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.

Structural and Functional Changes in Aged Skin Lymphatic Vessels.

Lymphatic structure and function play a critical role in fluid transport, antigen delivery, and immune homeostasis. A dysfunctional lymphatic system is associated with chronic low-grade inflammation of peripheral tissues, poor immune responses, and recurrent infections, which are also hallmarks of aging pathology. Previous studies have shown that aging impairs lymphatic structure and function in a variety of organ systems, including the intestines and central nervous system. However, previous studies are mostly limited to qualitative analysis of lymphatic structural changes and quantification of intestinal collecting vessel contractile function. It is not clear whether decreased lymphatic function contributes to pathological conditions related to aging, nor how it affects the skin immune microenvironment. Further, the effects of aging on skin initial and collecting lymphatic vessels, dendritic cell (DC) migration, cutaneous lymphatic pumping, and VEGFR-3 signaling in lymphatic endothelial cells (LECs) have not been quantitatively analyzed. Here, using fluorescent immunohistochemistry and flow cytometry, we confirm that aging decreases skin initial and collecting lymphatic vessel density. Indocyanine green (ICG) lymphangiography and DC migration assays confirm that aging decreases both fluid pumping and cell migration via lymphatic vessels. At the cellular level, aging causes decreased VEGFR-3 signaling, leading to increased LEC apoptosis and senescence. Finally, we determined that aging causes decreased lymphatic production of chemokines and alters LEC expression of junctional and adhesion molecules. This in turn leads to increased peri-lymphatic inflammation and nitrosative stress that might contribute to aging pathology in a feed-forward manner. Taken together, our study, in addition to quantitatively corroborating previous findings, suggests diverse mechanisms that contribute to lymphatic dysfunction in aging that in turn exacerbate the pathology of aging in a feed-forward manner.

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.

Lymphatic-specific intracellular modulation of receptor tyrosine kinase signaling improves lymphatic growth and function.

Exogenous administration of lymphangiogenic growth factors is widely used to study changes in lymphatic function in pathophysiology. However, this approach can result in off-target effects, thereby generating conflicting data. To circumvent this issue, we modulated intracellular VEGF-C signaling by conditionally knocking out the lipid phosphatase PTEN using the Vegfr3 promoter to drive the expression of Cre-lox in lymphatic endothelial cells (LECs). PTEN is an intracellular brake that inhibits the downstream effects of the activation of VEGFR3 by VEGF-C. Activation of Cre-lox recombination in adult mice resulted in an expanded functional lymphatic network due to LEC proliferation that was independent of lymphangiogenic growth factor production. Furthermore, compared with lymphangiogenesis induced by VEGF-C injection, LECPTEN animals had mature, nonleaky lymphatics with intact cell-cell junctions and reduced local tissue inflammation. Last, compared with wild-type or VEGF-C-injected mice, LECPTEN animals had an improved capacity to resolve inflammatory responses. Our findings indicate that intracellular modulation of lymphangiogenesis is effective in inducing functional lymphatic networks and has no off-target inflammatory effects.

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.

Regulation of lymphatic function and injury by nitrosative stress in obese mice.

OBJECTIVE: Obesity results in lymphatic dysfunction, but the cellular mechanisms that mediate this effect remain largely unknown. Previous studies in obese mice have shown that inducible nitric oxide synthase-expressing (iNOS+) inflammatory cells accumulate around lymphatic vessels. In the current study, we therefore tested the hypothesis that increased expression of iNOS results in nitrosative stress and injury to the lymphatic endothelial cells (LECs). In addition, we tested the hypothesis that lymphatic injury, independent of obesity, can modulate glucose and lipid metabolism. METHODS: We compared the metabolic changes and lymphatic function of wild-type and iNOS knockout mice fed a normal chow or high-fat diet for 16 weeks. To corroborate our in vivo findings, we analyzed the effects of reactive nitrogen species on isolated LECs. Finally, using a genetically engineered mouse model that allows partial ablation of the lymphatic system, we studied the effects of acute lymphatic injury on glucose and lipid metabolism in lean mice. RESULTS: The mesenteric lymphatic vessels of obese wild-type animals were dilated, leaky, and surrounded by iNOS+ inflammatory cells with resulting increased accumulation of reactive nitrogen species when compared with lean wild-type or obese iNOS knockout animals. These changes in obese wild-type mice were associated with systemic glucose and lipid abnormalities, as well as decreased mesenteric LEC expression of lymphatic-specific genes, including vascular endothelial growth factor receptor 3 (VEGFR-3) and antioxidant genes as compared with lean wild-type or obese iNOS knockout animals. In vitro experiments demonstrated that isolated LECs were more sensitive to reactive nitrogen species than blood endothelial cells, and that this sensitivity was ameliorated by antioxidant therapies. Finally, using mice in which the lymphatics were specifically ablated using diphtheria toxin, we found that the interaction between metabolic abnormalities caused by obesity and lymphatic dysfunction is bidirectional. Targeted partial ablation of mesenteric lymphatic channels of lean mice resulted in increased accumulation of iNOS+ inflammatory cells and increased reactive nitrogen species. Lymphatic ablation also caused marked abnormalities in insulin sensitivity, serum glucose and insulin concentrations, expression of insulin-sensitive genes, lipid metabolism, and significantly increased systemic and mesenteric white adipose tissue (M-WAT) inflammatory responses. CONCLUSIONS: Our studies suggest that increased iNOS production in obese animals plays a key role in regulating lymphatic injury by increasing nitrosative stress. In addition, our studies suggest that obesity-induced lymphatic injury may amplify metabolic abnormalities by increasing systemic and local inflammatory responses and regulating insulin sensitivity. These findings suggest that manipulation of the lymphatic system may represent a novel means of treating metabolic abnormalities associated with obesity.

