Epigenetic response in mice mastitis: Role of histone H3 acetylation and microRNA(s) in the regulation of host inflammatory gene expression during Staphylococcus aureus infection
- Rahul Modak†1, 5,
- Susweta Das Mitra†2, 4,
- Madavan Vasudevan3,
- Paramanandhan Krishnamoorthy2,
- Manoj Kumar1,
- Akshay V Bhat1,
- Mani Bhuvana2, 6,
- Sankar K Ghosh4,
- Bibek R Shome2 and
- Tapas K Kundu1Email author
© Modak et al.; licensee BioMed Central Ltd. 2014
Received: 29 March 2014
Accepted: 6 June 2014
Published: 30 June 2014
There is renewed interest towards understanding the host-pathogen interaction in the light of epigenetic modifications. Although epithelial tissue is the major site for host-pathogen interactions, there is handful of studies to show how epithelial cells respond to pathogens. Bacterial infection in the mammary gland parenchyma induces local and subsequently systemic inflammation that results in a complex disease called mastitis. Globally Staphylococcus aureus is the single largest mastitis pathogen and the infection can ultimately result in either subclinical or chronic and sometimes lifelong infection.
In the present report we have addressed the differential inflammatory response in mice mammary tissue during intramammary infection and the altered epigenetic context induced by two closely related strains of S. aureus, isolated from field samples. Immunohistochemical and immunoblotting analysis showed strain specific hyperacetylation at histone H3K9 and H3K14 residues. Global gene expression analysis in S. aureus infected mice mammary tissue revealed a selective set of upregulated genes that significantly correlated with the promoter specific, histone H3K14 acetylation. Furthermore, we have identified several differentially expressed known miRNAs and 3 novel miRNAs in S. aureus infected mice mammary tissue by small RNA sequencing. By employing these gene expression data, an attempt has been made to delineate the gene regulatory networks in the strain specific inflammatory response. Apparently, one of the isolates of S. aureus activated the NF-κB signaling leading to drastic inflammatory response and induction of immune surveillance, which could possibly lead to rapid clearance of the pathogen. The other strain repressed most of the inflammatory response, which might help in its sustenance in the host tissue.
Taken together, our studies shed substantial lights to understand the mechanisms of strain specific differential inflammatory response to S. aureus infection during mastitis. In a broader perspective this study also paves the way to understand how certain bacteria can evade the immune surveillance and cause sustained infection while others are rapidly cleared from the host body.
KeywordsEpigenetic modifications Histone Acetylation Mastitis S. aureus infection Micro-RNA Gene expression Inflammatory response Chromatin immunoprecipitation
Mastitis, characterized by inflammation of the mammary gland, is a major disease affecting dairy cattle worldwide. The severity of the inflammation is dependent on the causative agent and the host response to it. Although viruses, fungi, or protozoa can cause mastitis, the most common causes are Gram positive and negative bacteria. These bacterial infections can result in a spectrum of clinical outcomes ranging from acute and life-threatening to chronic and subclinical mastitis. Staphylococcus aureus and Escherichia coli are the two largest single pathogens isolated from milk. S. aureus induced mastitis is characterized by a more moderate and delayed Somatic Cell Count (SCC) increase and infection can ultimately result in subclinical, chronic, and sometimes lifelong infection. Outcome of infection can be influenced by the infecting strain. S. aureus potentially produces a variety of virulence factors that contribute to persistence and pathogenicity of the organism. Lipotechoic acid (LTA) is the major immune stimulatory molecule, identified in Gram positive organisms recognized by TLR-2. Activation of the pattern recognition receptors (PRR) initiates signaling cascades and finally leads to activation of NF-κB and MAP kinase pathways that culminate in the transcription of a wide range of immune genes including cytokines which are synthesized by infiltrating cells as well as resident cells in response to S. aureus infection[1, 5]. We have selected two predominant evolutionarily related but not identical clones of S. aureus from fresh milk samples collected from subclinical cases of mastitis cows to compare the host immune response during mastitis.
