The use of biological scaffold materials for wound healing and tissue remodeling has profoundly impacted regenerative medicine and tissue engineering. The porcine-derived small intestinal submucosa (SIS) is a licensed bioscaffold material regularly used in wound and tissue repair, often in contaminated surgical fields. Complications and failures due to infection of this biomaterial have therefore been a major concern and challenge. SIS can be colonized and infected by wound-associated bacteria, particularly Staphylococcus aureus. In order to address this concern and develop novel intervention strategies, the immune microenvironment orchestrated by the combined action of S. aureus and SIS should be critically evaluated. Since the outcome of tissue remodeling is largely controlled by the local immune microenvironment, we assessed the innate immune profile in terms of cytokine/chemokine microenvironment and inflammasome-responsive genes. BALB/c mice were injected intra-peritoneally with heat-killed S. aureus in the presence or absence of SIS. Analyses of cytokines, chemokines and microarray profiling of inflammasome-related genes were done using peritoneal lavages collected 24 hours after injection. Results showed that unlike SIS, the S. aureus-SIS interactome was characterized by a Th1-biased immune profile with increased expressions of IFN-γ, IL-12 and decreased expressions of IL-4, IL-13, IL-33 and IL-6. Such modulation of the Th1/Th2 axis can greatly facilitate graft rejections. The S. aureus-SIS exposure also augmented the expressions of pro-inflammatory cytokines like IL-1β, Tnf-α, CD30L, Eotaxin and Fractalkine. This heightened inflammatory response caused by S. aureus contamination could enormously affect the biocompatibility of SIS. However, the mRNA expressions of many inflammasome-related genes like Nlrp3, Aim2, Card6 and Pycard were down-regulated by heat-killed S. aureus with or without SIS. In summary, our study explored the innate immune microenvironment induced by the combined exposure of SIS and S. aureus. These results have practical implications in developing strategies to contain infection and promote successful tissue repair.
Citation: Roy Chowdhury R, Aachoui Y, Ghosh SK (2012) Effects of Small Intestinal Submucosa (SIS) on the Murine Innate Immune Microenvironment Induced by Heat-Killed Staphylococcus aureus. PLoS ONE 7(11): e48724. doi:10.1371/journal.pone.0048724
Editor: Stefan Bereswill, Charité-University Medicine Berlin, Germany
Received: July 31, 2012; Accepted: September 28, 2012; Published: November 26, 2012
Copyright: © 2012 Roy Chowdhury et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was initiated under the sponsorship of Cook Biotech, the Fraternal Order of Eagles Grant, and the School of graduate and professional studies, Indiana state university. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: This research was initiated with a grant support from Cook Biotech. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
The porcine-derived small intestinal submucosa is a clinically approved surgical scaffold routinely used as a biological tissue support for wound healing and tissue remodeling –. SIS is essentially an organic biomaterial consisting primarily of collagen (Types I, III and V) –, a constituent of the extracellular matrix (ECM). Other ECM components like glycosaminoglycans, proteoglycans, fibronectin and b-FGF are present in SIS in small amounts , , . In spite of its xenogeneic origin, SIS has been successfully used in the management of complex abdominal wall defects  and Peyronie's disease , as dural substitutes , anal fistula plugs  and many others. This has been possible since SIS comprises evolutionarily conserved proteins of the ECM (nature's ideal biological scaffold) that do not elicit any vigorous and adverse immune response. Furthermore, SIS is completely degraded and replaced by the host tissue over time , . The SIS biomaterial has also been evaluated as a vaccine carrier and adjuvant . It has shown promise as a cancer vaccine adjuvant against human prostate cancer by evoking effective cell-mediated immunity . In an earlier study, we have reported the efficiency of SIS in augmenting the immunogenic potential of soluble protein antigens like ovalbumin and keyhole-limpet hemocyanin . We also described that SIS exhibits adjuvanticity without inducing any adverse pathologic inflammatory response even in autoimmune prone NZB/WF1 mice . In this respect, SIS is better than some commercial adjuvants that often induce inflammatory tissue damage at sites of injection. The particulate nature of SIS may also promote phagocytosis, antigen uptake and retention by antigen presenting cells (APCs) .
The efficacy of SIS as a bioscaffold has been largely attributed to a Th2-biased immune response and a mild or non-inflammatory immune profile . Analysis of the Th1/Th2 balance induced by SIS has shown a robust IL-4 (Th2) expression with a characteristic IgG1 antibody response , . The Th2 response, characterized by the expression of cytokines like IL-4, IL-10, IL-13, favors transplant acceptance. On the other hand, the hallmark cytokine profile of Th1 response (IFN-γ, IL-12, TNF-α), activates macrophages and complement-fixing antibodies leading to xenogeneic transplant rejection . Analysis of the early inflammatory events is critical in assessing the potential of a bioscaffold material since the innate immune system is known to play a vital role in determining the biocompatibility of biomaterials –. It is also important in the evaluation of adjuvanticity since the initial innate immune microenvironment orchestrates the subsequent development of a robust adaptive immune response. The early innate immune proteome (cytokines, chemokines and growth factors) also shapes the wound healing process by stimulating the influx of inflammatory and connective tissue cells . Alterations in the localized innate immune profile may therefore impact the adjuvant properties, promote transplant rejection and decrease the likelihood of wound healing.
Potential complications at the surgical site due to infection remain a major challenge in wound healing and tissue remodeling. Infection of bioscaffold materials has been a serious problem ,  and studies have reported SIS to be susceptible to bacterial colonization and infection , . Staphylococcus aureus, a gram positive bacterium, is the primary cause of postsurgical wound infections  and therefore a major contaminant of biological scaffolds. It is a versatile and ubiquitous microbe that survives as a commensal on the nose, throat and skin of nearly 50% of the adult population . However, it is also the source of various life-threatening conditions such as endocarditis, meningitis and osteomyelitis . In order to evaluate the initial S. aureus-SIS-host interactome without inducing infection, we used heat-killed S. aureus (HKSA) since both live and HKSA are known to bind collagen with very high affinities . This ability of S. aureus to bind to elements of the ECM with high affinity is an essential attribute of the bacterium's invasive properties. HKSA and SIS were injected intra-peritoneally for two major reasons: first, SIS being derived from the small intestine is routinely used for gastrointestinal healing and cecal wound repair ,  and second, S. aureus-induced peritonitis remains a major postoperative complication .
