Breast cancer-related lymphedema results in impaired epidermal differentiation and tight junction dysfunction.

Breast cancer-related lymphedema (BCRL) is characterized by skin changes, swelling, fibrosis, and recurrent skin infections. Clinical studies have suggested that lymphedema results in skin barrier defects; however, the underlying cellular mechanisms and the effects of bacterial contamination on skin barrier function remain unknown. In matched biopsies from patients with unilateral BCRL, we observed decreased expression of filaggrin and the tight junction protein zona occludens-1 (ZO-1) in skin affected by moderate lymphedema, or by subclinical lymphedema in which dermal backflow of lymph was identified by indocyanine green lymphography, relative to controls (areas without backflow and from the unaffected arm). In vitro stimulation of keratinocytes with lymph fluid obtained from patients undergoing lymphedema surgery led to the same changes, as well as increased expression of keratin 14, a marker of immature keratinocytes. Finally, using mouse models of lymphedema, we showed that like the clinical scenario, the expression of skin barrier proteins was decreased relative to normal skin and that colonization with S. epidermidis bacteria amplified this effect, as well as lymphedema severity. Taken together, our findings suggest that lymphatic fluid stasis contributes to skin barrier dysfunction in lymphedema.

Skin microbiome alterations in upper extremity secondary lymphedema.

Lymphedema is a chronic condition that commonly occur from lymphatic injury following surgical resection of solid malignancies. While many studies have centered on the molecular and immune pathways that perpetuate lymphatic dysfunction, the role of the skin microbiome in lymphedema development remains unclear. In this study, skin swabs collected from normal and lymphedema forearms of 30 patients with unilateral upper extremity lymphedema were analyzed by 16S ribosomal RNA sequencing. Statistical models for microbiome data were utilized to correlate clinical variables with microbial profiles. Overall, 872 bacterial taxa were identified. There were no significant differences in microbial alpha diversity of the colonizing bacteria between normal and lymphedema skin samples (p = 0.25). Notably, for patients without a history of infection, a one-fold change in relative limb volume was significantly associated with a 0.58-unit increase in Bray-Curtis microbial distance between paired limbs (95%CI = 0.11,1.05, p = 0.02). Additionally, several genera, including Propionibacterium and Streptococcus, demonstrated high variability between paired samples. In summary, we demonstrate high compositional heterogeneity in the skin microbiome in upper extremity secondary lymphedema, supporting future studies into the role of host-microbe interactions on lymphedema pathophysiology.

Tumor-Associated Lymphatics Upregulate MHC-II to Suppress Tumor-Infiltrating Lymphocytes.

Steady-state lymphatic endothelial cells (LECs) can induce peripheral tolerance by presenting endogenous antigens on MHC class I (MHC-I) molecules. Recent evidence suggests that lymph node LECs can cross-present tumor antigens on MHC-I to suppress tumor-specific CD8+ T cells. Whether LECs can act as immunosuppressive cells in an MHC-II dependent manner in the local tumor microenvironment (TME) is not well characterized. Using murine heterotopic and spontaneous tumor models, we show that LECs in the TME increase MHC-II expression in the context of increased co-inhibitory signals. We provide evidence that tumor lymphatics in human melanoma and breast cancer also upregulate MHC-II compared to normal tissue lymphatics. In transgenic mice that lack LEC-specific MHC-II expression, heterotopic tumor growth is attenuated, which is associated with increased numbers of tumor-specific CD8+ and effector CD4+ T cells, as well as decreased numbers of T regulatory CD4+ cells in the TME. Mechanistically, we show that murine and human dermal LECs can take up tumor antigens in vitro. Antigen-loaded LECs in vitro can induce antigen-specific proliferation of CD8+ T cells but not CD4+ T cells; however, these proliferated CD8+ T cells have reduced effector function in the presence of antigen-loaded LECs. Taken together, our study suggests LECs can act as immunosuppressive cells in the TME in an MHC-II dependent manner. Whether this is a result of direct tumor antigen presentation on MHC-II requires additional investigation.

TGF-ß1 mediates pathologic changes of secondary lymphedema by promoting fibrosis and inflammation.