Bovine mammary epithelial cells (MEC) is a good model system to study host immune response during bacteria-induced mastitis, but recent reports have shown that it poorly mimics whole animal data. Inbred mice strains, like BALB/c, have been extensively used to study mastitis. Our previous study had shown that E. coli infection induced severe mastitis in mice, which inspired us to use the same model system to study S. aureus induced mastitis. The alteration of global mRNA profile during S. aureus induced mastitis in bovine and goat has been reported earlier[9, 10]. Recently Lawless et al. reported alteration of microRNA (miRNA) profile in MECs upon Streptococcus uberis infection. However the alteration of miRNA upon S. aureus infection in the host mammary tissue has not been studied. Vanselow et al. reported DNA remethylation at the alphaS1-casein promoter during E. coli infection induced bovine mastitis. Wang et al. also showed aberrant DNA methylation at the CD4 gene promoter in the peripheral blood cells of the mastitis cow.
In the present study we have developed a mice model for S. aureus induced mastitis. S. aureus infection induced hyperacetylation at histone H3K9 and H3K14 residues in mice mammary tissue. Hyperacetylated histones were selectively enriched at the promoters of overexpressed proinflammatory genes. S. aureus infection also altered the microRNA profile of the host tissue. We have used two S. aureus isolates to compare the strain specific response of mice mammary tissue. Remarkably, it was observed that the two strains of S. aureus induced differential expression of inflammatory genes. The strain relative/specific alteration in gene expression in mice tissue seems to be linked to the histone acetylation pattern. The RNA sequence analysis of the infected tissue revealed that small microRNAs expression also changed upon infection, suggesting a complex network of coding and non-coding RNA expression (in the context specific epigenetic state) in the process of pathogen clearance or sustained infection.
Staphylococcus aureus is the major mastitis pathogen isolated from milk samples
Distribution of identified Staphylococcus spp. from bovine milk samples
Total species identified
Total no. submitted to gene bank
Gene bank accession no.
HM451958-1991; HM451994-1999; HM452101-2103
HM452000; HM452100; HM462053
Mice mammary tissue differentially responds to S. aureus infection
The SA2 inoculated mice showed no observable histological changes after 2, 4, and 8 h of intramammary inoculation. At 12 h, mild infiltration of alveolar macrophages in the interstitial space with vacuolar degeneration of alveolar epithelial cells was observed. After 24 h the alveolar lumen showed moderate infiltration of neutrophils and mononuclear cells (Figure 2A-III, blue arrow) with necrosis of alveolar epithelial cells were observed. The adjacent alveoli coalesce to form large alveoli and thinning of alveolar epithelial cells was also observed (Figure 2A-III, black arrow). After 48 h of inoculation, severe infiltration of mononuclear cells, occupying the entire alveolar lumen and interalveolar space along with necrosis of the alveolar epithelial cells was observed. The loss of alveolar architecture was seen with few areas showing intact alveoli and few fat cells were observed. Based on the histopathological changes observed in the mammary glands, the SA1 caused more severe histological changes when compared to SA2 infected mice (Additional file1: Figure S1A and Additional file2: Figure S1B).
Gram-positive bacterium like S. aureus stimulates innate immune response through a wide variety of bacterial components like peptidoglycan, lipoteichoic acid, lipopeptides, and bacterial DNA. These pathogen associated molecular patterns (PAMPs) bind to TLR2 and other PAMP receptors and activate MyD88 complex mediated signalling cascades like classical NF-κB, MAPK, phosphoinositide 3-kinase (PI3K)/AKT pathways. Activation of TLR2-MyD88 cascade leads secretion of several pro-inflammatory mediators like interleukins, interferon (IFN)γ, TNFα, and cytokines that are essential for the recruitment and survival of polymorphonuclear neutrophils (PMN). Quantitative RT-PCR (qRT-PCR) analysis of a set of pro-inflammatory genes had shown gradual increase in their expression from 4 h to 24 h post infection by both SA1 and SA2 (Figure 2B,C). Interestingly, at 48 h post infection most of these genes showed significant decrease in expression level that is essential for sustained S. aureus infection. We also observed markedly higher expression of pro-inflammatory genes in SA1 infected samples in comparison to SA2 infected tissues. This further highlights the differential host tissue response to different pathogen strains.