Regulation of Lymphatic Function in Obesity.

The lymphatic system has many functions, including macromolecules transport, fat absorption, regulation and modulation of adaptive immune responses, clearance of inflammatory cytokines, and cholesterol metabolism. Thus, it is evident that lymphatic function can play a key role in the regulation of a wide array of biologic phenomenon, and that physiologic changes that alter lymphatic function may have profound pathologic effects. Recent studies have shown that obesity can markedly impair lymphatic function. Obesity-induced pathologic changes in the lymphatic system result, at least in part, from the accumulation of inflammatory cells around lymphatic vessel leading to impaired lymphatic collecting vessel pumping capacity, leaky initial and collecting lymphatics, alterations in lymphatic endothelial cell (LEC) gene expression, and degradation of junctional proteins. These changes are important since impaired lymphatic function in obesity may contribute to the pathology of obesity in other organ systems in a feed-forward manner by increasing low-grade tissue inflammation and the accumulation of inflammatory cytokines. More importantly, recent studies have suggested that interventions that inhibit inflammatory responses, either pharmacologically or by lifestyle modifications such as aerobic exercise and weight loss, improve lymphatic function and metabolic parameters in obese mice. The purpose of this review is to summarize the pathologic effects of obesity on the lymphatic system, the cellular mechanisms that regulate these responses, the effects of impaired lymphatic function on metabolic syndrome in obesity, and the interventions that may improve lymphatic function in obesity.

Lipoteichoic acid of Enterococcus faecalis interferes with Porphyromonas gingivalis lipopolysaccharide signaling via IRAK-M upregulation in human periodontal ligament cells.

Periodontitis is a chronic inflammatory disease of the gum caused by infection with multispecies oral bacteria. Since the periodontopathic bacteria, Porphyromonas gingivalis together with Enterococcus faecalis are frequently detected in patients with a severe form of periodontitis, interactions between their virulence factors might play an important role in progression of the disease. P. gingivalis and E. faecalis possess lipopolysaccharide (Pg.LPS) and lipoteichoic acid (Ef.LTA), respectively, as the major virulence factors inducing inflammatory responses. However, the combinatorial effect of these virulence factors on chemokine expression was poorly understood. Here, we examined the interaction between Ef.LTA and Pg.LPS on IL-8 induction in human periodontal ligament (PDL) cells. Pg.LPS, but not Ef.LTA, induced IL-8 expression at both mRNA and protein levels, which was suppressed in the presence of Ef.LTA. Although Ef.LTA and Pg.LPS could stimulate Toll-like receptor 2 (TLR2), Ef.LTA did not interfere with Pg.LPS induced-TLR2 activation. However, Ef.LTA decreased Pg.LPS-induced phosphorylation of ERK, JNK, and p38 kinase. Furthermore, Ef.LTA suppressed Pg.LPS-induced IL-8 promoter activity as well as AP-1, NF-IL6 and NF-κB transcription factors, which are indispensable for IL-8 expression. Interestingly, Ef.LTA enhanced only IL-1 receptor-associated kinase-M (IRAK-M) expression among the tested negative regulators of TLR intracellular signaling cascades in the presence of Pg.LPS. In addition, silencing IRAK-M restored the decreased IL-8 expression by Ef.LTA in the presence of Pg.LPS. Collectively, these results suggest that Ef.LTA inhibits Pg.LPS-induced IL-8 expression in human PDL cells via inducing the expression of a negative regulator of TLR signaling cascades, IRAK-M.

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".

Lipoproteins in Streptococcus gordonii are critical in the infection and inflammatory responses.

