

EMT is a phenomenon where a polarized epithelialcell undergoes multiple biochemical changes that enable itto transdifferentiate into a mesenchymal cell phenotype orfibroblast-like cells, which includes enhanced migratorycapacity, invasiveness, elevated resistance to apoptosis, andgreatly increased production of extra-cellular matrix (ECM)components (Schneider et al., 2008). EMT is a normalprocess during implantation, embryogenesis, and organdevelopment (Type I EMT), EMT associated with tissue

regeneration and organ fibrosis (Type 2 EMT) and EMTassociated with cancer progression and metastasis (Type 3EMT) (Kalluri and Weinberg, 2009).

Alpha smooth muscle actin (alpha-SMA), acontractile protein and actin isoform,are expressed mainlyin smooth-muscle cells of blood vessels and plays animportant role in fibrogenesis (Kawasaki et al., 2008). S-100proteins are EF-hand Ca2+-binding proteins which areessential for various array of both intracellular andextracellular regulatory functions (Donato et al., 2013).S-100 proteins have strong antimicrobial properties whichare an essential part of the immune response to invadingpathogens (Donato et. al., 2013). Caspase-3 is a member ofthe endoproteases family important for maintaining cellregulatory networks controlling cell death and inflammation(Porter and Jänicke, 1996).

The aim of this study was to investigate throughimmunohistochemistry the expression and localization ofalpha-SMA, S-100 and caspase-3 in bovine mammary glandtissue of both normal and mastitic animals for assessment ofinvolvement of epithelial to mesenchymal transition inpathogenesis of bovine mastitis.MATERIALS AND METHODS

Mammary gland samples (2 normal and 5 mastitis)were collected from animals (cattle) brought for postmortemin Department of Veterinary Pathology, GADVASU, Punjab.Animal showing changes in mammary gland were includedin the study. Detailed necropsy was done, gross lesions

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Fig 1: Photomicrograph of normal mammary gland showing normal aveolar areas. (H&E, bar=100µm).

recorded, and accordingly, mammary gland tissues werecollected and fixed in 10% buffered formalin and processedfor routine histopathology and special stain like picro-siriusred stain were employed.

For picro-sirius red staining, the slides were stainedwith picro-sirius red stain (0.1% Sirius red in aqueoussaturated picric acid) for 1 hour, washed in two changes ofacidified water (0.5% glacial acetic acid), dehydrated andmounted with DPX. Collagen was red in colour while non-collagen components were orange.

For immunohistochemistry, 4-μm sections offormalin fixed tissue were taken on poly-L-lysine coatedslides. After heat antigen retrieval in citrate buffer (pH 6)using microwave followed by non-specific blocking, theslides were incubated with primary antibodies to mousemonoclonal alpha-smooth muscle actin (1:500, Abcam, UK),to rabbit polyclonal S-100 (1:500, Dako, Denmark) andcaspase-3 (1:250, Abcam, UK) at 4C overnight. This isfollowed by blocking of endogenous peroxidase using 3%H2O2 solution. Washing with PBS was followed by incubationwith secondary antibody (ABC, Universal, Vector). Colourdevelopment was done using diaminobenzidine substrate(DAB) and counterstained with hematoxylin. In negativecontrol, tissue section was processed without application ofprimary antibody. All slides were examined underOlympus™ BX51 microscope (Olympus Life Science,Hamburg Germany) attached with camera.

The images were analyzed using Image J (Fiji)software (http://fiji.sc). The intensity of the S-100 IHCpositive reaction was expressed as mean OD while alpha-SMA IHC and picro-sirius red was expressed as percentagearea (µm2). The data are represented as Mean±SE. Statisticalanalysis was performed using the SPSS software. Thedifferences in the mean OD between normal and mastitisanimals were compared using student’s t test.RESULTS AND DISCUSSION

In the present study, out of five cases of mastitis,one showed features of acute mastitis while four showedchronic mastitis. The normal mammary tissue was soft anduniform in texture whereas the mastitic mammary tissue washard and thickened in consistency. In this study only fivemastitic cows in which Staphylococcus aureus was the solepathogen isolated were used. Based on histopathology, thenormal mammary tissue showed normal alveolar luminalareas with no inflammatory cells infiltration (Fig 1). In acutemastitis (Fig 2), there were infiltrations of inflammatory cellswithin the alveolar parenchyma. There was alsodesquamation of epithelium into the lumen of acini. Inchronic mastitis (Fig 3), there was deposition of ECMproteins leading to fibrosis and infiltration with mononuclearcells. The alveolar epithelial cells were replaced by fibroustissues in various areas. Picrosirius red staining is a

Fig 4: Photomicrograph of mastitis showing red stained collagen within the tissue parenchyma (Picrosirius red, bar=100 µm).