S. aureus infection induces specific histone H3 hyperacetylation in alveolar epithelial cells
S. aureus infection induces differential expression of coding and non-coding (miRNA) transcripts
As RNA Polymerase-II-driven transcription also synthesize small non-coding RNAs, including microRNAs (miR), we further selected the small RNAs from the total RNA isolated from the PBS, SA1 and SA2 (one each) inoculated mice mammary tissue. cDNA library was prepared for these samples and then sequenced through next generation sequencing (NGS). After performing the initial quality check the small RNAseq data were analyzed through a workflow (Additional file6: Figure S4) that included mapping sequences to different small RNA annotation, identifying differentially expressed microRNAs and determining the mRNA targets.
In order to understand the functions of these DE miRNAs we performed correlation analysis with mRNA expression data. We used three different algorithms, Targetscan, miRANDA, and PITA[18–20], to predict microRNAs complementary to 3’-untranslated regions (3’UTRs) of the DE mRNA in our data. Then we compared these predicted miRNAs to our DE microRNAs and identified annotated and novel DE miRNAs. We have identified 6 and 23 of anti-correlated DE miRNAs and mRNAs in SA1 and SA2 infected tissues, respectively, through negative correlation analysis. Interestingly many of the DE mRNA and miRNA did not show any correlation, which indicates involvement of other regulatory mechanisms of gene expression. As expected there was large variation in the number of predicted interactions of microRNAs and mRNAs (Additional file8: Table S2).
Histone H3K14 acetylation is selectively enriched at the overexpressed gene promoters
Mastitis is a complex disease-involving multitude of host and pathogen factors. The outcome of bacterial infection ranges from unnoticeable subclinical mastitis to life-threating severe infection. The growth and pathogenesis of any bacterium is largely influenced by the host immune system but the detailed understanding of pathogenesis in mastitis is sketchy. Our previous studies have shown that staphylococci are the largest pathogen group associated with mastitis, which is as per with the global scenario. We have observed that S. aureus is the largest single mastitis pathogen isolated from fresh cow milk samples (Figure 1A and Mitra et al.). S. aureus infection induces less virulent subclinical mastitis that persists for a long time. Recently it has been shown that E. coli and S. aureus induced mastitis elicit very different inflammatory responses, both in mammary tissue and in MECs[7, 36, 37].
In the present study we have successfully established S. aureus induced mastitis in mice. Our data clearly demonstrate that two closely related strains of S. aureus induced very different inflammatory responses in mice mammary tissue (Figure 2A). At 24 h post infection, SA1 infected tissue showed drastic induction in inflammatory genes expression (Figure 2B), which is associated with infiltration of neutrophils and alveolar macrophages. SA2 infection induced significantly lower inflammatory response in the mice mammary tissue (Figure 2C) and there were fewer numbers of macrophages. Interestingly, in both cases the expression of inflammatory genes drastically decreased at 48 h post infection that is contrary to our previous observations in the case of E. coli induced mastitis. This is not surprising, given the fact that S. aureus infection often leads to asymptomatic but sustained mastitis whereas E. coli infection often gets cleared rapidly. This observation encouraged us to investigate the regulatory mechanisms of gene expression during S. aureus induced mastitis.