In this study, we sought to understand the dynamics of the innate immune profile orchestrated by the combined action of SIS and S. aureus. The immune microenvironment induced as a result of infection or presence of inflammatory bacterial products such as endotoxins or lipotechoic acids could determine the success or failure of an implant or surgical manipulations as well as wound healing. We therefore addressed the following: how does SIS, in the presence of S. aureus, modulate (a) the Th1/Th2 cytokine profile; (b) the milieu of cytokines, chemokines and growth factors; and (c) consequently the inflammasome-responsive genes? Our results demonstrated a Th1-biased cytokine profile (with increased expression of IFN-γ and IL-12) in the presence of S. aureus together with SIS. This clearly represents a shift from the Th2 cytokine profile known to be induced by SIS alone , . Previously, we have reported that SIS by itself is mildly inflammatory . Addition of S. aureus to SIS was found to alter the expression patterns of a number of inflammatory cytokines, like IL-6, IL-1α, IL-1β, Tnf-α and sTNFRs (I and II), chemokines, like CD30L, Eotaxin, Fractalkine, I-TAC, MIG, TECK, LIX and MCP-1, and growth factors, like M-CSF and GM-CSF. Such alterations in the innate immune profile may have significant consequences on inflammation, wound healing and tissue remodeling. This report provides insights into the innate immune microenvironment induced by the dual action of S. aureus and SIS, and will therefore be helpful in developing novel strategies for wound-associated infection containment and successful tissue remodeling.
Materials and Methods
Bacterial inoculum preparation
S. aureus (ATCC number: 25923), kindly provided by Dr. H. K. Dannelly of the Department of Life Sciences, Indiana State University, was used for all experiments since this strain has been shown to exhibit high-affinity, specific-binding to collagen . S. aureus was grown overnight in Luria-Bertani (LB) broth (Difco, Detroit, MI) at 37°C for 14–16 hours and harvested with phosphate buffered saline (PBS). Cells were washed in PBS; the culture concentration was determined by spectrophotometry (OD600) and then suspended to the appropriate density in PBS. Bacteria were killed by heating suspensions at 60°C for 1 hour. Killing was confirmed by plating the suspensions on agar plates and incubating them at 37°C overnight.
Experimental animals and design
The use of mice has been guided by strict adherence to the protocols (ID# 09-15-2010:SKG/YA; ID# 01-03-2011: SKG/RR), approved specifically for this study, by the Indiana State University Animal Care and Use Committee (IACUC). Female BALB/c mice (6–8 weeks of age) were used for all experiments. Pathogen-free BALB/c mice were bought from Harlan Sprague-Dawley (Indiana, USA), kept in quarantine for two weeks and then relocated to specific mouse facility that is routinely monitored by veterinarians, IACUC and USDA inspectors. These mice were bred and maintained at the animal care facility of Indiana State University.
5 mg of particulate SIS-hydrated (SIS-H) alone, obtained from Cook Biotech (Bloomington, IN) and 5×106 CFU of HKSA alone or in combination with SIS-H, in a total volume of 500 µl was administered intra-peritoneally into the BALB/c mice. One group of mice received PBS only (control). All experiments were performed and repeated at least 3 times with 3–4 mice per test group. Peritoneal lavages were collected as described below, 24 hours later.
Collection of peritoneal cells and lavages
Peritoneal exudates were harvested using 3 ml of PBS with 19 gauge needles. The collected samples were centrifuged at 500×g for 10 minutes at 4°C and the supernatants were used for cytokine and chemokine analysis. Peritoneal cells were washed twice with PBS and used for profiling inflammation-related gene expression.
Determination of cytokines and chemokines secreted in the peritonea
Cytokines and chemokines in peritoneal fluids were assessed using the mouse inflammatory cytokine array kits (Ray Biotech Inc., GA, USA) following the manufacturer's instructions. Briefly, the cytokine array membranes provided were blocked with 2 ml of blocking buffer for 30 minutes and then incubated overnight with 1 ml of undiluted samples at 4°C. Samples were then decanted, and the membranes washed three times with wash buffer. Membranes were incubated with biotin-conjugated primary antibodies (1:250 dilutions) at room temperature for 2 hours, then washed and exposed to horseradish peroxidase-conjugated streptavidin (1:1000 dilution) for 1 hour. This was followed by treatment with 500 µl of peroxidase substrate for 2 minutes in the dark. The signal intensities were read by a chemiluminescence reader (Epi Chemi II Darkroom, UVP) and analyzed using the Ray Biotech cytokine expression analysis software. Positive and negative controls from six array spots were used to normalize the results and the net result for each spot was determined after subtraction of the background intensity. Data are expressed as the fold changes relative to the PBS control of each cytokine or chemokine protein detected using pooled peritoneal fluids of 3–4 mice per group. The experiment was repeated at least 3 times.
Real time polymerase chain reaction for transcriptome profiling of inflammasome-responsive genes
Peritoneal exudate cells isolated from the control (PBS) and experimental groups were used to profile inflammasome-responsive gene expression by semi-quantitative RT-PCR. Briefly, total RNA was extracted from the peritoneal exudate cells according to the manufacturer's protocol (Ambion, Austin, TX). The quality of the RNA preparation was first assessed by spectrophotometry. All samples had 260/280 ratios above 2.0 and 230/260 ratios above 1.7. Further assessment was done using quality control plates (PAMM-999A-1, SA Biosciences, Frederick, MD). Then equal amounts of RNA (1 µg) from all samples were subjected to first-strand cDNA synthesis using RT2 first-strand kit from SA Biosciences, followed by PCR amplification. We used the RT2 Profiler PCR inflammasome array (PAMM-097) from SA Biosciences for the transcriptome analysis of inflammasome-associated genes. The experiments were performed in a Stratagene Mx3000P cycler using a cycling program provided by the manufacturer. Data were analyzed and fold changes in values were calculated using the PCR array analysis tool available at their website (http://pcrdata-analysis.sabiosciences.com/pcr/arrayanalysis.php). Gene expressions were normalized with respect to five house-keeping genes included in the array kit and expressed as averages of log2 ratios. Fold regulation changes of genes that differed by ≤ or ≥1.5 compared to PBS controls was considered significant.