BACKGROUND: Secondary lymphedema is a common complication of cancer treatment, and previous studies have shown that the expression of transforming growth factor-beta 1 (TGF-ß1), a pro-fibrotic and anti-lymphangiogenic growth factor, is increased in this disease. Inhibition of TGF-ß1 decreases the severity of the disease in mouse models; however, the mechanisms that regulate this improvement remain unknown. METHODS: Expression of TGF-ß1 and extracellular matrix molecules (ECM) was assessed in biopsy specimens from patients with unilateral breast cancer-related lymphedema (BCRL). The effects of TGF-ß1 inhibition using neutralizing antibodies or a topical formulation of pirfenidone (PFD) were analyzed in mouse models of lymphedema. We also assessed the direct effects of TGF-ß1 on lymphatic endothelial cells (LECs) using transgenic mice that expressed a dominant-negative TGF-ß receptor selectively on LECs (LECDN-RII ). RESULTS: The expression of TGF-ß1 and ECM molecules is significantly increased in BCRL skin biopsies. Inhibition of TGF-ß1 in mouse models of lymphedema using neutralizing antibodies or with topical PFD decreased ECM deposition, increased the formation of collateral lymphatics, and inhibited infiltration of T cells. In vitro studies showed that TGF-ß1 in lymphedematous tissues increases fibroblast, lymphatic endothelial cell (LEC), and lymphatic smooth muscle cell stiffness. Knockdown of TGF-ß1 responsiveness in LECDN-RII resulted in increased lymphangiogenesis and collateral lymphatic formation; however, ECM deposition and fibrosis persisted, and the severity of lymphedema was indistinguishable from controls. CONCLUSIONS: Our results show that TGF-ß1 is an essential regulator of ECM deposition in secondary lymphedema and that inhibition of this response is a promising means of treating lymphedema.

Circulating IgA Antibodies Against Fusobacterium nucleatum Amyloid Adhesin FadA are a Potential Biomarker for Colorectal Neoplasia.

Fusobacterium nucleatum (Fn) is a gram-negative oral anaerobe and prevalent in colorectal cancer. Fn encodes a unique amyloid-like adhesin, FadA complex (FadAc), consisting of intact pre-FadA and cleaved mature FadA, to promote colorectal cancer tumorigenesis. We aimed to evaluate circulating anti-FadAc antibody levels as a biomarker for colorectal cancer. Circulating anti-FadAc IgA and IgG levels were measured by ELISA in two study populations. In study 1, plasma samples from patients with colorectal cancer (n = 25) and matched healthy controls (n = 25) were obtained from University Hospitals Cleveland Medical Center. Plasma levels of anti-FadAc IgA were significantly increased in patients with colorectal cancer (mean ± SD: 1.48 ± 1.07 µg/mL) compared with matched healthy controls (0.71 ± 0.36 µg/mL; P = 0.001). The increase was significant in both early (stages I and II) and advanced (stages III and IV) colorectal cancer. In study 2, sera from patients with colorectal cancer (n = 50) and patients with advanced colorectal adenomas (n = 50) were obtained from the Weill Cornell Medical Center biobank. Anti-FadAc antibody titers were stratified according to the tumor stage and location. Similar as study 1, serum levels of anti-FadAc IgA were significantly increased in patients with colorectal cancer (2.06 ± 1.47 µg/mL) compared with patients with colorectal adenomas (1.49 ± 0.99 µg/mL; P = 0.025). Significant increase was limited to proximal cancers, but not distal tumors. Anti-FadAc IgG was not increased in either study population, suggesting that Fn likely translocates through the gastrointestinal tract and interact with colonic mucosa. Anti-FadAc IgA, but not IgG, is a potential biomarker for early detection of colorectal neoplasia, especially for proximal tumors. Significance: Fn, an oral anaerobe highly prevalent in colorectal cancer, secretes the amyloid-like FadAc to promote colorectal cancer tumorigenesis. We report that circulating levels of anti-FadAc IgA, but not IgG, are increased in patients with both early and advanced colorectal cancer compared with the healthy controls, and especially in those with proximal colorectal cancer. Anti-FadAc IgA may be developed into a serological biomarker for early detection of colorectal cancer.