Forty-three genes were differentially expressed in the SA1 (fold change ≥2, FDR ≤0.05) (Figure 5B) infected tissue and 151 genes were dysregulated in SA2 infected tissue (Figure 5C). Similar numbers of genes were differentially expressed during S. aureus induced mastitis in goat and cow[9, 36], but their expression levels were different. The number of differentially expressed genes in S. aureus induced mastitis in mice are significantly lower than E. coli infection, which had been reported in bovine mastitis as well. Detailed analysis of gene expression data showed differential response between SA1 and SA2 infection (Figure 2B,C, Figure 5). Downregulation of tubulin expression indicated induction of tissue damage that was supported by histopathological analysis (Figure 2A). All these data clearly indicated that S. aureus infection induced various signaling pathways in mice mastitis. We have decided to study the role of epigenetic mechanisms in the regulation of gene expression during S. aureus induced mastitis in mice.
Alteration of miRNA expression during bacterial infection has been well documented. Lawless et al. reported differential expression of a set of microRNAs in bovine MEC upon Streptococcus uberis infection. Alteration of small RNA in S. aureus and its role in gene expression has been extensively studied[38, 39]. There are few reports to show the alteration of host microRNA profile by S. aureus cell wall components[2, 40]. Recently Jensen et al. had shown that S. aureus infection alters the gene expression profile of the uninfected quarter in bovine mastitis which underscores the necessity for the whole animal- live pathogen model to study host-pathogen interactions. To our knowledge there is no study to show the effect of live S. aureus infection on the host small RNA profile.
SA1 infection led to a two-fold increase in the total number of expressed microRNAs (Figure 6A), whereas SA2 did not significantly alter the number of microRNAs. There was no notable difference in the size distribution of miRNAs in all three samples indicating absence of size bias upon treatment (Figure 6B). DE analysis showed SA1 infection both induced as well as repressed expression of several microRNAs, whereas SA2 infection mostly led to repression of microRNA expression (Figure 6C). For detailed analysis of gene expression and miRNA expression data please refer to Additional file9.
Immuno-histochemical analysis (Figure 3) coupled with western blotting (Figure 4) showed significant hyperacetylation at histone H3K9 and H3K14 residues in the SA1 infected tissue, whereas there was hardly any difference between SA2 and PBS inoculated tissues. Unlike E. coli infection, there was no change in histone H4 acetylation and H3K36 trimethylation levels during the S. aureus induced mastitis. We did not observe any change in the H3S10 phosphorylation in the S. aureus infected tissue, which is contrary to previous reports during other Gram-positive bacterial infection. Taken together these data indicate that there are few epigenetic modifications (like H3K14 acetylation) that are commonly altered during many bacterial infections. It also strongly argues for the fact that there is pathogen specific alteration of other epigenetic modifications, which clearly highlights the complexity of the epigenetic regulation associated with the tissue response during bacterial infection. Selective enrichment of histone H3K14 acetylation at the overexpressed gene promoter (Figure 9) clearly shows involvement of histone modifications in the process. We have not detected significant enrichment of H3K14ac in many other promoters that could be due to overall low levels of histone hyperacetylation in the infected tissue. Although western blot and IHC analysis showed H3K9 hyperacetylation in SA2 infected tissue, we did not see any promoter specific enrichment of H3K9 acetylation. These data clearly shows that S. aureus infection induced both global as well as promoter specific H3K14 hyperacetylation, whereas H3K9 acetylation levels had been altered globally but may not be in a promoter specific manner. It is also possible that poor efficacy of the H3K9ac antibody in formaldehyde-fixed paraffin embedded (FFPE) ChIP applications interfered in detection of promoter specific enrichment. Our data argue for the fact that hyperacetylation of H3K14 residue is necessary but not sufficient for global transcription activation. A combination of several histone modifications (Figure 3 and Modak et al.) writes the epigenetic language of global transcription activation during bacterial infection.