All data are expressed as mean ± SD. Data were analyzed by one-way analysis of variance (ANOVA) followed by a Tukey's post hoc test or the Student's t-test wherever relevant. For all analysis GraphPad Prism version 5 (GraphPad Software, San Diego, CA) was used. A p-value<0.05 was considered statistically significant.
Results and Discussion
Impact of HKSA with or without SIS-H on the Th1/Th2 cytokine profile
It is now well documented that SIS-H alone primarily induces a Th2 response , , a signature profile promoting its broad application in tissue remodeling. In this study, we wanted to delineate how this Th1/Th2 profile changes in the presence of HKSA. We therefore determined the expression patterns of some key cytokines corresponding to Th1 (IFN-γ and IL-12) and Th2 (IL-4 and IL-13)-mediated immune response, using the cytokine antibody-array technique. Our results showed that HKSA in the presence of SIS-H (or in other words, SIS-H contaminated with HKSA) induced higher levels of IFN-γ and significantly higher IL-12, but significantly lower levels of IL-4 and IL-13 as compared to SIS-H alone (Figure 1). HKSA by itself registered a Th1-biased immune response (IFN-γ = 1.9 fold and IL-12 = 1.963 fold as compared to IL-4 = 0.912 fold and IL-13 = 1.05 fold). Such a Th1-type immune profile of HKSA clearly suppressed the Th2 response mediated by SIS-H, while augmenting the Th1 immune response. These results demonstrated a shift in the T helper profile from type 2 to type 1 induced in response to the exposure of SIS-H to HKSA. This switch to the Th1 response could have important consequences such as delayed wound healing and aggravated graft rejections, as illustrated in Figure 2.
Figure 1. Shift in Th1-Th2 balance induced by the combined action of HKSA and SIS-H.
HKSA in the presence of SIS-H shows higher expression levels of Th1-associated cytokines (IFN- γ and IL-12) than Th2-associated cytokines (IL-4 and IL-13) relative to SIS-H alone. HKSA by itself promotes a Th1-type immune response. Data for each group used in the figure are based on fold change values obtained by comparing to the PBS control group. (* = p<0.05).doi:10.1371/journal.pone.0048724.g001
Figure 2. Diagrammatic representation of Th1/Th2 imbalance caused by HKSA-SIS-H exposure on graft transplantation.
The addition of HKSA to SIS-H can promote graft rejection due to the higher expression levels of IFN-γ and IL-12 (Th1-type immune response) and lower expression levels of IL-4 and IL-13 (Th2-type immune response). The Th2 response is known to promote graft acceptance.doi:10.1371/journal.pone.0048724.g002
Effects of HKSA-SIS-H exposure on the milieu of cytokines, chemokines and growth factors
Early inflammatory events play a critical role in shaping the outcome of the wound healing or tissue remodeling process . The cytokine/chemokine cascades can drive the recruitment of various immune cells like neutrophils and macrophages that can either promote or aggravate wound healing. In this study, we examined the induction of some inflammatory cytokines and chemokines by SIS-H alone, and HKSA alone or in combination with SIS-H, and discussed their possible roles in tissue remodeling, graft-versus-host disease (GVHD) and wound healing. Our results showed that the addition of HKSA could impair the efficacy of SIS-H by promoting adverse inflammation and subsequent tissue damage. This is clearly evident in the augmented expressions of CD30L, Fractalkine and Eotaxin, relative to the levels induced by SIS-H alone (Figure 3). The addition of HKSA had a cumulative effect on the expression of these cytokines, particularly CD30L and Fractalkine. CD30L-CD30 signaling can promote CD4+ T-cell mediated GVHD , . The increased expression of Fractalkine may also be unfavorable for the host, since the blocking of Fractalkine, also called MAdCAM-1, is known to alleviate graft-versus-host reaction . Eotaxin is the primary chemoattractant for eosinophils and drives their maturation, migration and activation. Although acute graft rejections are primarily attributed to Th1 responses, Th2-biased eosinophilic inflammation can also mediate rejections. Eosinophils can promote detrimental tissue damage by the release of cationic granules and cytokines like IL-3 that stimulate further inflammation .
Figure 3. Effect of the combined action of HKSA and SIS-H on CD30L, Eotaxin and Fractalkine.
The expression levels of CD30L, Eotaxin and Fractalkine are up-regulated due to the combined exposure of HKSA and SIS-H relative to SIS-H alone. All three of these molecules are known to promote graft rejections. Data for each group used in the figure are based on fold change values obtained by comparing to the PBS control group (* = p<0.05, ** = p<0.01 and *** = p<0.001).doi:10.1371/journal.pone.0048724.g003
The augmented expression of CD30L, Fractalkine (a potent T-cell and monocyte chemoattractant) and Eotaxin due to the combined action of HKSA and SIS-H, compared to either HKSA or SIS-H alone, implies an increased mobilization and activation of leukocytes like eosinophils, monocytes, macrophages and mast cells that can lead to detrimental inflammatory response. However, the expression of a number of chemokines was found to be suppressed when SIS-H was combined or contaminated with HKSA. The chemokines modulated include GM-CSF, I-TAC, MIG, Leptin and TECK. The granulocyte-macrophage colony stimulating factor (GM-CSF) binds to receptors belonging to the gp140 family of proteins and stimulates hematopoiesis . Since granulocytes play important defensive roles in the course of S. aureus infection , GM-CSF is vital in bacterial clearance. GM-CSF also plays a key role in the development of dendritic cells that are specialized in antigen uptake and presentation . Furthermore, GM-CSF can be therapeutically useful during organ transplantation, as it does not induce graft rejection and help resist infections , . Our findings showed that SIS-H by itself induced high expression of GM-CSF, which was significantly suppressed upon the addition of HKSA (Figure 4). This suppression of GM-CSF could be detrimental to the host in terms of bacterial clearance, innate-adaptive immune system crosstalk and graft rejections.
Figure 4. Down-regulation of inflammatory cytokines due to exposure to HKSA together with SIS-H.