Pilot Study of Anti-Th2 Immunotherapy for the Treatment of Breast Cancer-Related Upper Extremity Lymphedema.

Recent studies suggest that Th2 cells play a key role in the pathology of secondary lymphedema by elaborating cytokines such as IL4 and IL13. The aim of this study was to test the efficacy of QBX258, a monoclonal IL4/IL13 neutralizing antibody, in women with breast cancer-related lymphedema (BCRL). We enrolled nine women with unilateral stage I/II BCRL and treated them once monthly with intravenous infusions of QBX258 for 4 months. We measured limb volumes, bioimpedance, and skin tonometry, and analyzed the quality of life (QOL) using a validated lymphedema questionnaire (Upper Limb Lymphedema 27, ULL-27) before treatment, immediately after treatment, and 4 months following treatment withdrawal. We also obtained 5 mm skin biopsies from the normal and lymphedematous limbs before and after treatment. Treatment was well-tolerated; however, one patient with a history of cellulitis developed cellulitis during the trial and was excluded from further analysis. We found no differences in limb volumes or bioimpedance measurements after drug treatment. However, QBX258 treatment improved skin stiffness (p < 0.001) and improved QOL measurements (Physical p < 0.05, Social p = 0.01). These improvements returned to baseline after treatment withdrawal. Histologically, treatment decreased epidermal thickness, the number of proliferating keratinocytes, type III collagen deposition, infiltration of mast cells, and the expression of Th2-inducing cytokines in the lymphedematous skin. Our limited study suggests that immunotherapy against Th2 cytokines may improve skin changes and QOL of women with BCRL. This treatment appears to be less effective for decreasing limb volumes; however, additional studies are needed.

Bacterial Lipoproteins Induce BAFF Production via TLR2/MyD88/JNK Signaling Pathways in Dendritic Cells.

B-cell activating factor (BAFF) plays a crucial role in survival, differentiation, and antibody secretion of B cells. Microbial products with B-cell mitogenic properties can indirectly promote expansion and activation of B cells by stimulating accessory cells, such as dendritic cells (DCs), to induce BAFF. Although bacterial lipoproteins are potent B-cell mitogen like lipopolysaccharides (LPSs), it is uncertain whether they can stimulate DCs to induce BAFF expression. Here, we evaluated the effect of bacterial lipoproteins on BAFF expression in mouse bone marrow-derived DCs. Lipoprotein-deficient Staphylococcus aureus mutant induced relatively low expression level of membrane-bound BAFF (mBAFF) and the mRNA compared with its wild-type strain, implying that bacterial lipoproteins can positively regulate BAFF induction. The synthetic lipopeptides Pam2CSK4 and Pam3CSK4, which mimic bacterial lipoproteins, dose-dependently induced BAFF expression, and their BAFF-inducing capacities were comparable to those of LPS in DCs. Induction of BAFF by the lipopeptide was higher than the induction by other microbe-associated molecular patterns, including peptidoglycan, flagellin, zymosan, lipoteichoic acid, and poly(I:C). Pam3CSK4 induced both mBAFF and soluble BAFF expression in a dose- and time-dependent manner. BAFF expression by Pam3CSK4 was completely absent in DCs from TLR2- or MyD88-deficient mice. Among various MAP kinase inhibitors, only JNK inhibitors blocked Pam3CSK4-induced BAFF mRNA expression, while inhibitors blocking ERK or p38 kinase had no such effect. Furthermore, Pam3CSK4 increased the DNA-binding activities of NF-κB and Sp1, but not that of C/EBP. Pam3CSK4-induced BAFF promoter activity via TLR2/1 was blocked by NF-κB or Sp1 inhibitor. Collectively, these results suggest that bacterial lipoproteins induce expression of BAFF through TLR2/MyD88/JNK signaling pathways leading to NF-κB and Sp1 activation in DCs, and BAFF derived from bacterial lipoprotein-stimulated DCs induces B-cell proliferation.

Tumor Lymphatic Function Regulates Tumor Inflammatory and Immunosuppressive Microenvironments.