In summary, we have demonstrated that S. aureus infection induces specific histone posttranslational modifications during mice mastitis. It also induces differential expression of a set of microRNAs during mice mastitis. Our data clearly show the strain specific alteration of epigenetic modulators, namely histone acetylation and microRNA expression, that results in differential expression of pro- and anti-inflammatory genes. This kind of strain specific alterations in host gene expression will subject some pathogens for rapid clearance whereas others can sustain for longer duration inside the host. This probably could partially explain why S. aureus vaccine has not been successful so far. Although the mechanism of differential inflammatory response is not very evident from this study, yet we speculate the involvement of activated p300 and/or PCAF/GCN5 in the entire process. This study also shows the role of host microRNAs in host-pathogen interaction and disease manifestation. In future these epigenetic modifications can emerge as potential therapeutic target for mastitis and/or other bacterial diseases.
Mastitis is one of the major bacterial infection induced pathogenic condition that affects all mammals including human. Globally bovine mastitis is the biggest challenge to the dairy industry that causes a financial burden of several million dollars. E. coli and S. aureus are the two major mastitis pathogens. In the present study we show that S. aureus infection altered host gene expression in a strain dependent as well as temporal manner. The change in host gene expression is regulated via alteration in a specific set of histone posttranslational modifications and microRNA expression. All these data indicate that S. aureus infection creates an environment favorable for sustenance of pathogen in the host tissue in a strain specific manner. Collectively these data show how bacterial infection alters host epigenetic landscape and provide new insights into epigenetic regulation of bacterial strain specific host inflammatory response.
Virulence gene profiling of S. aureus isolates
The genus and species-specific PCR conditions has been reported earlier. All the S. aureus isolates were screened for the presence of eight major virulence factors-fnb A, fnb B, clf A, cna, pvl, tst, nuc, and coa, in their genome. The detailed PCR amplification condition has been reported earlier.
PFGE of S. aureus isolates
Two S. aureus isolates,SA1-HF-14Y/t6877 and SA2-HF-16LY/t-267-each harboring seven virulent genes, were selected for further characterization by pulse field gel electrophoresis (PFGE) following the protocol published by Mitra et al..
The animal experiments have been approved by Institutional Animal Ethics Committee (IAEC) of PD_ADMAS where the animal experiments were carried out bearing Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) registration no. 881/03/ac/05/CPCSEA. The animal experiments protocol was approved during the IAEC meeting held on 22 October 2011 by the committee members. The animal experiments were carried out as per the guidelines of CPCSEA, Government of India, New Delhi.
Establishment of mastitis in Swiss albino mice
Timed pregnant Swiss albino mice procured from National Centre for Laboratory Animal Science (NCLAS), National Institute of Nutrition, Hyderabad, India were acclimatized under controlled conditions in individually ventilated cages (IVC).
Inoculum dose: The most predominant clones of S. aureus field isolates (SA1 and SA2) were selected based on characterization profile to challenge the mice. TheS. aureus was grown in brain heart infusion (BHI) broth overnight and centrifuged at 3,000 × g for 5 min at room temperature to pellet the cells. S. aureus pellet was washed with sterile cell culture tested PBS (HiMedia) twice to remove all the media components and finally resuspended in PBS. Standard plate count (SPC) method was employed to adjust the bacterial load at a cell density of 5 × 103 cfu/ 50 μL for intramammary inoculation.
Inoculation procedure:S. aureus challenge experiments were performed following the protocol described by Chandler[42–44] with modifications. Briefly, 7 days postpartum mice were anesthetized with intraperitoneal injection of 2 mg Ketamine per mouse. Mice teats were cleansed with 70% ethanol. A total of 50 μL of the bacterial culture were carefully inoculated with a sterile syringe fitted with 30G needle in two abdominal pairs of mammary glands (left fourth (L4), right fourth (R4), left fifth (L5), and right fifth (R5)) in 36 mice. Twelve mice were simultaneously inoculated with PBS as control. Mice recovered from anesthesia were left in the cage with pups to suckle 1 h post inoculation. The animals were examined at regular intervals for generalized and local reaction that include redness of the teat, swelling of mammary gland, and appearance of a sunken abdomen.