The growth factor GM-CSF, antibacterial chemokines I-TAC, MIG, TECK and adipokine Leptin are down-regulated in response to HKSA plus SIS-H. SIS-H by itself can induce high levels of these inflammatory molecules. Significant suppression of these molecules can impair bacterial clearance and delay wound healing. Data for each group used in the figure are based on fold change values obtained by comparing to the PBS control group. (* = p<0.05).doi:10.1371/journal.pone.0048724.g004
The chemokines MIG (Monokine induced by IFN-γ or CXCL9) and I-TAC (IFN-γ-inducible T-cell chemoattractant or CXCL11) regulate lymphocyte mobilization  and have antimicrobial properties . These CXCR3 chemokines have also been implicated in transplant rejections . The expression of MIG and I-TAC were significantly decreased due to the combined HKSA-SIS-H exposure, relative to HKSA alone and SIS-H respectively (Figure 4). The other cytokines suppressed by the HKSA-SIS-H combination were Leptin and TECK (Figure 4). Leptin is an adipokine, which is known to influence both the innate and adaptive arms of immunity. It can stimulate monocyte proliferation and lymphocyte activation. It is also known to promote a Th1 type immune response . TECK or thymus-expressed chemokine is an activator of dendritic cells and thymocytes  and promotes leukocyte migration or inflammation. It has been shown to play a critical role in wound healing . Thus, any suppression of TECK, due to the HKSA-SIS-H exposure, may also negatively impact the wound healing process. In addition to these chemokines, we observed that SIS-H by itself induced enhanced expressions of BLC (B-lymphocyte chemoattractant), LIX (Lipopolysaccharide induced CXC chemokine), MCP-1 (monocyte chemotactic protein-1), TCA-3 (T-cell activation-3) and M-CSF (macrophage-colony stimulating factor) (Figure 5A). However, the addition of HKSA to SIS-H significantly down-regulated the expressions of BLC, LIX, TCA-3 and M-CSF (Figure 5A). M-CSF is important in wound repair  and its suppression could therefore impair the wound healing response. Similarly, TCA-3 (or CCL1) and MCP-1 also have multiple functions in the process of wound healing , .
(A–C): Effect of HKSA-SIS-H exposure on the expression of inflammatory cytokines and chemokines involved in wound healing and GVHD. Data for each group used in the figure are based on fold change values obtained by comparing to the PBS control group. (* = p<0.05, ** = p<0.01 and *** = p<0.001).doi:10.1371/journal.pone.0048724.g005
Furthermore, a number of other inflammatory cytokines, such as Fas-ligand, IL-1α, IL-2, lymphotactin, MIP-1α, Rantes, SDF-1 and sTNFRs (I and II) that were down-regulated by the combined action of SIS-H and HKSA, compared to SIS-H or HKSA alone, are depicted in figures 5B and 5C. Fas-ligand, which is important for the host's ability to control GVHD , is significantly down-regulated by SIS-H plus HKSA, relative to SIS-H alone (Figure 5B). Like Fas-ligand, low-dose IL-2 has also been reported to be vital in the clinical management of GVHD for its ability to promote the growth and survival of T-regulatory cells . Additionally, the chemokines MIP-1α, Rantes and SDF-1 that play critical roles in accelerating wound healing , , were highly suppressed in the presence of HKSA together with SIS-H, compared to SIS-H alone (Figure 5C). The expression of sTNFRs has been shown to suppress clinical manifestations of GVHD . It is therefore clear from the above discussion that the contamination of SIS-H with HKSA could repress the expression of critical cytokines thereby impairing wound repair and exacerbating GVHD.
Effects of HKSA-SIS-H exposure on inflammasome-responsive genes
The members of the Nod-like receptor (NLR) family can form multi-protein complexes called inflammasomes that regulate the secretion of pro-inflammatory cytokines like IL-1β . IL-1β is a key mediator of inflammation and is known to be a major protective cytokine against S. aureus infection . However, increased production of IL-1β can also aggravate GVHD pathophysiology . Studies have identified different inflammasome pathways like Nlrp1, Nlrp3 and Ipaf. Apart from their role in infection, the inflammasomes have been linked to pyroptosis and autophagy . Additionally, the Nlrp3 inflammasome is known to mediate the adjuvanticity of alum , . The role of the inflammasome in wound healing is also becoming increasingly apparent –.
Because of the involvement of the inflammasome in infection, adjuvanticity, wound healing and GVHD, we analyzed the influence of HKSA with or without SIS-H on the mRNA expression patterns of various inflammasome-responsive genes. We have previously reported that SIS-H by itself does not activate any of the known inflammasome pathways . However, S. aureus has been reported to stimulate the Nlrp3-mediated inflammasome pathway . We therefore wanted to determine if the presence of SIS-H could alter the inflammasome-responsive immune profile induced by HKSA. It is clear from our above discussions that HKSA greatly impacted the immune microenvironment induced by SIS-H. It was therefore of interest to see if SIS-H could impact the pathophysiology due to HKSA. This would be particularly important while evaluating the adjuvant properties of SIS. Inflammasome-responsive genes, like IFN-γ, IL-1β, Tnf and Irf-1 that were highly induced (fold regulation ≥1.5) by both HKSA alone and in combination with SIS-H are presented in Table 1. IFN-γ, IL-1β and Tnf are pro-inflammatory cytokines that indicate the Th1-biased immune response engendered by HKSA alone and in the presence of SIS-H. These results support our cytokine antibody array data. Genes that were significantly up-regulated due to the combined exposure of HKSA and SIS-H, relative to HKSA alone, are listed in Table 2. Among these genes are IL-12b, MyD88 and NLR family proteins, Nlrp5, Nlrp6, Nlrp9b and Nlrx1. The enhanced expression of IL-12, as mentioned before, indicates a Th1-type response that may promote graft rejections but also S. aureus clearance . Nlrx1 is known to induce the production of reactive oxygen species  and this may aid in S. aureus killing. However, a number of inflammasome-related genes, like IL-6, IL-33, Cxcl1, Casp1 and Casp12 were significantly down-regulated by the combined action of HKSA and SIS-H, relative to HKSA alone (Table 3). The fact that the combined exposure of HKSA and SIS-H promotes a Th1-response is further supported by the suppression of the pro-inflammatory cytokines IL-33 and IL-6. Both these cytokines are known to promote Th2 and limit Th1 immune responses –. Their significant suppression therefore indicates the stimulation of a Th1-biased immune response, which could exacerbate GVHD. Additionally, IL-6 has been shown to exhibit protective roles against S. aureus infections , suggesting that reduced expression of IL-6 may impair bacterial clearance. Cxcl1 or KC is a potent chemoattractant for neutrophils. Since neutrophils are crucial in host defense against S. aureus  and also in the wound healing process , suppression of this chemokine might adversely affect infection control and the wound healing response.