Proliferation of aberrant, dysfunctional lymphatic vessels around solid tumors is a common histologic finding. Studies have shown that abnormalities in lymphatic function result in accumulation of inflammatory cells with an immunosuppressive profile. We tested the hypothesis that dysfunctional lymphatic vessels surrounding solid tumors regulate changes in the tumor microenvironment and tumor-specific immune responses. Using subcutaneously implanted mouse melanoma and breast cancer tumors in a lymphatic endothelial cell-specific diphtheria toxin receptor transgenic mouse, we found that local ablation of lymphatic vessels increased peritumoral edema, as compared with controls. Comparative analysis of the peritumoral fluid demonstrated increases in the number of macrophages, CD4+ inflammatory cells, F4/80+/Gr-1+ (myeloid-derived suppressor cells), CD4+/Foxp3+ (Tregs) immunosuppressive cells, and expression of inflammatory cytokines such as TNFα, IFNγ, and IL1ß following lymphatic ablation. Tumors grown in lymphatic ablated mice exhibited reduced intratumoral accumulation of cytotoxic T cells and increased tumor PD-L1 expression, causing rapid tumor growth, compared with tumors grown in nonlymphatic-ablated mice. Our study suggests that lymphatic dysfunction plays a role in regulating tumor microenvironments and may be therapeutically targeted in combination with immunotherapy to prevent tumor growth and progression.

Regulation of Immune Function by the Lymphatic System in Lymphedema.

The lymphatic vasculature has traditionally been thought to play a passive role in the regulation of immune responses by transporting antigen presenting cells and soluble antigens to regional lymph nodes. However, more recent studies have shown that lymphatic endothelial cells regulate immune responses more directly by modulating entry of immune cells into lymphatic capillaries, presenting antigens on major histocompatibility complex proteins, and modulating antigen presenting cells. Secondary lymphedema is a disease that develops when the lymphatic system is injured during surgical treatment of cancers or is damaged by infections. We have used mouse models of lymphedema in order to understand the effects of chronic lymphatic injury on immune responses and have shown that lymphedema results in a mixed T helper cell and T regulatory cell (Treg) inflammatory response. Prolonged T helper 2 biased immune responses in lymphedema regulate the pathology of this disease by promoting tissue fibrosis, inhibiting formation of collateral lymphatics, decreasing lymphatic vessel pumping capacity, and increasing lymphatic leakiness. Treg infiltration following lymphatic injury results from proliferation of natural Tregs and suppresses innate and adaptive immune responses. These studies have broad clinical relevance since understanding how lymphatic injury in lymphedema can modulate immune responses may provide a template with which we can study more subtle forms of lymphatic injury that may occur in physiologic conditions such as aging, obesity, metabolic tumors, and in the tumor microenvironment.

Fibrosis and secondary lymphedema: chicken or egg?

Secondary lymphedema is a common complication of cancer treatment resulting in progressive fibroadipose tissue deposition, increased risk of infections, and, in rare cases, secondary malignancies. Until recently, the pathophysiology of secondary lymphedema was thought to be related to impaired collateral lymphatic formation after surgical injury. However, more recent studies have shown that chronic inflammation-induced fibrosis plays a key role in the pathophysiology of this disease. In this review, we will discuss the evidence supporting this hypothesis and summarize recent publications demonstrating that lymphatic injury activates chronic immune responses that promote fibrosis and lymphatic leakiness, decrease collecting lymphatic pumping, and impair collateral lymphatic formation. We will review how chronic mixed T-helper cell inflammatory reactions regulate this process and how this response may be used to design novel therapies for lymphedema.

Fusobacterium nucleatum promotes colorectal cancer by inducing Wnt/ß-catenin modulator Annexin A1.