Fig 3: Photomicrograph of chronic mastitis showing fibrous connective tissue along with infiltration of lymphocytes, macrophages and plasma cells. (H&E, bar=100 µm).

Fig 2: Photomicrograph of acute mastitis showing infiltration of inflammatory cells mainly neutrophils, macrophages within the interstitial connective tissue and hyperemia of interstitial blood vessels. (H&E, bar=100 µm).

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Fig 8: Photomicrograph of chronic mastitis showing brown stained S-100 in the alveolar epithelial cells located within the fibrosed tissue parenchyma. (IHC, bar=100 µm).

commonly used histological technique to visualize thedistribution of collagen in healthy and fibrotic tissue sectionsby bright-field or polarization microscopy (Whittaker et al.,1994). In the present study, percentage areas of picro-siriusred colored stained collagen were observed more in the areasof fibrosis (µm2) in mastitis (27.80±0.51) (Fig 4) as comparedto that of normal (11.23±1.11).

Fibrosis is the replacement of normal tissue bythe formation of excess fibrous connective tissue (ECMproteins e.g. collagen) in an organ or tissue in a reparativeor reactive process. It can also be a complex pathologicalresponse, common to many tissues in response to injury(Kisseleva and Brenner, 2008). Advanced stages of fibrosisresult in organ dysfunction and eventually organ failure(Guarino et al., 2009). Kalluri and Weinberger (2009) statedthat type 2 EMT is associated with tissue damage,inflammation and fibrosis. EMT plays a prominent role infibrogenesis in adult tissues such as the kidney, lungs andliver (Schneider et al., 2008). Okada et al.,(1997) have shownthat the epithelial cells migrate from the epithelial layer, travelthrough the basement membrane and accumulate in theinterstitium of the tissue where they loss the epithelialmarkers and gain a fully fibroblastic phenotype.

Further, by IHC, in normal mammary gland, alpha-SMA was observed in the myoepithelial cells of the alveoliand vascular smooth muscle cells (SMCs) (Fig 5) whereasin mastitis tissue, along with myoepithelial cells and vascularsmooth muscle, fibroblastic cells in areas of fibrosis withinthe interstitial space also expressed alpha-SMA (Fig 6). Thepercentage area of alpha-SMA expression (µm2) was higherin mastitis (14.87 ±1.19) than that of normal (8.94±0.54).S-100 was easily detectable in the alveolar epithelium.Higher concentration of S-100 was detected in the mammaryalveolar epithelium as well as myoepithelial cells of mastitisanimals (Fig 7, 8). S-100 staining intensity was measured asmean optical density and showed that mastitic animals havehigher intensity (1.20±0.03) than that of normal (0.69± 0.03).Caspase-3 was observed in the nucleus of the alveolarepithelial cells in acute mastitis tissue (Fig 9).

Fig 5: Photomicrograph of normal mammary gland showing brown stained alpha-SMA in the myoepithelial cells and blood vessels. (IHC, bar=100 µm).

Fig 6: Photomicrograph of chronic mastitis showing brown stained alpha-SMA in the areas of fibrosis along with myoepithelial cells and blood vessels. (IHC, bar=100 µm).

Fig 7: Photomicrograph of normal mammary gland showing brown stained S-100 in the alveolar epithelial cells. (IHC, bar=100 µm).

Fig 9: Photomicrograph of mammary gland showing brown stained caspase-3 in the alveolar epithelial cells. (IHC, bar=100 µm).

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Hellmén and Isaksson (1997) and Alkafafy et al(2012) showed strong immunostaining for alpha-SMA in thevascular SMCs and myoepithelial cells. Functionally,myoepithelial cells exhibit phenotypes of smooth muscle andepithelial cells. Chen et al (2016) demonstrated that alpha-SMA exhibited increased protein expression in inflammation-associated stromal fibroblasts compared with normalfibroblast (Chen et al., 2016). Alpha-SMA is expressedmainly in vascular SMCs and plays an important role infibrogenesis (Kawasaki et al., 2008).