Table 1. Genes highly expressed by HKSA alone and in combination with SIS-H.doi:10.1371/journal.pone.0048724.t001
Table 2. Genes significantly up-regulated by HKSA-SIS-H combination relative to HKSA alone.doi:10.1371/journal.pone.0048724.t002
Table 3. Genes significantly down-regulated by HKSA-SIS-H combination relative to HKSA alone.doi:10.1371/journal.pone.0048724.t003
Most of the inflammasome-associated genes like Aim2, Bcl2, Card6, Naip1, Naip5, Nlrp1a, Nlrp3, Nlrp4b, Nlrp4e, Ptgs2 and others, listed in Table 4, did not show significant change in their expression patterns when exposed to HKSA together with SIS-H, versus HKSA alone. It is noteworthy that neither HKSA alone, nor HKSA with SIS-H induced the activation of the Nlrp3 inflammasome after 24 hours. Interestingly, the other common inflammasome pathways like Nlrp1 and Aim2 were also not activated by HKSA in the presence or absence of SIS-H.
Table 4. Genes whose expressions do not change significantly due HKSA-SIS-H exposure.doi:10.1371/journal.pone.0048724.t004
It is therefore clear from our cytokine-chemokine and inflammasome array studies that the combined action of HKSA and SIS-H evokes a unique local immune microenvironment that is distinct from those induced by either HKSA or SIS-H alone. Based on the cytokine-chemokine and inflammation-responsive gene array data, the picture that emerges of the immune microenvironment in BALB/c mice from the combined action of HKSA and SIS-H has been depicted in Figure 6.
Figure 6. A simplistic model of the local microenvironment (cytokines and infiltrating cells) induced by the combined action of HKSA and SIS-H.
The uptake of HKSA and SIS-H by APCs can release IL-12, which can stimulate NK cells and T-cells to produce IFN-γ. The IFN-γ can in turn activate macrophages to produce pro-inflammatory cytokines IL-1β and Tnf-α and endothelial cells to produce the chemokines Eotaxin and Fractalkine. Eosinophil is a potent chemoattractant of eosinophils and the induced Fractalkine can cause chemotaxis of NK cells, macrophages, CD4+ and CD8+ T-cells. The Th1-cells can CD30L, which can influence both the innate and acquired arms of immunity. Such an immune microenvironment can cause heightened local inflammation. = Suppression, = Activation.doi:10.1371/journal.pone.0048724.g006
Our studies revealed that S. aureus can severely compromise the efficacy of SIS as a biomaterial, primarily because of its impact on the immune microenvironment surrounding the affected host tissues. S. aureus in conjunction with SIS promotes a Th1-biased immune response (IL-12, IFN-γ), suppresses the Th2 cytokine profile (IL-4, IL-13, IL-33 and IL-6), induces the expression of pro-inflammatory cytokines like IL-1β and Tnf and down regulates the expression of a number of inflammasome-responsive genes, Nlrp3, Aim2 etc. Other important pro-inflammatory molecules such as CD30L, Fractalkine and Eotaxin are also augmented from the S. aureus-SIS exposure. A variety of chemokines and growth factors like GM-CSF, Cxcl1, MIG, I-TAC and Leptin are also modulated. Such an immune proteome implying heightened inflammation and Th1 response can aggravate graft survival, hinder bacterial clearance and therefore be detrimental to the host. Overall, the information gained about the modulation of the host innate immune microenvironment by the S. aureus-SIS exposure will help define new therapeutic targets for developing effective intervention strategies for wound-related infections.
SIS is an FDA approved  and highly biocompatible tissue scaffold material that is also an effective immunoadjuvant. However, one of the major concerns associated with SIS has been surgical site infection and wound contamination. A number of studies have therefore focused on improving the anti-bacterial property of SIS by incorporating molecules like silver nanoparticles  and bismuth thiol . However, the first step to finding a potential intervention to this clinical problem should be the analysis of the complex host-SIS-bacteria interactome. The issue for future studies would be to determine if the adversarial immune microenvironment engendered due to contamination by S. aureus or other bacterial products could be mitigated by including antibacterial compounds in SIS biomaterials.
Conceived and designed the experiments: RRC YA SKG. Performed the experiments: RRC YA. Analyzed the data: RRC YA SKG. Contributed reagents/materials/analysis tools: RRC YA SKG. Wrote the paper: RRC YA SKG.
- 1. Ahn HH, Kim KS, Lee JH, Lee MS, Song IB, et al. (2007) Porcine small intestinal submucosa sheets as a scaffold for human bone marrow stem cells. Int J Biol Macromol 41: 590–596. doi: 10.1016/j.ijbiomac.2007.07.019
- 2. Crapo PM, Wang Y (2010) Small intestinal submucosa gel as a potential scaffolding material for cardiac tissue engineering. Acta Biomater 6: 2091–2096. doi: 10.1016/j.actbio.2009.10.048
- 3. Hodde JP, Badylak SF, Brightman AO, Voytik-Harbin SL (1996) Glycosaminoglycan content of small intestinal submucosa: a bioscaffold for tissue replacement. Tissue Eng 2: 209–217. doi: 10.1089/ten.1996.2.209
- 4. Cimini M, Boughner DR, Ronald JA, Johnston DE, Rogers KA (2005) Dermal fibroblasts cultured on small intestinal submucosa: Conditions for the formation of a neotissue. J Biomed Mater Res A 75: 895–906. doi: 10.1002/jbm.a.30493
- 5. Badylak SF, Freytes DO, Gilbert TW (2009) Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater 5: 1–13. doi: 10.1016/j.actbio.2008.09.013
- 6. Musahl V, Abramowitch SD, Gilbert TW, Tsuda E, Wang JHC, et al. (2004) The use of porcine small intestinal submucosa to enhance the healing of the medial collateral ligament—a functional tissue engineering study in rabbits. J Orthop Res 22: 214–220. doi: 10.1016/s0736-0266(03)00163-3
- 7. Graham MF, Diegelmann RF, Elson CO, Lindblad WJ, Gotschalk N, et al. (1988) Collagen content and types in the intestinal strictures of Crohn's disease. Gastroenterology 94: 257–265.