Fusobacterium nucleatum, a Gram-negative oral anaerobe, is a significant contributor to colorectal cancer. Using an in vitro cancer progression model, we discover that F. nucleatum stimulates the growth of colorectal cancer cells without affecting the pre-cancerous adenoma cells. Annexin A1, a previously unrecognized modulator of Wnt/ß-catenin signaling, is a key component through which F. nucleatum exerts its stimulatory effect. Annexin A1 is specifically expressed in proliferating colorectal cancer cells and involved in activation of Cyclin D1. Its expression level in colon cancer is a predictor of poor prognosis independent of cancer stage, grade, age, and sex. The FadA adhesin from F. nucleatum up-regulates Annexin A1 expression through E-cadherin. A positive feedback loop between FadA and Annexin A1 is identified in the cancerous cells, absent in the non-cancerous cells. We therefore propose a "two-hit" model in colorectal carcinogenesis, with somatic mutation(s) serving as the first hit, and F. nucleatum as the second hit exacerbating cancer progression after benign cells become cancerous. This model extends the "adenoma-carcinoma" model and identifies microbes such as F. nucleatum as cancer "facilitators".

Streptococcus gordonii induces nitric oxide production through its lipoproteins stimulating Toll-like receptor 2 in murine macrophages.

Streptococcus gordonii, a Gram-positive commensal in the oral cavity, is an opportunistic pathogen that can cause endodontic and systemic infections resulting in infective endocarditis. Lipoteichoic acid (LTA) and lipoprotein are major virulence factors of Gram-positive bacteria that are preferentially recognized by Toll-like receptor 2 (TLR2) on immune cells. In the present study, we investigated the effect of S. gordonii LTA and lipoprotein on the production of the representative inflammatory mediator nitric oxide (NO) by the mouse macrophages. Heat-killed S. gordonii wild-type and an LTA-deficient mutant (ΔltaS) but not a lipoprotein-deficient mutant (Δlgt) induced NO production in mouse primary macrophages and the cell line, RAW 264.7. S. gordonii wild-type and ΔltaS also induced the expression of inducible NO synthase (iNOS) at the mRNA and protein levels. In contrast, the Δlgt mutant showed little effect under the same condition. Furthermore, S. gordonii wild-type and ΔltaS induced NF-κB activation, STAT1 phosphorylation, and IFN-ß expression, which are important for the induction of iNOS gene expression, with little activation by Δlgt. S. gordonii wild-type and ΔltaS showed an increased adherence and internalization to RAW 264.7 cells compared to Δlgt. In addition, S. gordonii wild-type and ΔltaS, but not Δlgt, substantially increased TLR2 activation while none of these induced NO production in TLR2-deficient macrophages. Triton X-114-extracted lipoproteins from S. gordonii were sufficient to induce NO production. Collectively, we suggest that lipoprotein is an essential cell wall component of S. gordonii to induce NO production in macrophages through TLR2 triggering NF-κB and STAT1 activation.

Sodium Hypochlorite Inactivates Lipoteichoic Acid of Enterococcus faecalis by Deacylation.

INTRODUCTION: Enterococcus faecalis is a pathogenic gram-positive bacterium closely associated with apical periodontitis. Although sodium hypochlorite (NaOCl) has been used as a common endodontic irrigant to eradicate bacteria in the root canal, it has not been elucidated whether NaOCl attenuates the inflammatory response induced by the E. faecalis virulence factor lipoteichoic acid (EfLTA). METHODS: Structurally intact EfLTA purified from E. faecalis was treated with NaOCl at various concentrations and time periods. Murine macrophage cell line RAW 264.7 was treated with interferon gamma followed by treatment with intact or NaOCl-treated EfLTA to determine the inducibility of inflammatory mediators such as nitric oxide, interferon gamma-inducible protein 10, and macrophage inflammatory protein-1α. Reporter gene assays assessed by flow cytometry were used to examine the ability of intact or NaOCl-treated EfLTA to activate Toll-like receptor 2 (TLR2), which is known to recognize EfLTA on host cells. Structural damage of EfLTA by NaOCl was examined using silver staining and thin-layer chromatography. RESULTS: NaOCl-treated EfLTA showed markedly less induction of nitric oxide, interferon gamma-inducible protein 10, and macrophage inflammatory protein-1α in RAW 264.7 cells compared with intact EfLTA. In contrast to intact EfLTA that potently stimulated TLR2 activation, NaOCl-treated EfLTA did not activate TLR2. Structural analysis showed that NaOCl damaged EfLTA structure by deacylation. CONCLUSIONS: NaOCl deacylates the glycolipid moiety of EfLTA, which fails to activate TLR2, leading to the reduced production of inflammatory mediators.