Tissue collection: Mice were sacrificed with overdose of Ketamine and laid on their back; the abdominal skin was opened by mid ventral incision. The mammary glands were carefully dissected out using sterile scissor and forceps at 2, 4, 8, 12, 24, and 48 h post inoculation. Gross examination of mammary gland tissues was carried out and was collected in protease inhibitor cocktail (Sigma), RNA- later® solution (Ambion), and 10% formalin (Merck). The samples collected in RNA-later were incubated at 4°C overnight. RNA stabilized tissues were flash frozen in liquid nitrogen after discarding the RNA-later and stored at -80°C. The formalin fixed tissues were processed for histopathology, stained with hematoxylin and eosin and were observed under light microscope for recording histological changes. The same tissues were used for immunohistochemical analysis following the protocol published by Modak et al.. Tissues collected in protease inhibitor were incubated on ice for 1 h and then flash frozen before storing at -80°C till further processing. For western blot analysis, tissue lysates were prepared following the previously published protocol.
RNA isolation, microarray, and small RNA sequencing
Total RNA was isolated from mouse mammary tissues using TRIzol® Reagent (Invitrogen) as per manufacturer’s instructions. RNA preparations were stored at -80°C till further use. The RNA samples were further processed for genes expression analysis by microarray and small RNA analysis by deep sequencing. For detailed procedure please refer to the Additional file9.
Computational analysis of sequencing and microarray data
Small RNA sequencing
Small 35 nt RNA reads were produced using an IlluminaGAIIx Genome Analyzer. The detailed methodologies for the microarray and small RNA data processing and pathway analysis have been included in Additional file9.
Integration of microarray and small RNA sequencing data
miRNA: mRNA integrome was performed by using differentially expressed miRNA and its target genes. Further, miRScape plugin (http://ferrolab.dmi.unict.it/mirscape.html) for Cytoscape v 8.0 was used to visualize the enriched miRNA: mRNA integrome to understand the key regulatory circuits.
Real time qRT-PCR analysis
qRT-PCR to study the temporal expression of inflammatory genes was performed using gene specific probes (Roche) (Additional file7: Table S1) using Roche Lightcycler® 480 real-time PCR system. Total mRNA present in the samples was converted to cDNA by First Strand cDNA Synthesis Kit (Thermo Scientific) using oligo-dT primer. Primer3 software was used to design gene specific primers for qRT-PCR (Additional file8: Table S2). Total RNA isolated from mice mammary tissue were converted to cDNA by NCode™ VILO™ miRNA cDNA Synthesis Kit (Invitrogen). For qRT-PCR, miRNA specific forward primers (Additional file10: Table S3) were designed as per manufacturer’s protocol and universal qPCR supplied with the kit was used as reverse primer. SensiFAST SYBR No-ROX Kit (Bioline) was used for qRT-PCR.
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed with the FFPE mice mammary tissue samples using H3K9ac, H3K14ac, and H4K8ac antibodies. The detailed ChIP- qRT-PCR protocol has been reported earlier. Primers to amplify the promoters of the selective genes have been listed in Additional file9: Table S4. NCBI GEO accession no.: GSE54230, GSE54231, GSE54232.
Formalin Fixed Paraffin Embedded
- qRT PCR:
Quantitative Real Time Polymerase chain reaction
Sodium dodecyl sulphate polyacrylamide gel electrophoresis.
The research was supported by grants from National Agricultural Innovative Project (NAIP), Indian Council of Agricultural Research, New Delhi, Govt. of India under component 4: Basic and Strategic Research vide NAIP/Comp-4/C-30017/2008-09Dt. 23.12.2008 to BRS (PD_ADMAS) and TKK (JNCASR). TKK also acknowledges the Department of Biotechnology, Govt. of India and JNCASR for partial financial support. TKK is a Sir J.C. Bose National Fellow, Department of Science and Technology, Govt. of India.
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