- 8. McPherson TB, Badylak SF (1998) Characterization of fibronectin derived from porcine small intestinal submucosa. Tissue Eng 4: 75–83. doi: 10.1089/ten.1998.4.75
- 9. Voytik-Harbin SL, Brightman AO, Kraine MR, Waisner B, Badylak SF (1997) Identification of extractable growth factors from small intestinal submucosa. J Cell Biochem 67: 478–491. doi: 10.1002/(sici)1097-4644(19971215)67:4<478::aid-jcb6>3.3.co;2-c
- 10. Badylak S, Kokini K, Tullius B, Simmons-Byrd A, Morff R (2002) Morphologic study of small intestinal submucosa as a body wall repair device. J Surg Res 103: 190–202. doi: 10.1006/jsre.2001.6349
- 11. Knoll LD (2007) Use of small intestinal submucosa graft for the surgical management of Peyronie's disease. J Urol 178: 2474–2478; discussion 2478. doi: 10.1016/j.juro.2007.08.044
- 12. Bejjani GK, Zabramski J (2007) Safety and efficacy of the porcine small intestinal submucosa dural substitute: results of a prospective multicenter study and literature review. J Neurosurg 106: 1028–1033. doi: 10.3171/jns.2007.106.6.1028
- 13. Champagne BJ, O'Connor LM, Ferguson M, Orangio GR, Schertzer ME, et al. (2006) Efficacy of anal fistula plug in closure of cryptoglandular fistulas: long-term follow-up. Dis Colon Rectum 49: 1817–1821. doi: 10.1007/s10350-006-0755-3
- 14. Record RD, Hillegonds D, Simmons C, Tullius R, Rickey FA, et al. (2001) In vivo degradation of 14C-labeled small intestinal submucosa (SIS) when used for urinary bladder repair. Biomaterials 22: 2653–2659. doi: 10.1016/s0142-9612(01)00007-2
- 15. Gilbert TW, Stewart-Akers AM, Simmons-Byrd A, Badylak SF (2007) Degradation and remodeling of small intestinal submucosa in canine Achilles tendon repair. J Bone Joint Surg Am 89: 621–630. doi: 10.2106/jbjs.e.00742
- 16. Suckow MA, Hall P, Wolter W, Sailes V, Hiles MC (2008) Use of an extracellular matrix material as a vaccine carrier and adjuvant. Anticancer Res 28: 2529–2534.
- 17. Suckow MA, Rosen ED, Wolter WR, Sailes V, Jeffrey R, et al. (2007) Prevention of human PC-346C prostate cancer growth in mice by a xenogeneic tissue vaccine. Cancer Immunol Immunother 56: 1275–1283. doi: 10.1007/s00262-006-0278-8
- 18. Aachoui Y, Ghosh SK (2011) Extracellular matrix from porcine small intestinal submucosa (SIS) as immune adjuvants. PLoS One 6: e27083. doi: 10.1371/journal.pone.0027083
- 19. Aachoui Y, Ghosh SK (2011) Immune enhancement by novel vaccine adjuvants in autoimmune-prone NZB/W F1 mice: relative efficacy and safety. BMC Immunol 12: 61. doi: 10.1186/1471-2172-12-61
- 20. Allman AJ, McPherson TB, Merrill LC, Badylak SF, Metzger DW (2002) The Th2-restricted immune response to xenogeneic small intestinal submucosa does not influence systemic protective immunity to viral and bacterial pathogens. Tissue Eng 8: 53–62. doi: 10.1089/107632702753503054
- 21. Badylak SF (2004) Xenogeneic extracellular matrix as a scaffold for tissue reconstruction. Transpl Immunol 12: 367–377. doi: 10.1016/j.trim.2003.12.016
- 22. Nikolic B, Lee S, Bronson RT, Grusby MJ, Sykes M (2000) Th1 and Th2 mediate acute graft-versus-host disease, each with distinct end-organ targets. J Clin Invest 105: 1289–1298. doi: 10.1172/jci7894
- 23. Kim MS, Ahn HH, Shin YN, Cho MH, Khang G, et al. (2007) An in vivo study of the host tissue response to subcutaneous implantation of PLGA- and/or porcine small intestinal submucosa-based scaffolds. Biomaterials 28: 5137–5143. doi: 10.1016/j.biomaterials.2007.08.014
- 24. Vince DG, Hunt JA, Williams DF (1991) Quantitative assessment of the tissue response to implanted biomaterials. Biomaterials 12: 731–736. doi: 10.1016/0142-9612(91)90021-2
- 25. Remes A, Williams DF (1992) Immune response in biocompatibility. Biomaterials 13: 731–743. doi: 10.1016/0142-9612(92)90010-l
- 26. Hodde JP, Johnson CE (2007) Extracellular matrix as a strategy for treating chronic wounds. Am J Clin Dermatol 8: 61–66. doi: 10.2165/00128071-200708020-00001
- 27. Harth KC, Rosen MJ (2009) Major complications associated with xenograft biologic mesh implantation in abdominal wall reconstruction. Surg Innov 16: 324–329. doi: 10.1177/1553350609353609
- 28. Shah BC, Tiwari MM, Goede MR, Eichler MJ, Hollins RR, et al. (2011) Not all biologics are equal!. Hernia 15: 165–171. doi: 10.1007/s10029-010-0768-7
- 29. Bellows CF, Wheatley BM, Moroz K, Rosales SC, Morici LA (2011) The effect of bacterial infection on the biomechanical properties of biological mesh in a rat model. PLoS One 6: e21228. doi: 10.1371/journal.pone.0021228
- 30. Carbonell AM, Matthews BD, Dreau D, Foster M, Austin CE, et al. (2005) The susceptibility of prosthetic biomaterials to infection. Surg Endosc 19: 430–435. doi: 10.1007/s00464-004-8810-4
- 31. Perl TM (2003) Prevention of Staphylococcus aureus infections among surgical patients: Beyond traditional perioperative prophylaxis. Surgery 134: S10–S17. doi: 10.1016/s0039-6060(03)00391-x
- 32. Holtfreter S, Kolata J, Broker BM (2010) Towards the immune proteome of Staphylococcus aureus - The anti-S. aureus antibody response. Int J Med Microbiol 300: 176–192. doi: 10.1016/j.ijmm.2009.10.002
- 33. Fluit AC, Schmitz FJ, Verhoef J (2001) Frequency of isolation of pathogens from bloodstream, nosocomial pneumonia, skin and soft tissue, and urinary tract infections occurring in European patients. Eur J Clin Microbiol Infect Dis 20: 188–191. doi: 10.1007/s100960100455
- 34. Holderbaum D, Hall GS, Ehrhart LA (1986) Collagen binding to Staphylococcus aureus. Infect Immun 54: 359–364.