Human salivary proteins with affinity to lipoteichoic acid of Enterococcus faecalis.

Enterococcus faecalis is associated with refractory apical periodontitis and its lipoteichoic acid (Ef.LTA) is considered as a major virulence factor. Although the binding proteins of Ef.LTA may play an important role for mediating infection and immunity in the oral cavity, little is known about Ef.LTA-binding proteins (Ef.LTA-BPs) in saliva. In this study, we identified salivary Ef.LTA-BPs with biotinylated Ef.LTA (Ef.LTA-biotin) through mass spectrometry. The biotinylation of Ef.LTA was confirmed by binding capacity with streptavidin-FITC on CHO/CD14/TLR2 cells. The biological activity of Ef.LTA-biotin was determined based on the induction of nitric oxide and macrophage inflammatory protein-1α in a macrophage cell-line, RAW 264.7. To identify salivary Ef.LTA-BPs, the Ef.LTA-biotin was mixed with a pool of human saliva obtained from nine healthy subjects followed by precipitation with a streptavidin-coated bead. Ef.LTA-BPs were then separated with 12% SDS-PAGE and subjected to the mass spectrometry. Six human salivary Ef.LTA-BPs including short palate lung and nasal epithelium carcinoma-associated protein 2, zymogen granule protein 16 homolog B, hemoglobin subunit α and ß, apolipoprotein A-I, and lipocalin-1 were identified with statistical significance (P<0.05). Ef.LTA-BPs were validated with lipocalin-1 using pull-down assay. Hemoglobin inhibited the biofilm formation of E. faecalis whereas lipocalin-1 did not show such effect. Collectively, the identified Ef.LTA-BPs could provide clues for our understanding of the pathogenesis of E. faecalis and host immunity in oral cavity.

Lipoteichoic Acid of Enterococcus faecalis Inhibits the Differentiation of Macrophages into Osteoclasts.

INTRODUCTION: Enterococcus faecalis is associated with persistent endodontic infection and refractory apical periodontitis. Recently, we have shown that heat-killed E. faecalis attenuates osteoclast differentiation. Because lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria, we investigated the effect of LTA from E. faecalis (EfLTA) on osteoclast differentiation. METHODS: EfLTA was purified through organic solvent extraction, hydrophobic interaction column chromatography, and ion exchange column chromatography. Bone marrow cells from C57BL/6 or Toll-like receptor 2-deficient mice were incubated with macrophage colony-stimulating factor (M-CSF) for 5 days to generate macrophages (bone marrow-derived macrophages [BMMs]). The cells were differentiated into osteoclasts with M-CSF and receptor activator of NF-κB ligand (RANKL) in the presence or absence of EfLTA. The degree of osteoclast differentiation was determined by tartrate-resistant acid phosphatase staining. The expression of NFATc1 and c-Fos transcription factors was determined by Western blotting. A phagocytosis assay was performed by measuring the uptake of carboxyfluorescein diacetate succinimidyl ester-stained E. faecalis. An enzyme-linked immunosorbent assay was used to determine the amount of cytokines and chemokines. RESULTS: When BMMs were treated with EfLTA, osteoclast differentiation was attenuated. EfLTA inhibited the RANKL-induced expression of NFATc1 and c-Fos. EfLTA inhibition of osteoclast differentiation was not observed in TLR2-deficient BMMs. In addition, EfLTA sustained the phagocytic capacity of BMMs even after the differentiation into osteoclasts, whereas it induced the expression of inflammatory cytokines and chemokines. CONCLUSIONS: EfLTA inhibits the differentiation of macrophages into osteoclasts and thereby maintains the phagocytic and inflammatory capacities of macrophages, potentially contributing to refractory apical periodontitis.

Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus.