In tissue injury, the local connective tissuefibroblasts are recruited, activated and becomemyofibroblasts which show expression of alpha-SMA(Sandbo and Dulin, 2011; Higashiyama et al., 2011).Myofibroblasts has a phenotype between a fibroblast and asmooth muscle cell and is regarded as a main players fororchestrating physiological tissue injury and in generatingthe pathological tissue fibrosis (Gabbiani, 2003; Desmoulièreet al., 2005). Further in normal physiological wound healing,myofibroblasts disappear via apoptosis whereas in manypathological fibrotic conditions, myofibroblasts fail toundergo apoptosis, and persist thereby leading to furtherpathology (Darby et al., 2014).

The appearance of alpha-SMA in the trans-differentiating cells during EMT is a hallmark of myofibroblast formation. Alpha-SMA expression is an excellentmarker of EMT and myofibroblasts. Alpha-SMA positivemyofibroblasts have been demonstrated in type 2 EMT(Zeisberg et al., 2007). In the present study, besides bloodvessels and myoepithelial cells, the cells positive foralpha-SMA were myofibroblasts. Their presence inmastitis may be suggestive of their role in fibrosis throughtype 2 EMT.

S-100 proteins are EF-hand Ca2-binding proteinsinvolved in a diverse array of both intracellular andextracellular regulatory functions (Donato et al., 2013).During infection, extracellular S-100 proteins can act asDAMP proteins and initiate a pro-inflammatory immuneresponse through interaction with pattern recognitionreceptors RAGE and TLR4 (Zackular et al., 2005). This leadsto inflammatory reaction through NF-B-mediatedinflammatory cascade thereby leading to increased S-100proteins expression and initiate a positive feedback loop(Zackular et al., 2005). Janjanam et al. (2014) stated that S-

100 proteins may be playing a major role in decreasing milkproduction in low producing animals. And this S-100 proteinbinds to calcium inside mammary epithelial cells therebymaking calcium unavailable for secretion activity resultingin decreased milk yield (Janjanam et al., 2014). Moreover,there is also correlation of a calcium binding protein S-100A4(fibroblast specific protein) with EMT (Okada et al., 1997).

In the present study, S-100 was highly expressed inthe mammary epithelial cells lining the alveoli as well as inalveolar epithelial cells within the areas of fibrosis indicatingthat there was loss of the epithelial cells and replaced byfibrous tissue. Alkafafy et al (2012) showed immunostainingof S-100 in the luminal epithelial cells in alveoli and ducts.Lutzow et al (2008) showed marked upregulation ofS100A12 in bovine mammary epithelial cells stimulated withlipopolysaccharides and lipoteichoic acid.

During EMT, epithelial cells detach from the ECMand trigger the apoptotic process (Wu and Zhou, 2008).Caspase-3 is required for typical hallmarks of apoptosis, andis essential for apoptotic chromatin condensation and DNAfragmentation in all cell types (Porter and Janicke, 1999).The present study showed the presence of caspase-3 positiveepithelial cells lining the alveoli indicating apoptosis of thealveolar epithelial cells. Hu et al (2014) stated that S. aureus-induced apoptosis in primary bovine mammary epithelialcells was associated with activation of caspase-3 and caspase-8. Chronic S. aureus intra-mammary infection significantlyincreased immuno-expression of active caspase-3 inmammary tissue (Andreotti et al., 2017). Moreover, up-regulation of S-100 proteins caused apoptosis and decreasesthe ability to maintain the optimum mammary epithelial cellnumbers (Janjanam et al., 2014).

Thus, the present study provides information on thepossible role played by EMT in the pathogenesis of mastitis.Further studies are required with more samples and alsoemploy other EMT markers to understand the EMTmechanism. These markers could be used as a target inpreventing EMT in mastitis through therapeutic strategy.ACKNOWLEDGEMENT

The first author acknowledged Department ofScience and Technology, Government of India for providingthe necessary financial support to carry out this work throughWomen Scientist Scheme, DST-WOS-A vide Ref. No. SR/WOS - A/LS-1026/2015.

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