- 35. Delafuente S, Gottfried M, Lawson D, Harris M, Mantyh C, et al. (2003) Evaluation of Porcine-Derived Small Intestine Submucosa as a Biodegradable Graft for Gastrointestinal Healing. J Gastrointest Surg 7: 96–101. doi: 10.1016/s1091-255x(02)00050-1
- 36. Ueno T, Oga A, Takahashi T, Pappas TN (2007) Small intestinal submucosa (SIS) in the repair of a cecal wound in unprepared bowel in rats. J Gastrointest Surg 11: 918–922. doi: 10.1007/s11605-007-0171-6
- 37. Szeto CC, Chow KM, Kwan BC, Law MC, Chung KY, et al. (2007) Staphylococcus aureus peritonitis complicates peritoneal dialysis: review of 245 consecutive cases. Clin J Am Soc Nephrol 2: 245–251. doi: 10.2215/cjn.03180906
- 38. Allman AJ, McPherson TB, Badylak SF, Merrill LC, Kallakury B, et al. (2001) Xenogeneic Extracellular Matrix Grafts Elicit A Th2-Restricted Immune Response1. Transplantation 71: 1631–1640. doi: 10.1097/00007890-200106150-00024
- 39. Li J, Chen J, Kirsner R (2007) Pathophysiology of acute wound healing. Clin Dermatol 25: 9–18. doi: 10.1016/j.clindermatol.2006.09.007
- 40. Blazar BR, Levy RB, Mak TW, Panoskaltsis-Mortari A, Muta H, et al. (2004) CD30/CD30 ligand (CD153) interaction regulates CD4+ T cell-mediated graft-versus-host disease. J Immunol 173: 2933–2941.
- 41. Hutter G, Neumann M, Nowak D, Klein S, Kluter H, et al. (2011) The effect of the CCR5-delta32 deletion on global gene expression considering immune response and inflammation. J Inflamm (Lond) 8: 29. doi: 10.1186/1476-9255-8-29
- 42. Ueha S, Murai M, Yoneyama H, Kitabatake M, Imai T, et al. (2007) Intervention of MAdCAM-1 or fractalkine alleviates graft-versus-host reaction associated intestinal injury while preserving graft-versus-tumor effects. J Leukoc Biol 81: 176–185. doi: 10.1189/jlb.0306231
- 43. LaRosa DF, Rahman AH, Turka LA (2007) The Innate Immune System in Allograft Rejection and Tolerance. J Immunol 178: 7503–7509. doi: 10.4049/jimmunol.178.12.7503
- 44. Donahue RE, Seehra J, Metzger M, Lefebvre D, Rock B, et al. (1988) Human IL-3 and GM-CSF act synergistically in stimulating hematopoiesis in primates. Science 241: 1820–1823. doi: 10.1126/science.3051378
- 45. Verdrengh M, Tarkowski A (1998) Granulocyte-macrophage colony-stimulating factor in Staphylococcus aureus-induced arthritis. Infect Immun 66: 853–855.
- 46. van de Laar L, Coffer PJ, Woltman AM (2012) Regulation of dendritic cell development by GM-CSF: molecular control and implications for immune homeostasis and therapy. Blood 119: 3383–3393. doi: 10.1182/blood-2011-11-370130
- 47. Xu J, Lucas R, Schuchmann M, Kühnle S, Meergans T, et al. (2003) GM-CSF Restores Innate, But Not Adaptive, Immune Responses in Glucocorticoid-Immunosuppressed Human Blood In Vitro. J Immunol 171: 938–947.
- 48. Budde K, Waiser J, Neumayer HH (1994) The diagnostic value of GM-CSF and IL-6 determinations in patients after renal transplantation. Transpl Int 7Suppl 1: S97–101. doi: 10.1111/j.1432-2277.1994.tb01320.x
- 49. Lasagni L, Francalanci M, Annunziato F, Lazzeri E, Giannini S, et al. (2003) An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC, and acts as functional receptor for platelet factor 4. J Exp Med 197: 1537–1549. doi: 10.1084/jem.20021897
- 50. Braff MH, Bardan A, Nizet V, Gallo RL (2005) Cutaneous defense mechanisms by antimicrobial peptides. J Invest Dermatol 125: 9–13. doi: 10.1111/j.0022-202x.2004.23587.x
- 51. Zhao DX, Hu Y, Miller GG, Luster AD, Mitchell RN, et al. (2002) Differential expression of the IFN-gamma-inducible CXCR3-binding chemokines, IFN-inducible protein 10, monokine induced by IFN, and IFN-inducible T cell alpha chemoattractant in human cardiac allografts: association with cardiac allograft vasculopathy and acute rejection. J Immunol 169: 1556–1560.
- 52. Matarese G, Moschos S, Mantzoros CS (2005) Leptin in immunology. J Immunol 174: 3137–3142.
- 53. Murdoch C, Finn A (2000) Chemokine receptors and their role in inflammation and infectious diseases. Blood 95: 3032–3043.
- 54. McGrory K, Flaitz CM, Klein JR (2004) Chemokine changes during oral wound healing. Biochem Biophys Res Commun 324: 317–320. doi: 10.1016/j.bbrc.2004.09.056
- 55. Wu L, Yu YL, Galiano RD, Roth SI, Mustoe TA (1997) Macrophage colony-stimulating factor accelerates wound healing and upregulates TGF-beta1 mRNA levels through tissue macrophages. J Surg Res 72: 162–169. doi: 10.1006/jsre.1997.5178
- 56. DiPietro LA, Polverini PJ, Rahbe SM, Kovacs EJ (1995) Modulation of JE/MCP-1 expression in dermal wound repair. Am J Pathol 146: 868–875.