A Gram-positive bacterium, Staphylococcus aureus is known to be one of the major pathogenic bacteria responsible for causing bovine mastitis. Among the various cell wall components of S. aureus, lipoteichoic acid (LTA) and peptidoglycan (PGN) are closely associated with inflammatory responses. However, the role of LTA and PGN derived from S. aureus in bovine mastitis has not been clearly elucidated. In this study, we characterized the gene expression profile of a bovine mammary gland epithelial cell line, MAC-T cells, in the presence of LTA and PGN from S. aureus. LTA plus PGN, but not LTA or PGN alone, activated MAC-T cells. The analysis of transcriptional profiles using an Affymetrix genechip microarray showed that stimulation with LTA plus PGN produced a total of 2019 (fold change >1.2) differentially expressed genes (DEGs), with 801 up-regulated genes and 1218 down-regulated genes. Of the up-regulated genes, 14 inflammatory mediator-related DEGs, 22 intra-cellular signaling molecule-related DEGs, and 15 transcription factor-related DEGs were observed, whereas among the down-regulated DEGs 17 inflammation-related DEGs were found. The microarray results were confirmed using real-time RT-PCR of 18 genes with substantial changes in expression (9 each from the up-regulated and down-regulated DEGs). These results provide a comprehensive analysis of gene-expression profiles elicited by S. aureus LTA and PGN in MAC-T cells, contributing to an understanding of the pathogenesis for S. aureus-induced bovine mastitis.

Lipoteichoic acid of Streptococcus mutans interacts with Toll-like receptor 2 through the lipid moiety for induction of inflammatory mediators in murine macrophages.

Streptococcus mutans is a pathogenic Gram-positive bacterium that is closely associated with dental caries and subsequent pulpal inflammation. Although lipoteichoic acid (LTA) is considered a major virulence factor of Gram-positive bacteria, little is known about the innate immunity to S. mutans LTA. In this study, we purified LTA from S. mutans (Sm.LTA) through n-butanol extraction, hydrophobic interaction column chromatography, and ion-exchange column chromatography to investigate its immunological properties using murine macrophages. The Sm.LTA preparation had no detectable contamination with endotoxins, proteins, or nucleic acids. Upon exposure to Sm.LTA, the murine macrophage cell-line RAW 264.7 cells produced TNF-α and nitric oxide (NO) in a dose-dependent manner. Sm.LTA preferentially bound to and activated CHO/CD14/TLR2 cells rather than CHO/CD14/TLR4 cells, which are stable transfectants expressing CD14 and TLR2 or CD14 and TLR4, respectively. Sm.LTA could not induce TNF-α or NO production in macrophages derived from TLR2-deficient mice whereas it dose-dependently induced those inflammatory mediators in wild-type macrophages. TLR2-dependent induction of NO by Sm.LTA was also confirmed in RAW 264.7 cells using specific antibodies blocking TLR2. Furthermore, Sm.LTA deacylated by alkaline hydrolysis neither stimulated TLR2 nor induced TNF-α or NO production, suggesting that Sm.LTA lipid moieties are crucial for the immuno-stimulatory activity of Sm.LTA. Unlike Staphylococcus aureus LTA, which has potent immuno-stimulating activity, Sm.LTA showed a modest induction of NO production comparable to LTAs of other oral bacteria Enterococcus faecalis and Lactobacillus plantarum. In conclusion, our results suggest that the Sm.LTA interacts with TLR2 through the lipid moiety for the induction of inflammatory mediators in macrophages.

Muramyl dipeptide potentiates staphylococcal lipoteichoic acid induction of cyclooxygenase-2 expression in macrophages.

Gram-positive bacteria contain lipoteichoic acid (LTA) and peptidoglycan (PGN) layers, both of which are considered as major virulence factors associated with inflammation. Cyclooxygenase-2 (COX-2) plays an important role in the inflammation by generating prostaglandins at infections. Since LTA and PGN are thought to cooperate in the establishment of inflammation, we examined the ability of staphylococcal LTA (Sa.LTA) to induce COX-2 expression in the presence of muramyl dipeptide (MDP), which is the minimal structural unit of PGN required for inflammation, in macrophages. While MDP failed to induce COX-2 expression, Sa.LTA alone was sufficient to induce COX-2 production. Treatment with MDP enhanced Sa.LTA-induced COX-2 and prostaglandin E2 production. The cooperative effect between Sa.LTA and MDP was not observed in COX-2 expression by macrophages derived from Toll-like receptor 2 (TLR2)- or nucleotide-binding oligomerization domain 2 (NOD2)-deficient mice. In addition, MDP enhanced Sa.LTA-induced activation of the transcription factors NF-κB and CRE, which are known to modulate COX-2 gene transcription. Conclusively, these results suggest that MDP and Sa.LTA cooperatively induce inflammatory response by overproducing COX-2 through NOD2 and TLR2.