Gram-positive bacteria such as Streptococcus gordonii causing life-threatening infective endocarditis are mainly recognized by Toll-like receptor 2 (TLR2). Lipoteichoic acid (LTA) and lipoproteins are representative TLR2 ligands that play important roles in bacterial infection and in host inflammatory responses. In the present study, we generated an LTA-deficient mutant (ΔltaS) and a lipoprotein-deficient mutant (Δlgt) and investigated the contributions of LTA and lipoproteins to bacterial morphology and their effect on induction of proinflammatory cytokines in THP-1 and mouse bone-marrow derived macrophages (BMDMs). Deletion of ltaS and lgt was confirmed by PCR analysis of genomic DNA from each mutant. The mutants with absence of LTA or lipoproteins were examined by SDS-PAGE followed by Western blotting with anti-LTA antibodies and silver staining, respectively. Interestingly, scanning and transmission electron microscopies showed no difference in the bacterial cell morphology or size between the wild-type and the mutants even though substantial changes in the cell size and/or morphology have been reported in other Gram-positive bacteria such as Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis. However, S. gordonii wild-type and ΔltaS potently induced the expression of proinflammatory cytokines including TNF-α, IL-8, and IL-1ß at the mRNA and protein levels, while Δlgt did not have these effects. Furthermore, lipoproteins purified from S. gordonii also induced the expression of the aforementioned cytokines more potently than the purified LTA. Neither LTA nor lipoprotein induced TNF-α, KC (IL-8 counterpart in mouse), and IL-1ß in TLR2-deficient BMDMs. S. gordonii Δlgt was less virulent than the wild-type or ΔltaS in a mouse intraperitoneal infection model. Collectively, these results suggest that S. gordonii lipoproteins, but not LTA, are mainly responsible for the infection and inflammatory responses.

Lipoteichoic Acid Inhibits Staphylococcus aureus Biofilm Formation.

A biofilm is an aggregate of microorganisms in which cells adhere to biological or non-biological surfaces and is responsible for various infectious diseases. Infections caused by Staphylococcus aureus, including pneumonia, endocarditis, and osteomyelitis, are often associated with colonization and biofilm formation. Although lipoteichoic acid (LTA) is involved in biofilm formation, the specific role of LTA is not clearly understood. In this study, we demonstrated that LTA released from Lactobacillus plantarum could inhibit S. aureus biofilm formation and aggregation without affecting the growth of S. aureus in various in vitro and in vivo models. L. plantarum LTA (Lp.LTA) also inhibited biofilm formation of S. aureus clinical isolates, including a methicillin-resistant strain. Remarkably, Lp.LTA not only interfered with S. aureus biofilm formation, but it also disrupted a pre-formed biofilm. Mechanism studies demonstrated that Lp.LTA inhibited expression of the ica-operon, which is responsible for the production of poly-N-acetylglucosamine, a key molecule required for S. aureus biofilm development. Lp.LTA increased the release of autoinducer-2 from S. aureus, which contributed to the inhibition of S. aureus biofilm formation. Moreover, Lp.LTA treatment enhanced susceptibility of the biofilm to various antibiotics and to macrophages. Interestingly, Lp.LTA without D-alanine moieties was not able to inhibit biofilm formation by S. aureus. In conclusion, the present study suggests that LTA can inhibit S. aureus biofilm formation, and therefore could be applied for preventing and/or treating infectious diseases caused by S. aureus biofilms.

Lactobacillus plantarum lipoteichoic acid inhibits biofilm formation of Streptococcus mutans.

Dental caries is a biofilm-dependent oral disease and Streptococcus mutans is the known primary etiologic agent of dental caries that initiates biofilm formation on tooth surfaces. Although some Lactobacillus strains inhibit biofilm formation of oral pathogenic bacteria, the molecular mechanisms by which lactobacilli inhibit bacterial biofilm formation are not clearly understood. In this study, we demonstrated that Lactobacillus plantarum lipoteichoic acid (Lp.LTA) inhibited the biofilm formation of S. mutans on polystyrene plates, hydroxyapatite discs, and dentin slices without affecting the bacterial growth. Lp.LTA interferes with sucrose decomposition of S. mutans required for the production of exopolysaccharide, which is a main component of biofilm. Lp.LTA also attenuated the biding of fluorescein isothiocyanate-conjugated dextran to S. mutans, which is known to have a high affinity to exopolysaccharide on S. mutans. Dealanylated Lp.LTA did not inhibit biofilm formation of S. mutans implying that D-alanine moieties in the Lp.LTA structure were crucial for inhibition. Collectively, these results suggest that Lp.LTA attenuates S. mutans biofilm formation and could be used to develop effective anticaries agents.

Staphylococcal LTA antagonizes the B cell-mitogenic potential of LPS.

Lipoteichoic acid (LTA) of Gram-positive bacteria is regarded as the counterpart biomolecule of lipopolysaccharide (LPS) of Gram-negative bacteria because of their structural and immunological similarities. Although LPS induces a strong polyclonal expansion of B cells, little is known about the effect of LTA on B cell proliferation. In the present study, we prepared LTAs from Gram-positive bacteria and examined their effect on splenic B cell proliferation. Unlike LPS, LTA did not induce B cell proliferation. Instead, Staphylococcus aureus LTA (Sa.LTA) appeared to inhibit LPS-induced B cell proliferation in vitro, ex vivo, and in vivo models. Such effect was observed neither in splenocytes from Toll-like receptor 2 (TLR2)-deficient mice nor in the purified splenic B cells. Furthermore, decreased ERK phosphorylation appeared to be responsible for this phenomenon. Collectively, our results support that Sa.LTA inhibited LPS-induced B cell proliferation through the decrease of ERK phosphorylation via TLR2 signaling pathway.