- 57. Liaskou E, Zimmermann HW, Li KK, Htun Oo Y, Suresh S, et al. (2012) Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics. Hepatology doi: 10.1002/hep.26016
- 58. van den Brink MR, Moore E, Horndasch KJ, Crawford JM, Murphy GF, et al. (2000) Fas ligand-deficient gld mice are more susceptible to graft-versus-host-disease. Transplantation 70: 184–191.
- 59. Koreth J, Matsuoka K, Kim HT, McDonough SM, Bindra B, et al. (2011) Interleukin-2 and regulatory T cells in graft-versus-host disease. N Engl J Med 365: 2055–2066. doi: 10.1056/nejmoa1108188
- 60. Gillitzer R, Goebeler M (2001) Chemokines in cutaneous wound healing. J Leukoc Biol 69: 513–521.
- 61. Loh SA, Chang EI, Galvez MG, Thangarajah H, El-ftesi S, et al. (2009) SDF-1 alpha expression during wound healing in the aged is HIF dependent. Plast Reconstr Surg 123: 65S–75S. doi: 10.1097/prs.0b013e318191bdf4
- 62. Sakata N, Yasui M, Okamura T, Inoue M, Yumura-Yagi K, et al. (2001) Kinetics of plasma cytokines after hematopoietic stem cell transplantation from unrelated donors: the ratio of plasma IL-10/sTNFR level as a potential prognostic marker in severe acute graft-versus-host disease. Bone Marrow Transplant 27: 1153–1161. doi: 10.1038/sj.bmt.1703060
- 63. Schroder K, Tschopp J (2010) The Inflammasomes. Cell 140: 821–832. doi: 10.1016/j.cell.2010.01.040
- 64. Miller LS, Pietras EM, Uricchio LH, Hirano K, Rao S, et al. (2007) Inflammasome-Mediated Production of IL-1β Is Required for Neutrophil Recruitment against Staphylococcus aureus In Vivo. J Immunol 179: 6933–6942. doi: 10.4049/jimmunol.179.10.6933
- 65. Shin OS, Harris JB (2011) Innate immunity and transplantation tolerance: the potential role of TLRs/NLRs in GVHD. Korean J Hematol 46: 69–79. doi: 10.5045/kjh.2011.46.2.69
- 66. Franchi L, Nunez G (2008) The Nlrp3 inflammasome is critical for aluminium hydroxide-mediated IL-1beta secretion but dispensable for adjuvant activity. Eur J Immunol 38: 2085–2089. doi: 10.1002/eji.200838549
- 67. Li H, Willingham SB, Ting JP, Re F (2008) Cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3. J Immunol 181: 17–21. doi: 10.4049/jimmunol.181.1.17
- 68. Faustin B, Reed JC (2008) Sunburned skin activates inflammasomes. Trends Cell Biol 18: 4–8. doi: 10.1016/j.tcb.2007.10.004
- 69. Vegesna V, McBride WH, Taylor JM, Withers HR (1995) The effect of interleukin-1 beta or transforming growth factor-beta on radiation-impaired murine skin wound healing. J Surg Res 59: 699–704. doi: 10.1006/jsre.1995.1226
- 70. Graves DT, Nooh N, Gillen T, Davey M, Patel S, et al. (2001) IL-1 Plays a Critical Role in Oral, But Not Dermal, Wound Healing. J Immunol 167: 5316–5320.
- 71. Shimada T, Park BG, Wolf AJ, Brikos C, Goodridge HS, et al. (2010) Staphylococcus aureus evades lysozyme-based peptidoglycan digestion that links phagocytosis, inflammasome activation, and IL-1beta secretion. Cell Host Microbe 7: 38–49. doi: 10.1016/j.chom.2009.12.008
- 72. Hultgren OH, Stenson M, Tarkowski A (2001) Role of IL-12 in Staphylococcus aureus-triggered arthritis and sepsis. Arthritis Res 3: 41–47. doi: 10.1186/ar138
- 73. Tattoli I, Carneiro LA, Jehanno M, Magalhaes JG, Shu Y, et al. (2008) NLRX1 is a mitochondrial NOD-like receptor that amplifies NF-kappaB and JNK pathways by inducing reactive oxygen species production. EMBO Rep 9: 293–300. doi: 10.1038/sj.embor.7401161
- 74. Komai-Koma M, Xu D, Li Y, McKenzie AN, McInnes IB, et al. (2007) IL-33 is a chemoattractant for human Th2 cells. Eur J Immunol 37: 2779–2786. doi: 10.1002/eji.200737547
- 75. Louten J, Rankin AL, Li Y, Murphy EE, Beaumont M, et al. (2011) Endogenous IL-33 enhances Th2 cytokine production and T-cell responses during allergic airway inflammation. Int Immunol 23: 307–315. doi: 10.1093/intimm/dxr006
- 76. Dodge IL, Carr MW, Cernadas M, Brenner MB (2003) IL-6 Production by Pulmonary Dendritic Cells Impedes Th1 Immune Responses. J Immunol 170: 4457–4464.
- 77. Hume EB, Cole N, Garthwaite LL, Khan S, Willcox MD (2006) A protective role for IL-6 in staphylococcal microbial keratitis. Invest Ophthalmol Vis Sci 47: 4926–4930. doi: 10.1167/iovs.06-0340
- 78. Robertson CM, Perrone EE, McConnell KW, Dunne WM, Boody B, et al. (2008) Neutrophil depletion causes a fatal defect in murine pulmonary Staphylococcus aureus clearance. J Surg Res 150: 278–285. doi: 10.1016/j.jss.2008.02.009
- 79. Dovi JV, Szpaderska AM, DiPietro LA (2004) Neutrophil function in the healing wound: adding insult to injury? Thromb Haemost 92: 275–280. doi: 10.1160/th03-11-0720
- 80. Zhou HY, Zhang J, Yan RL, Wang Q, Fan LY, et al. (2011) Improving the antibacterial property of porcine small intestinal submucosa by nano-silver supplementation: a promising biological material to address the need for contaminated defect repair. Ann Surg 253: 1033–1041. doi: 10.1097/sla.0b013e31821260f3
- 81. Bates BL, Hiles MC, Johnson CE (2008) Biofilm-inhibiting medical products. In: Organization WIP, editor. United States.