Lipoteichoic acid of Enterococcus faecalis induces the expression of chemokines via TLR2 and PAFR signaling pathways.

Enterococcus faecalis is one of the most common opportunistic pathogens responsible for nosocomial infections, and its LTA is known as an important virulence factor causing inflammatory responses. As chemokines play a key role in inflammatory diseases by triggering leukocyte infiltration into the infection site, we purified EfLTA and investigated its effect on the expression of chemokines, IP-10, MIP-1α, and MCP-1, in murine macrophages. EfLTA induced the expression of these chemokines at the mRNA and protein levels. TLR2, CD14, and MyD88 were involved in the EfLTA-induced chemokine expression, as the expression was reduced remarkably in macrophages derived from TLR2-, CD14-, or MyD88-deficient mice. EfLTA induced phosphorylation of MAPKs and enhanced the DNA-binding activity of NF-κB, AP-1, and NF-IL6 transcription factors. The induction of IP-10 required ERK, JNK, p38 MAPK, PKC, PTK, PI3K, and ROS. We noticed that all of these signaling molecules, except p38 MAPK and ROS, were indispensable for the induction of MCP-1 and MIP-1α. Interestingly, the EfLTA-induced chemokine expression was mediated through PAFR/JAK/STAT1 signaling pathways without IFN-ß involvement, which is different from LPS-induced chemokine expression requiring IFN-ß/JAK/STAT1 signaling pathways. Furthermore, the culture supernatant of EfLTA-treated RAW 264.7 cells promoted the platelet aggregation, and exogenous PAF induced the chemokine expression in macrophages derived from WT and TLR2-deficient mice. These results suggest that EfLTA induces the expression of chemokines via signaling pathways requiring TLR2 and PAFR, which is distinct from that of LPS-induced chemokine expression.

Lipoteichoic acid of Staphylococcus aureus enhances IL-6 expression in activated human basophils.

At allergic inflammation, cross-linking of FcɛRI with multivalent antigen-bound IgE triggers the signaling pathways via activation of protein kinase C (PKC) and mobilization of intracellular Ca(2+) leading to the production of various mediators such as interleukin-6 (IL-6). Accumulating reports demonstrated that interaction of Toll-like receptor 2 (TLR2) expressed on basophils with a TLR2 ligand, lipoteichoic acid (LTA) of Staphylococcus aureus, exacerbated allergic inflammation. Here, we showed that staphylococcal LTA (Sa.LTA) substantially enhanced IL-6 expression at both the protein and the mRNA levels in the human basophil line, KU812, in the presence of a PKC activator (phorbol 12-myristrate 13-acetate; PMA), and a calcium ionophore (A23187), whereas Sa.LTA alone could not induce IL-6 expression. PMA/A23187 augmented the expression of CD14 and TLR2 on the surface of KU812 cells and concomitantly increased the binding of fluorochrome-labeled Sa.LTA to the cells. Sa.LTA enhanced the phosphorylation of mitogen-activated protein (MAP) kinases in PMA/A23187-stimulated KU812 cells. Notably, Sa.LTA could not enhance PMA/A23187-induced IL-6 expression in the presence of inhibitors of MAP kinases, reactive oxygen species, or protein kinase C. Furthermore, Sa.LTA enhanced the PMA/A23187-increased DNA-binding activities of the transcription factors NF-κB and AP-1. Experiments using human peripheral blood mononuclear cells demonstrated that not only PMA/A23187 but also Sa.LTA increased the intracellular IL-6 expression in the basophil population and Sa.LTA plus PMA/A23187 further enhanced the IL-6 expression. Collectively, these results suggest that Sa.LTA exacerbates allergic inflammation by potentiating IL-6 production from activated basophils.